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WO2009030153A1 - The roles of mirnas in the diagnosis and treatment of systemic lupus erythematosus - Google Patents

The roles of mirnas in the diagnosis and treatment of systemic lupus erythematosus Download PDF

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Publication number
WO2009030153A1
WO2009030153A1 PCT/CN2008/072159 CN2008072159W WO2009030153A1 WO 2009030153 A1 WO2009030153 A1 WO 2009030153A1 CN 2008072159 W CN2008072159 W CN 2008072159W WO 2009030153 A1 WO2009030153 A1 WO 2009030153A1
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ribonucleic acid
expression
seq
small ribonucleic
mir
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PCT/CN2008/072159
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French (fr)
Chinese (zh)
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Nan Shen
Yuanjia Tang
Huijuan Cui
Xiaobing Luo
Xuming Ni
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Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
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Publication of WO2009030153A1 publication Critical patent/WO2009030153A1/en

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    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111General methods applicable to biologically active non-coding nucleic acids
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/14Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141MicroRNAs, miRNAs
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    • C12N2320/00Applications; Uses
    • C12N2320/10Applications; Uses in screening processes
    • C12N2320/12Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
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    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/178Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • microRNA gene The role of microRNA gene in the diagnosis and treatment of systemic lupus erythematosus
  • the invention belongs to the field of biotechnology; in particular, the invention relates to a method and a kit for diagnosing systemic lupus erythematosus, and the application of a microRNA gene in the prevention and treatment of a disease associated with abnormal activation of type I interferon pathway (such as systemic lupus erythematosus) .
  • Background technique a technique for diagnosing systemic lupus erythematosus, and the application of a microRNA gene in the prevention and treatment of a disease associated with abnormal activation of type I interferon pathway (such as systemic lupus erythematosus) .
  • SLE Systemic Lupus Erythematosus
  • SLE Systemic Lupus Erythematosus
  • its clinical and immunological phenotypes are extremely complex and diverse, including immune tolerance defects, lymphocyte function regulation and apoptotic disorders, complement defects.
  • immune complex clearance disorder, cytokine secretion regulation disorder, etc. almost covering the entire immune system disorder, is recognized as the prototype of autoimmune disease.
  • the pathogenesis of SLE has not been fully elucidated. Because the etiology and pathogenesis are unknown, and there is no specific treatment, it is impossible to fundamentally improve the level of disease prevention and treatment.
  • Small RNA is a type of single-stranded small-molecule RNA with a length of about 21-25 nt that is not encoded. It is widely distributed in eukaryotes and regulates target mRNA by complementary binding of nucleic acid sequences to specific target mRNAs. Translation or degradation of target mRNA is a negatively regulated molecule.
  • miRNAs are involved in physiological processes such as development, differentiation, growth, and immune response, and their expression and dysfunction may lead to a variety of pathological phenomena such as tumorigenesis, leukemia and viral infection.
  • the 2' base of the 5' end of the miRNA plays a key role in its function. It is required that this region is completely paired with the target gene, and the matching requirements of other regions with the target sequence are not very strict.
  • Another object of the present invention is to provide the reagent or kit for diagnosing systemic lupus erythematosus.
  • a small ribonucleic acid for the preparation of a reagent or kit for detecting systemic lupus erythematosus (SLE);
  • small ribonucleic acid has the sequence shown by SEQ ID NO: 2CmiR-146a).
  • the small ribonucleic acid further comprises a small ribonucleic acid selected from the group consisting of:
  • RNA sequence of SEQ ID NO: 4 a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 4,
  • miR99a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 5
  • RNA molecules having the nucleic acid sequence of SEQ ID NO: 7,
  • a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 8
  • a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 9.
  • the reagent or kit is for detecting the degree of systemic lupus erythematosus activity or the degree of renal involvement.
  • kits for detecting systemic lupus erythematosus comprising:
  • the kit further comprises a primer or probe that specifically targets a small ribonucleic acid selected from the group consisting of:
  • RNA sequence of SEQ ID NO: 4 a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 4,
  • miR99a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 5
  • miR-10a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 6,
  • RNA molecules having the nucleic acid sequence of SEQ ID NO: 7,
  • miR-31 having the nucleic acid sequence of SEQ ID NO: 8, or
  • a small ribonucleic acid (miR-95) having the nucleic acid sequence of SEQ ID NO: 9.
  • the probe is a Taqman probe.
  • a small ribonucleic acid for the preparation of a composition for regulating a type I interferon pathway; or for screening for a substance which modulates a type I interferon pathway,
  • R is selected from A or G; Y is selected from C, U, or D.
  • the small ribonucleic acid is used to prepare a composition that inhibits aberrant activation of the type I interferon pathway; or for screening for a substance that inhibits abnormal activation of the type I interferon pathway.
  • composition is also useful for the prevention and treatment of systemic lupus erythematosus.
  • the small RNA-regulated type I interferon pathway is selected from, but not limited to, (a) inhibiting expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), and (b) inhibiting interleukin 1 expression of receptor-associated kinase 1 (IRAKI), (c) inhibition of expression of interferon-inducible factor 5 (IRF5); (d) inhibition of signal transduction and expression of activator of transcription (STAT1) (e) inhibition of IFN a (f) inhibition of expression; (g) inhibition of mucinous virus resistance factor 1 (MX1) expression; (h) inhibition of 5, oligonucleotide synthetase (0AS1) expression; or (e) inhibition of lymphocyte antigen 6 (Ly6E) expression.
  • the regulation includes: direct regulation or indirect regulation.
  • a method of screening for a potential substance that modulates a type I interferon pathway comprising the steps of:
  • R is selected from A or G; Y is selected from C, U, or T;
  • the candidate substance can increase the expression of the small ribonucleic acid, it indicates that the candidate substance is a latent substance that inhibits the type I interferon pathway; if the candidate substance can reduce the expression of the small ribonucleic acid, it indicates that the candidate substance is Potential substances that promote the type I interferon pathway.
  • step (a) comprises: adding a candidate substance to the system containing the small RNA in the test group; and/or
  • the step (b) comprises: detecting the expression of the system ribonucleic acid of the test group, and comparing the control group to the control group containing the small RNA-containing system without adding the candidate substance;
  • test picorna is statistically higher (preferably significantly higher than, for example, 20% higher, more preferably 40% higher, further preferably 60% higher or higher) in the control group, it indicates that the candidate is inhibition I a substance of the type interferon pathway; if the expression of the test small RNA is statistically lower (preferably significantly lower than, for example, 20% lower, more preferably 40% lower, further preferably 60% lower or lower), This candidate is indicated to be a substance that promotes the type I interferon pathway.
  • the system is a cell system (e.g., Hela cells (or cell culture), HEK 293 cells (or cell culture), or primary cultured PBMC cells (or cell culture).
  • a cell system e.g., Hela cells (or cell culture), HEK 293 cells (or cell culture), or primary cultured PBMC cells (or cell culture).
  • Figure 1 shows the decrease in miR-146a expression levels in SLE patients.
  • A A clustering analysis of 42 miRNAs with significant differences in expression levels between SLE patients and normal controls, with arrows pointing to 7 miRNAs with a greater than six-fold decline.
  • FIG. 2 shows that miR-146a expression levels are associated with disease activity. among them,
  • A: miR-146a was compared in the normal control, stable SLE patients and active SLE patients.
  • Figure 4 shows the role of miR-146a in the activation of the type I interferon pathway.
  • A Overexpression of miR-146a is able to inhibit the production of type I interferons, which are capable of recognizing the most IFN a subtype.
  • B The PBMC was firstly transfected with the miR-146a inhibitory sequence or the random unrelated sequence, and then stimulated with R837 (5ug/ml). Two hours later, the difference in expression levels of miR-146a between the two different treatment methods was detected.
  • Type I interferon 1000 U/ml was incubated with 293T/ISRE for 6 hours and transfected into miR_146a. The results showed that miR-146a inhibited the activity of I SRE reporter gene, and the data was displayed in the SEM format.
  • Type I interferon 1000 U/ml was incubated with PBMC for 6 hours and transfected into miR_146a.
  • MiR-146a in PBMC primary cells inhibited the production of interferon-inducible genes downstream of type I interferon.
  • FIG. 5 shows the identification of two target genes miR-146a, IRF5 and STAT1.
  • Figure 6 shows the decrease in the expression level of interferon-inducible genes after artificially increasing the expression level of miR-146a in patient-derived PBMC.
  • Figure 7 shows the effect of different stimulants on the expression level of miR-146a in normal human-derived PBMCs.
  • the stimulant concentrations were LPS (10 ug/ml), R837 (5 ug/ml), CpG-A (5 uM) and type I interferon (1000 U/ml), and the stimulating time was 6 hours.
  • Figure 8 shows the miR-146a negative feedback regulation of the type I interferon pathway. detailed description
  • miRNAs small ribonucleotides having the sequence represented by the general formula (I) are closely related to systemic lupus erythematosus (SLE). The inventors have further verified the target gene of the miRNA. Furthermore, the inventors have also found that the small ribonucleic acid can inhibit the type I interferon pathway and can be used to prevent diseases associated with abnormal activation of the type I interferon pathway (e.g., systemic lupus erythematosus). The present invention has been completed on this basis. As used herein, unless otherwise defined, the term "miR-146" refers to miR-146a or miR-146b.
  • the inventors have found that a small ribonucleic acid can regulate the type I interferon pathway.
  • the small ribonucleic acid has the following formula:
  • R is selected from A or G; Y is selected from C, U, or D.
  • the small ribonucleic acid represented by the general formula (I) differs only in two bases at the 3' end, and the key sequence in which the small ribonucleic acid functions is located at the 5' end 2-8 bases; and, the general formula (I)
  • the small ribonucleic acids shown have the same target genes, so they have substantially the same function of regulating the type I interferon pathway.
  • the small ribonucleic acid preferably has the sequence shown by SEQ ID NO: 2 (miR-146a) or SEQ ID NO: 3 (miR_146b).
  • the small ribonucleic acid is a small ribonucleic acid (miR_146a) having the nucleic acid sequence of SEQ ID NO: 2, which is expressed very low in systemic lupus erythematosus, and further studies have found that The expression signal of the small ribonucleic acid is inversely related to the degree of activity of the systemic lupus erythematosus and the degree of renal involvement; in systemic lupus erythematosus, the target gene of the small ribonucleic acid is significantly higher than the normal control group and the small ribose sugar The expression signal of the nucleic acid is negatively correlated.
  • miR_146a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 2, which is expressed very low in systemic lupus erythematosus
  • small ribonucleic acids selected from the group consisting of very low expression in systemic lupus erythematosus (more than 6 times lower than normal human levels):
  • RNA sequence of SEQ ID NO: 4 a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 4,
  • miR99a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 5
  • miR-10a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 6,
  • RNA molecules having the nucleic acid sequence of SEQ ID NO: 7,
  • miR-31 having the nucleic acid sequence of SEQ ID NO: 8, or
  • a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 9.
  • the picornacle nucleic acid has many new uses. These uses include (but are not limited to):
  • tumor necrosis factor receptor-associated factor 6 interleukin-1 receptor-associated kinase 1, interferon-inducible factor 5 or signal transduction and activator of transcription 1, or preventing or treating tumor necrosis factor receptor a related factor 6, an interleukin 1 receptor-associated kinase 1, an interferon-inducible factor 5 or a signal transduction and a transcriptional activator 1 expression or activity disorder associated with a disease;
  • a substance which regulates the type I interferon pathway which is used, for example, to prepare a drug for inhibiting a disease associated with abnormal activation of the type I interferon pathway, is selected.
  • various methods well known in the art can be used for screening and regulation.
  • a substance of the type I interferon pathway A substance of the type I interferon pathway.
  • a screening method comprising: contacting a candidate substance with a system containing a small ribonucleic acid represented by the general formula (I); and observing the candidate substance for the general formula (I) Effect of expression of the displayed small ribonucleic acid; if the candidate substance can be increased (preferably significantly increased, such as by 20% or less; more preferably by 40%) Or higher) Expression of the small ribonucleic acid represented by the general formula (I) indicates that the candidate substance is a latent substance that inhibits the type I interferon pathway; conversely, the candidate substance is a latent substance that promotes the type I interferon pathway.
  • the influence of the candidate substance on the expression of the small ribonucleic acid represented by the general formula (I) can be observed by setting a control group; the control group is not containing the candidate substance and contains the formula (I) A system of small RNAs.
  • the agonist or antagonist of the small ribonucleic acid represented by the general formula (I) can modulate the expression of the small ribonucleic acid represented by the general formula (I)
  • the antagonist can modulate the type I interferon pathway by affecting the small ribonucleic acid represented by the general formula (I).
  • the agonist of the small ribonucleic acid represented by the general formula (I) means any one which can maintain the stability of the small ribonucleic acid represented by the general formula (I), promote the expression of the small ribonucleic acid represented by the general formula (I), and prolong the passage.
  • the substance having a small ribonucleic acid effective time as shown in the formula (I), which can be used in the present invention, is useful as a substance useful for regulating (especially inhibiting) the type 1 interferon pathway.
  • the antagonist of the small ribonucleic acid represented by the general formula (I) is any substance which can reduce the stability of the small ribonucleic acid represented by the general formula (I) and inhibit the expression of the small ribonucleic acid represented by the general formula (I). These materials can be used in the present invention as a useful substance for regulating (especially promoting) type 1 interferon pathways.
  • the antagonist is, for example, an antisense oligonucleotide chain of miR-146a or miR-146b or an analog thereof; the antagonist can be developed into some tumors and infectious diseases by enhancing the type I interferon pathway. Therapeutic drugs.
  • the small ribonucleic acid represented by the general formula (I) or a gene encoding the same can be used by various methods well known in the art, or
  • the pharmaceutical composition is administered to a subject in need of treatment (e.g., a patient with SLE). Preferably, it can be carried out by means of gene therapy.
  • the small ribonucleic acid represented by the general formula (I) or an analog thereof can be directly administered to a subject by a method such as injection; or, the small ribonucleic acid represented by the general formula (I) can be carried by a certain route.
  • the expression unit of the analog or analog thereof is delivered to the target and expressed to express the small ribonucleic acid represented by the general formula (I), which are well known to those skilled in the art.
  • the analog of the small ribonucleic acid means that the key region is identical to the sequence of the formula (I) (ie, positions 2-8 in SEQ ID NO: 1) and has the same sequence as the formula (I). Or close proximity to the small RNA that regulates the type I interferon pathway.
  • the analog has 70% or more homology with the sequence of SEQ ID NO: 2, more preferably 85% or more homologous, and most preferably has 95% or more homology.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intramuscular, intravenous, or subcutaneous administration.
  • the small ribonucleic acid or an agonist or antagonist thereof can be directly used for the treatment of diseases, for example, for the treatment of systemic lupus erythematosus.
  • other agents for treating systemic lupus erythematosus can be used at the same time.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a safe and effective amount of a small ribonucleic acid of the invention, or an agonist or antagonist thereof, and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 0.1 microgram per kilogram body weight to about 10 milligrams per kilogram body weight per day.
  • a safe and effective amount of the picorucleic acid or an agonist thereof is administered to a mammal, wherein the safe and effective amount is usually at least about 0.1 microgram per kilogram of body weight, and in most cases no more than about 10
  • the mg/kg body weight preferably the dose is from about 0.1 microgram per kilogram body weight to about 100 micrograms per kilogram body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the small ribonucleic acid such as miR-146a or miR-146b
  • the content and expression of the small ribonucleic acid in the subject can be increased, thereby preventing or treating the small Ribonucleotide-related diseases, such as disorders associated with abnormal activation of the interferon pathway.
  • the small Ribonucleotide-related diseases such as disorders associated with abnormal activation of the interferon pathway.
  • the present invention also relates to a diagnostic test method for quantitatively and qualitatively detecting said small ribonucleic acid levels to determine the occurrence or development of systemic lupus erythematosus.
  • diagnostic test method for quantitatively and qualitatively detecting said small ribonucleic acid levels to determine the occurrence or development of systemic lupus erythematosus.
  • assays are well known in the art and include, for example, but not limited to: real-time fluorescent quantitative PCR, cluster analysis, non-parametric Mann-Whitney test, Spearman correlation analysis, and the like.
  • a method for detecting the presence or absence of the small ribonucleic acid and the amount thereof in a tissue or sample to be tested is detected by real-time fluorescent quantitative PCR, which comprises: preparing a specific probe for each miRNA (the probe preferably carries a probe The detection signal), specific primers for each miRNA are prepared, PCR is performed, and the level of the small RNA is judged by detecting the intensity of the detectable signal after the end of the PCR.
  • detection is based on TaqMan fluorescence technology, i.e., based on a TaqMan fluorescent probe.
  • the TaqMan fluorescent probe is an oligonucleotide having a fluorescent emitting group (as a detectable signal) and a fluorescence quenching group, respectively, at both ends.
  • the fluorescent signal emitted by the fluorescent emitting group is absorbed by the quenching group;
  • the 5'_3' exonuclease activity of the Taq enzyme degrades the probe, and the fluorescent emitting group and The fluorescence quenching group is separated, so that the fluorescence signal can be accepted by the fluorescence detecting system, that is, every time one DNA strand is amplified, one fluorescent molecule is formed, that is, the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product, thereby achieving qualitative and Quantitative.
  • the small ribonucleic acid-specific primers and/or probes are also included in the present invention for detecting the presence or absence of the small ribonucleic acid in a sample and for determining whether the subject has a system. Lupus erythematosus or risk of illness.
  • the probes can be immobilized on a microarmy or gene chip for analysis of differential expression analysis and genetic diagnosis of miRNAs in tissues or samples.
  • the invention also includes a kit for detecting the expression level of a miRNA, thereby diagnosing systemic lupus erythematosus, which kit may comprise specifically amplifying said miRNA (selected from at least one of the group consisting of: miR-146a Primers for miR-130b, miR99a, miR_10a, miR_134, miR_31, miR-95).
  • it further comprises: a probe that specifically binds to said miRNA; more preferably said probe carries a detectable signal.
  • the small ribonucleic acid is a small ribonucleic acid represented by the general formula (I).
  • the kit further contains instructions for clinical detection of systemic lupus erythematosus.
  • a total of 52 patients with SLE were selected, including the outpatient and ward patients of Shanghai Renji Hospital, which were from Shanghai and many other provinces and cities in China.
  • the diagnosis was in accordance with the 11 criteria recommended by the American College of Rheumatology SLE classification criteria. At least 4 of them, including 4 males and 48 females, with an average age of 33.5 years (12-60 years), 47 patients were divided into SLE inactive groups according to SLE disease activity scores (SLEDAI 2K). 0 to 4 points, ie SLE stable group) 18 cases and SLE activity group (5 points) 29 cases. Of these, 26 met the criteria for ARA lupus nephritis.
  • miRNA reverse transcription primers and Real-time PCR reaction probes were purchased directly from ABI (Applied Biosystems, Inc., TaqMan® MicroRNA (miRNA) Assays); in addition, one skilled in the art can conveniently design the miRNA sequences according to the described miRNA sequences. Primers and probes.
  • TRAF6 antisense strand 5 ' ATTGATGCAGCACAGTTGTC 3 ' (SEQ ID NO: 13);
  • Ly6E antisense strand 5 ' GCACACATCCCTACTGACAC 3 ' (SEQ ID NO: 15);
  • MX1 sense chain 5 ' GGGTAGCCACTGGACTGA 3 ' (SEQ ID NO: 18),
  • MX1 antisense strand 5 ' AGGTGGAGCGATTCTGAG 3 ' (SEQ ID NO: 19);
  • IFN a sense strand 5 ' -TCCATGAGATGATCCAGCAG-3 ' (SEQ ID NO: 24), IFNa antisense strand: 5, -ATTTCTGCTCTGACAACCTCCC-3' (SEQ ID NO: 25); IFNP sense strand: 5, - TCTAGCACTGGCTGGAATGAG-3, (SEQ ID NO: 26),
  • IFNP antisense strand 5'-GTTTCGGAGGTAACCTGTAAG-3' (SEQ ID NO: 27).
  • the target fragment sequence of the amplification was prevented from being non-specific by BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) and synthesized by Shanghai Biotech Co., Ltd.
  • RPL13A The quantitative reference primer for the internal reference gene-ribosomal protein L13a (RPL13A) was derived from the sequence reported on the Quantitative PCR Primer Database (QPPD) website, and was also synthesized by Shanghai Shenggong Company:
  • RPL13A sense strand 5' CCTGGAGGAGAAGAGGAAAGAGA 3' (SEQ ID NO: 20),
  • RPL13A antisense strand 5' TTGAGGACCTCTGTGTATTTGTCAA 3' SEQ ID NO: 21.
  • RNA of the blood sample was extracted by Trizol phenol chloroform method, and the obtained RNA was identified by capillary electrophoresis (NanoDrop Specthophotometer), and its concentration was determined by an ultraviolet spectrophotometer.
  • RNA was used as oligo dT reverse transcription, using the Superscript II reverse transcriptase kit
  • RNA-specific primer Another part of total RNA was used as a miRNA-specific primer for reverse transcription.
  • the loading system was:
  • dNTP 0.03ul, MMLV 0.2ul, 10X buffer 0 ⁇ 3ul, RNase inhibitor 0.02ul, dd3 ⁇ 40 (no RNase) 0.45ul, primer lul, RNA lul.
  • the loading system using miRNA Taqman method was Master Mix 2ul, specific probe lul and template lul. Two negative wells were made for each sample, and the difference of CT values between negative holes was controlled at 0.5. Within the CT, there is an inter-plate control on each board.
  • the reaction conditions were 50 ° C for 2 min, 95 ° C lOmin; then 95 ° C for 15 s, 60 ° C lmin for a total of 40 cycles.
  • the target gene SYBR Green quantitative loading system is SYBR Green Master Mix 2.5ul, R0X 0. lu primer (sense strand 0. lul, antisense strand 0. lul), dd3 ⁇ 40 1.2ul and cDNA template lul, also do two for each sample
  • the difference between the negative and negative CT values is controlled within 0.5 CT, and each plate is provided with an inter-plate control.
  • the reaction conditions were 95 ° C for 15 s; then a total of 40 cycles were carried out at 95 ° C for 5 s and 60 ° C for 30 s; then 95 ° C for 15 s, 60 V for 15 s, and 95 ° C for 15 s. 5.
  • IFN integral calculations are based on existing related research reports [Xuebing Feng, Hui Wu, Bevra H. Hahn, and Betty P. Tsao, et al Association of Increased Interferon-Inducible Gene Expression With Disease Activity and Lupus Nephritis in Patients With Systemic Lupus Erythematosus .
  • Gene i ctr gene expression level in the normal control group.
  • the mean value of IFN score in SLE patients was 65. 3, the minimum and maximum values were _0. 45 and 412. 6 respectively; the three values in the normal control group were 0, -1.88 and 6.55, respectively. 6.
  • the CT values of each well were analyzed, and the samples with CT values less than 33 and good repeatability between the two wells were selected, and the 2 ( -AA CT ) value after standardization with internal parameters was calculated, which is the number of initial copy numbers of the gene.
  • the first batch of expression data was first analyzed using SAM 2. 20 software, and then the data with different expressions were imported into HCE 3. 0 software for cluster analysis. The latter batch of expanded sample data was analyzed with Graph Pad 4. 03 software, and two sets of independent sample data were compared for non-parameters.
  • a miR-146a precursor fragment of about 280 bp in length was amplified by PCR, and the primers were as follows:
  • Upstream primer 5 ' GTGAGATCTGCATTGGATTTACC 3 ' (SEQ ID NO: 22);
  • Downstream primer 5 'GACCTCGAGACTCTGCCTTCTGT 3 ' (SEQ ID NO: 23).
  • the enzyme was digested with Bgl l l/Xho I and inserted into the pSUPER basic vector (OligoEngine) which was similarly digested.
  • the integrity and accuracy of the insert was determined by conventional sequencing methods to obtain the correct miR-146a expression vector.
  • Interferon-inducible factor 5 IRF5
  • UTR 3 ' untranslated region
  • 293T and P SMMC-7721 cells were cultured in DMEM supplemented with 10% FBS, and 293T/ISRE (293T cells stably transfected with ISRE) were cultured in hygromycin with 10% FBS and 20 ⁇ / ⁇ 1.
  • these three cell lines were transfected with liposome 2000 (l nv itrog en ).
  • Peripheral blood mononuclear cells (PBMC) were sorted from human peripheral blood by density gradient centrifugation (Cedarlane), and then cultured for 2 hours in RPMI 1640 supplemented with 10% FBS using an electrorotator (Amaxa, programme T -16) The 1.
  • 5 ⁇ empty vector or miR-146 expression vector was separately transferred into 3 ⁇ 10 6 PBMC, otherwise electroporated into 3 ⁇ M RIDIANTM (with miR-146a inhibitor) or a random unrelated sequence (Dharmacon Inc.) ).
  • RIDIANTM with miR-146a inhibitor
  • a random unrelated sequence Dharmacon Inc.
  • stimulating cells first change the cells 18 or 24 hours after transfection, then culture the cells separately in fresh medium and add type I interferon (PBL Interferonsource) or various TLR ligands (Invivogen). In fresh medium.
  • PBL Interferonsource type I interferon
  • TLR ligands Invivogen.
  • the SMMC-7721 cell line was plated in a 96-well plate, and 20 ng of 3' UTR fluorescent reporter vector, 10 ng of pRL-TK vector (Promega) and 270 ng of empty vector (pSUPER basic) or miR_146a expression vector were mixed and transfected into each.
  • Well S ⁇ C-7721 cells were cultured for 24 hours after transfection. After lysis, they were assayed according to the dual fluorescence reporter system (Promega) (TR717, Applied Biosystems). For each well, The ratio of the fluorescence signal of an ISRE to Reni l la.
  • the 293T/ISRE cell line was subjected to the same procedure as above, except that 300 ng of empty vector or miR-146a expression vector was transferred. Each experiment was a quadruple well and each experiment was repeated three times.
  • the 293T cells were first cultured in a 6-well plate, and then transfected into 3 g of miR-146a expression vector in each well. After transfection for 24 hours, the cells were lysed by lysis. The supernatant was subjected to electrophoresis by adding SDS-PAGE gel, and immunoblotted with a direct antibody, and detected by Luminol/Enhancer reagent (Pierce). IRF5 and GAPDH antibodies were purchased from Abeam and Chen icon, respectively, and SATAl and HRP-labeled secondary antibodies were sourced from Santa Cruz. The relative expression of protein was obtained by calculation and analysis by Quantity One 4. 52 software (Bio-Rad). Example 1 Difference in expression levels between a group of miRNAs in SLE patients and normal controls
  • the inventors used the same method to detect the expression level of miR-146a in 47 patients with SLE, 6 patients with BD, and 21 normal controls.
  • the expression level of miR_146a in SLE patients was significantly lower than that of normal controls.
  • miR-146a the degree of disease activity and renal involvement
  • the miR-146a level was significantly lower in the SLE patient activity group (SLEDAI score 5) than in the stable group (SLEDAI score 0 to 4 points), and the nonparametric Mann-Whitney test showed a statistically significant difference (7). 0080), see Figure 2A. .
  • the inventors divided the SLE into a urine protein-positive group (24-hour urine protein greater than 0.5 g) and a urine protein-negative group (24-hour urine protein less than 0.5 g) according to the 24-hour urine protein level. After analysis, it was found that the level of miR-146a was also significantly different between the two groups (7). 0271), see Figure 2B.
  • IRAKI and TRAF6 can be transmitted through multiple signaling pathways leading to the production of downstream type I interferon, and that type I interferon pathway plays a key role in the pathogenesis of lupus.
  • Example 4 miR-146a Negative Feedback Regulation Type I Interferon Pathway
  • miR-146a was electroporated into a normal human-derived PBMC and then stimulated with TLR7 ligand R837 (purchased from invivogen) to detect type I interferon mRNA levels.
  • type I interferon After type I interferon is induced, it binds to cell membrane surface interferon receptors (such as IFNAR1 or IFNAR2) to phosphorylate downstream STAT.
  • Activated STAT1 and STAT2 together with the DNA-binding protein interferon-inducible factor 9 (IRF9), form interferon-stimulating gene factor 3 (ISGF3), which binds to ISRE to generate an activation signal that stimulates downstream interferon-inducible gene production [ Platanias IX.
  • miR-146a can effectively regulate the activation of type I interferon downstream.
  • Example 5 miR-146a regulates type I interferon pathway through multiple key signaling molecules
  • miR-146a is a negative feedback regulator of the type I interferon pathway.
  • the inventors used bioinformatics to analyze the potential target genes of miR-146a. A number of software analyses have shown that miRNAs act primarily on the positive regulatory motif of the gene and its downstream network of signal transduction molecules. Therefore, the inventors predicted the key signaling proteins of the type I interferon pathway and the miR-146a sequence to find potential binding sites. Use miRBase
  • Example 6 Human elevation of miR-146a expression levels in SLE patients can partially reverse type I interferon activation levels miR-146a regulates innate immune responses by modulating key signaling molecules on the type I interferon pathway, then artificially elevated SLE patients Whether the level of miR-146a expression in vivo reverses the extent of over-activation of the type I interferon pathway. Transferring miR-146a to PBMC from a patient with SLE, elevated levels of miR-146a reduced the level of some interferon-inducible genes, as shown in Figure 6.
  • Test group Recombinant HEK 293T with candidate substance added (for preparation method, see item 8 in Materials and Methods) Culture;
  • Control group Recombinant HEK 293T (see Section 8 of Materials and Methods) cultures without the addition of candidate substances.
  • miR-146a can inhibit the type I interferon pathway. Decreasing the level of miR_146a or inhibiting the action of miR_146a with the antisense oligonucleotide chain of miR-146a and its analogs would enhance the type I interferon pathway. As shown in Figure 4B, the antisense oligonucleotides of miR-146a inhibited miR_146a, and the expression of IFN a was significantly higher than that of the control group. Discussion
  • the invention firstly detects the difference of miRNA expression in SLE patients, and analyzes the differential expression of miR_146a in the activation of type I interferon pathway, and further expands the pathogenesis of autoimmune diseases. miR-146a
  • the apparent correlation between expression and disease activity also suggests that miR-146a can be used as a new biomarker for SLE.
  • the invention excludes the influence of infectious factors in the sample screening stage, and secondly, the difference between the expression level of miR-146a and the dose of different drugs (including hormones and immunosuppressive agents) is not found, so the drug factor is excluded. The interference with the results indicates that the difference in results is indeed due to the disease itself.
  • miR-146 a The level of miR-146 a was significantly lower in patients than in normal controls, and its level was negatively correlated with SLEDAI score and Renal score reflecting the degree of disease activity and renal damage, suggesting that miR-146 a can be used as a measure of disease activity and kidney.
  • a biomarker of severity of damage provides a good basis for early and convenient diagnosis of SLE, assessing the extent of disease activity and the severity of kidney involvement.
  • the interferon pathway is known to play an important role in the pathogenesis of SLE. Abnormal activation of the interferon pathway is a major molecular phenotype of SLE [Pasciml V et al., Banchereau J Systemi c lupus erythematosus: al l roads lead to type I interferons. Curr Op in Immunol. 2006 Dec; 18 (6): 676-82. Epub 2006 Oct 2].
  • TRAF6/IRAK1 acts as a linker protein downstream of the TLR pathway and plays a key role in the induction of type I IFN production [Uematsu S, Sato S Interleukin-1 receptor-assocated kinase-1 plays an essent ial role for Tol ll receptor (TLR) 7- and TLR9-mediated
  • TLR plays an important role in autoimmune response by binding to its corresponding ligand.
  • P DC plasma-derived dendritic cells
  • RNA-binding nucleoprotein and self-DNA bind to the corresponding autoantibodies.
  • the immune complex can promote the large-scale production of type I interferon by binding to TLR7 or TLR9, which requires the activation of the sequence of signal adaptor proteins and transcription proteins including MyD88, IRAKI, TRAF6, IRF5 and IRF7.
  • the current study found that autoimmune complexes containing RNA mainly activate TRF7 and TLR9, which are constitutively expressed on the endosomal membrane of cells, to activate downstream IRF5 and play an important role in the production of type I interferon.
  • interferon Once interferon is produced, it can bind to the interferon receptor, and the activation of the STAT protein ultimately leads to the transcription of the interferon-inducible gene.
  • immune cells Under physiological conditions, immune cells can spontaneously regulate the TLR signaling pathway through a variety of mechanisms to ensure that the immune system is in a relatively balanced state, including intracellular constitutive expression or induction of negative regulators (such as MyD88s, IRAKM, S0CS1), a defect in negative feedback regulation causes excessive conduction of the positive signal and ultimately causes human disease.
  • negative regulators such as MyD88s, IRAKM, S0CS1
  • miRNAs also belong to a class of negative regulators.
  • miR-146 was found to regulate TLR4 signaling by forming a negative feedback regulatory pathway.
  • R837 TLR7 ligand
  • CpG TLR9 ligand
  • type I interferon and LPS TLR4 ligand
  • miR-146a does participate in a complex regulatory network of innate immune responses.
  • TLR7 and TLR9 in THP-1 cell lines do not stimulate miR_146a expression, it may be due to differences in TLR expression patterns and different stimuli. Because studies have shown that single cell reactions in simple medium in vitro have no way to replicate the experimental results of multiple interactions between human peripheral blood hormones and cytokines.
  • miR-146a inhibits the production of downstream type I interferon and the reactivity downstream of interferon through the TLR7 pathway.
  • the inventors used bioinformatics methods to predict, based on the original findings, predicted two new target genes: IRF5 and STAT1, all of which are upstream and downstream of the interferon pathway.
  • a key signaling molecule therefore miR-146a is able to modulate multiple components of the type I interferon pathway (see Figure 8).
  • miR-146a has a limited regulatory effect on a target gene, but the synergy of several target genes significantly amplifies the regulation of miR-146a.
  • miR-181a effectively reduces the excitatory threshold of T cell recognition by antigen by mild regulation of multiple phosphatases, which is a powerful regulation of siRNA single target. incomparable.
  • Defective expression of miR-146a leads to a large accumulation of its target gene, which can further promote the large-scale production of type I interferon and the excessive activation of downstream signals.
  • the results show a negative relationship between miR-146a expression and type I interferon. The correlation is also consistent with the above hypothesis, and this study reveals the role of miRNA dysregulation in autoimmune diseases.
  • the inventors applied bioinformatics analysis to find a potential methylation site in the miR-146a promoter region (http: cp cpgislands. usc. edu/). The location of the CpG island overlaps significantly with a predicted STAT1 binding site and a corroborating NF K B binding site.
  • TLR and IFN antagonists are targets for the treatment of SLE, but they have to be treated with caution because of their potential threat to innate and adaptive immunity.
  • miRNAs regulate the target genes quantitatively. Therefore, artificial regulation of miRNA expression levels in vivo may develop into a new means of combating SLE.
  • the results of this study also showed that when the expression level of miR-146a in PBMC of SLE patients was artificially increased, the expression of interferon-inducible genes in response to the activation of interferon pathway was significantly down-regulated.
  • miR-146a can be used as a target for new SLE treatment.
  • miR-146a expression defects in SLE patients are closely related to disease biology and clinical phenotype.
  • the inventors' results suggest that miR-146a can be used as a new biomarker for SLE, and artificially changing the expression level of miR-146a in patients can be developed into a new treatment.

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Abstract

The present invention belongs to biotechnical field, which discloses the uses of a series of miRNAs in the preparation of reagents or kits for the detection of systemic lupus erythematosus (SLE). Reagents or kits for the detections of expression levels of these miRNAs and thus for diagnosis of SLE are also disclosed. It is the first time to reveal and demonstrate the close correlation between these miRNAs and SLE. These miRNAs are new drug targets of the prophylaxis and treatment of SLE, and the regulations of the expression levels of the same are novel measurements for the treatment of the disease.

Description

微小 RNA基因在系统性红斑狼疮疾病诊断和治疗中的作用 技术领域  The role of microRNA gene in the diagnosis and treatment of systemic lupus erythematosus
本发明属于生物技术领域; 具体地, 本发明涉及诊断系统性红斑狼疮的方法和试剂 盒, 以及微小 RNA基因在 I型干扰素通路异常激活相关疾病(如系统性红斑狼疮疾病)防 治中的应用。 背景技术  The invention belongs to the field of biotechnology; in particular, the invention relates to a method and a kit for diagnosing systemic lupus erythematosus, and the application of a microRNA gene in the prevention and treatment of a disease associated with abnormal activation of type I interferon pathway (such as systemic lupus erythematosus) . Background technique
系统性红斑狼疮(Systemic Lupus Erythematosus , SLE)是典型的非器官特异性自 身免疫病, 其临床及免疫学表型极为复杂多样, 包括免疫耐受缺陷, 淋巴细胞功能调节 和凋亡障碍, 补体缺陷和免疫复合物清除障碍, 细胞因子分泌调节障碍等, 几乎覆盖整 个免疫系统的紊乱, 被公认为是自身免疫病的原型。 目前 SLE的发病机制还没有完全阐 明, 由于病因和发病机理未明, 缺乏特异的治疗手段, 无法从根本上提高疾病防治水平。  Systemic Lupus Erythematosus (SLE) is a typical non-organ-specific autoimmune disease, and its clinical and immunological phenotypes are extremely complex and diverse, including immune tolerance defects, lymphocyte function regulation and apoptotic disorders, complement defects. And immune complex clearance disorder, cytokine secretion regulation disorder, etc., almost covering the entire immune system disorder, is recognized as the prototype of autoimmune disease. At present, the pathogenesis of SLE has not been fully elucidated. Because the etiology and pathogenesis are unknown, and there is no specific treatment, it is impossible to fundamentally improve the level of disease prevention and treatment.
人类疾病很多是由于一些基因表达紊乱或失控所引起的。小 RNA (microRNA, mi RNA) 是一类长度约 21-25nt的不编码蛋白质的单链小分子 RNA, 广泛存在于真核生物中, 通 过核酸序列互补性结合到特定的靶 mRNA上来调节靶 mRNA翻译或降解靶 mRNA,是一种起 负调控作用的分子。 目前研究显示许多 miRNA参与了生物体发育、 分化、 生长、 免疫应 答等生理过程, 且其表达及功能失调可能导致肿瘤发生, 白血病以及病毒感染等多种病 理现象。 目前研究认为, miRNA的 5 ' 端 2-8个碱基对其发挥作用起着关键作用, 要求这 区域与靶基因完全配对, 其它区域与靶序列的配对要求不是很严格。  Many human diseases are caused by some gene expression disorders or loss of control. Small RNA (microRNA, mi RNA) is a type of single-stranded small-molecule RNA with a length of about 21-25 nt that is not encoded. It is widely distributed in eukaryotes and regulates target mRNA by complementary binding of nucleic acid sequences to specific target mRNAs. Translation or degradation of target mRNA is a negatively regulated molecule. At present, many miRNAs are involved in physiological processes such as development, differentiation, growth, and immune response, and their expression and dysfunction may lead to a variety of pathological phenomena such as tumorigenesis, leukemia and viral infection. At present, it is believed that the 2' base of the 5' end of the miRNA plays a key role in its function. It is required that this region is completely paired with the target gene, and the matching requirements of other regions with the target sequence are not very strict.
尽管目前有报道显示 miR-146在免疫应答中起着重要的作用,然而本领域仍然不知 道其对于一些具体的免疫疾病的影响状况, 更没有报导其与系统性红斑狼疮的关系。 发明内容  Although miR-146 has been reported to play an important role in the immune response, it is still unknown in the field about the effects of specific immune diseases, and its relationship with systemic lupus erythematosus has not been reported. Summary of the invention
本发明的目的在于提供小核糖核酸的用途,用于诊断系统性红斑狼疮或制备诊断系 统性红斑狼疮的试剂或试剂盒, 或作为筛选疾病治疗药物的靶点, 或直接用于治疗相关 疾病。  It is an object of the present invention to provide a use of small RNA for the diagnosis of systemic lupus erythematosus or to prepare a reagent or kit for diagnosing systemic lupus erythematosus, or as a target for screening for a therapeutic drug, or for directly treating a related disease.
本发明的另一目的在于提供所述诊断系统性红斑狼疮的试剂或试剂盒。  Another object of the present invention is to provide the reagent or kit for diagnosing systemic lupus erythematosus.
在本发明的第一方面, 提供一种小核糖核酸的用途, 用于制备检测系统性红斑狼疮 (SLE) 的试剂或试剂盒;  In a first aspect of the invention, there is provided a use of a small ribonucleic acid for the preparation of a reagent or kit for detecting systemic lupus erythematosus (SLE);
其中, 所述的小核糖核酸具有 SEQ ID NO: 2CmiR-146a)所示的序列。  Wherein the small ribonucleic acid has the sequence shown by SEQ ID NO: 2CmiR-146a).
在另一优选例中, 所述的小核糖核酸还包括选自下组的小核糖核酸:  In another preferred embodiment, the small ribonucleic acid further comprises a small ribonucleic acid selected from the group consisting of:
具有 SEQ ID NO: 4所示核酸序列的小核糖核酸 (miR-130b),  a small ribonucleic acid (miR-130b) having the nucleic acid sequence of SEQ ID NO: 4,
具有 SEQ ID NO: 5所示核酸序列的小核糖核酸 (miR99a),  a small ribonucleic acid (miR99a) having the nucleic acid sequence of SEQ ID NO: 5,
具有 SEQ ID NO: 6所示核酸序列的小核糖核酸 (miR-lOa),  a small ribonucleic acid (miR-lOa) having the nucleic acid sequence of SEQ ID NO: 6,
具有 SEQ ID NO: 7所示核酸序列的小核糖核酸 (miR-134),  a small ribonucleic acid (miR-134) having the nucleic acid sequence of SEQ ID NO: 7,
具有 SEQ ID NO: 8所示核酸序列的小核糖核酸 (miR-31), 或 具有 SEQ ID NO: 9所示核酸序列的小核糖核酸 (miR-95)。 a small ribonucleic acid (miR-31) having the nucleic acid sequence of SEQ ID NO: 8, or A small ribonucleic acid (miR-95) having the nucleic acid sequence of SEQ ID NO: 9.
在另一优选例中,所述的试剂或试剂盒用于检测系统性红斑狼疮疾病活动程度或肾 脏受累程度。  In another preferred embodiment, the reagent or kit is for detecting the degree of systemic lupus erythematosus activity or the degree of renal involvement.
在本发明的第二方面, 提供一种可用于检测系统性红斑狼疮的试剂盒, 所述的试剂 盒包含:  In a second aspect of the invention, a kit for detecting systemic lupus erythematosus is provided, the kit comprising:
容器;  Container
位于容器中的特异性地针对小核糖核酸的引物或探针,所述的小核糖核酸具有 SEQ ID NO: 2(miR-146a)所示的序列; 以及  a primer or probe specific for a small ribonucleic acid in a container having the sequence shown by SEQ ID NO: 2 (miR-146a);
用于检测系统性红斑狼疮的说明书。  Instructions for detecting systemic lupus erythematosus.
在另一优选例中,所述试剂盒中还包括特异性地针对选自下组的小核糖核酸的引物 或探针:  In another preferred embodiment, the kit further comprises a primer or probe that specifically targets a small ribonucleic acid selected from the group consisting of:
具有 SEQ ID NO: 4所示核酸序列的小核糖核酸 (miR-130b),  a small ribonucleic acid (miR-130b) having the nucleic acid sequence of SEQ ID NO: 4,
具有 SEQ ID NO: 5所示核酸序列的小核糖核酸 (miR99a),  a small ribonucleic acid (miR99a) having the nucleic acid sequence of SEQ ID NO: 5,
具有 SEQ ID NO: 6所示核酸序列的小核糖核酸 (miR-10a),  a small ribonucleic acid (miR-10a) having the nucleic acid sequence of SEQ ID NO: 6,
具有 SEQ ID NO: 7所示核酸序列的小核糖核酸 (miR-134),  a small ribonucleic acid (miR-134) having the nucleic acid sequence of SEQ ID NO: 7,
具有 SEQ ID NO: 8所示核酸序列的小核糖核酸 (miR-31), 或  a small ribonucleic acid (miR-31) having the nucleic acid sequence of SEQ ID NO: 8, or
具有 SEQ ID NO: 9所示核酸序列的小核糖核酸 (miR-95)。  A small ribonucleic acid (miR-95) having the nucleic acid sequence of SEQ ID NO: 9.
在另一优选例中, 所述的探针是 Taqman探针。 在本发明的第三方面, 提供一种小核糖核酸的用途, 用于制备调控 I型干扰素通路 的组合物; 或用于筛选调控 I型干扰素通路的物质,  In another preferred embodiment, the probe is a Taqman probe. In a third aspect of the invention, there is provided a use of a small ribonucleic acid for the preparation of a composition for regulating a type I interferon pathway; or for screening for a substance which modulates a type I interferon pathway,
其中, 所述的小核糖核酸具有通式(I)所示的序列:  Wherein the small ribonucleic acid has the sequence represented by the general formula (I):
5 ' UGAGAACUGAAUUCC AURGGYU 3 ' (I);  5 ' UGAGAACUGAAUUCC AURGGYU 3 ' (I);
其中, R选自 A或 G; Y选自 C、 U、 或丁。  Wherein R is selected from A or G; Y is selected from C, U, or D.
在另一优选例中,所述的小核糖核酸用于制备抑制 I型干扰素通路异常激活的组合 物; 或用于筛选抑制 I型干扰素通路异常激活的物质。  In another preferred embodiment, the small ribonucleic acid is used to prepare a composition that inhibits aberrant activation of the type I interferon pathway; or for screening for a substance that inhibits abnormal activation of the type I interferon pathway.
在另一优选例中, 所述的组合物还用于防治系统性红斑狼疮。  In another preferred embodiment, the composition is also useful for the prevention and treatment of systemic lupus erythematosus.
在另一优选例中, 所述的小核糖核酸调控 I型干扰素通路选自(但不限于): (a)抑 制肿瘤坏死因子受体相关因子 6 (TRAF6)的表达、(b)抑制白介素 1受体相关激酶 1 (IRAKI) 的表达、 (c)抑制干扰素诱导因子 5 (IRF5)的表达; (d)抑制信号转导和转录激活子 l (STATl)的表达(e)抑制 IFN a的表达; (f)抑制 的表达; (g)抑制粘液病毒抗性因 子 1 (MX1)表达; (h)抑制 5, 寡核苷酸合成酶 (0AS1)表达;或 (e)抑制淋巴细胞抗原 6 (Ly6E) 表达。 所述的调控包括: 直接调控或间接调控。  In another preferred embodiment, the small RNA-regulated type I interferon pathway is selected from, but not limited to, (a) inhibiting expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), and (b) inhibiting interleukin 1 expression of receptor-associated kinase 1 (IRAKI), (c) inhibition of expression of interferon-inducible factor 5 (IRF5); (d) inhibition of signal transduction and expression of activator of transcription (STAT1) (e) inhibition of IFN a (f) inhibition of expression; (g) inhibition of mucinous virus resistance factor 1 (MX1) expression; (h) inhibition of 5, oligonucleotide synthetase (0AS1) expression; or (e) inhibition of lymphocyte antigen 6 (Ly6E) expression. The regulation includes: direct regulation or indirect regulation.
在本发明的第四方面, 提供一种筛选调控 I型干扰素通路的潜在物质的方法, 所述 方法包括步骤:  In a fourth aspect of the invention, a method of screening for a potential substance that modulates a type I interferon pathway is provided, the method comprising the steps of:
(a) 将候选物质与含有小核糖核酸的体系接触, 所述的小核糖核酸具有通式(I)所 示的序列: 5 ' UGAGAACUGAAUUCC AURGGYU 3 ' (I) ; (a) contacting the candidate substance with a system containing a small ribonucleic acid having the sequence of the formula (I): 5 ' UGAGAACUGAAUUCC AURGGYU 3 '(I);
其中, R选自 A或 G; Y选自 C、 U、 或 T;  Wherein R is selected from A or G; Y is selected from C, U, or T;
(b) 观察候选物质对于小核糖核酸的表达的影响;  (b) observing the effect of candidate substances on the expression of small RNA;
其中, 若所述候选物质可提高小核糖核酸的表达, 则表明该候选物质是抑制 I型干 扰素通路的潜在物质; 若所述候选物质可降低小核糖核酸的表达, 则表明该候选物质是 促进 I型干扰素通路的潜在物质。  Wherein, if the candidate substance can increase the expression of the small ribonucleic acid, it indicates that the candidate substance is a latent substance that inhibits the type I interferon pathway; if the candidate substance can reduce the expression of the small ribonucleic acid, it indicates that the candidate substance is Potential substances that promote the type I interferon pathway.
在另一优选例中, 步骤(a)包括: 在测试组中, 将候选物质加入到含有小核糖核酸 的体系中; 和 /或  In another preferred embodiment, step (a) comprises: adding a candidate substance to the system containing the small RNA in the test group; and/or
步骤 (b)包括: 检测测试组的体系小核糖核酸的表达, 并与对照组比较, 其中所述 的对照组是不添加所述候选物质的含有小核糖核酸的体系;  The step (b) comprises: detecting the expression of the system ribonucleic acid of the test group, and comparing the control group to the control group containing the small RNA-containing system without adding the candidate substance;
如果测试小核糖核酸的表达在统计学上高于(优选显著高于, 如高 20%, 更优选高 40%, 进一步优选高 60%或更高)对照组, 就表明该候选物是抑制 I型干扰素通路的物质; 如果测试小核糖核酸的表达在统计学上低于(优选显著低于, 如低 20%, 更优选低 40%, 进一步优选低 60%或更低)对照组, 就表明该候选物是促进 I型干扰素通路的物质。  If the expression of the test picorna is statistically higher (preferably significantly higher than, for example, 20% higher, more preferably 40% higher, further preferably 60% higher or higher) in the control group, it indicates that the candidate is inhibition I a substance of the type interferon pathway; if the expression of the test small RNA is statistically lower (preferably significantly lower than, for example, 20% lower, more preferably 40% lower, further preferably 60% lower or lower), This candidate is indicated to be a substance that promotes the type I interferon pathway.
在另一优选例中, 所述的体系是细胞体系 (如 Hela细胞(或细胞培养物), HEK 293 细胞(或细胞培养物), 或原代培养的 PBMC细胞(或细胞培养物) )。  In another preferred embodiment, the system is a cell system (e.g., Hela cells (or cell culture), HEK 293 cells (or cell culture), or primary cultured PBMC cells (or cell culture).
本发明的其它方面由于本文的公开内容, 对本领域的技术人员而言是显而易见的。 附图说明  Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS
图 1显示了 SLE患者中 miR-146a表达水平下降。  Figure 1 shows the decrease in miR-146a expression levels in SLE patients.
A:将在 SLE患者和正常对照组之间表达水平有明显差异的 42个 miRNA聚类分析作 图, 其中箭头所指代表下降幅度大于六倍的 7个 miRNA。  A: A clustering analysis of 42 miRNAs with significant differences in expression levels between SLE patients and normal controls, with arrows pointing to 7 miRNAs with a greater than six-fold decline.
B: miR-146a在 47例 SLE患者, 6例白塞病患者(BD)和 21例正常对照(NC)中表达 水平比较, 数据以平均值 (mean)士平均标准误(SEM)格式显示。  B: miR-146a was compared in 47 SLE patients, 6 Behcet's patients (BD) and 21 normal controls (NC). Data were presented in mean (Mean) standard error (SEM) format.
图 2显示了 miR-146a表达水平与疾病活动相关。 其中,  Figure 2 shows that miR-146a expression levels are associated with disease activity. among them,
A: miR-146a在正常对照、 稳定性 SLE患者和活动性 SLE患者三组中的表达差异比 较。  A: miR-146a was compared in the normal control, stable SLE patients and active SLE patients.
B: 根据有无蛋白尿将病人分成两组, 有蛋白尿组 miR-146a表达水平明显低于无蛋 白尿组。  B: Patients were divided into two groups according to the presence or absence of proteinuria. The expression level of miR-146a in the proteinuria group was significantly lower than that in the no-proteinuria group.
C: miR-146a表达与 SLE患者 SLEDAI积分之间的相关性, 根据前面结果, 此处采 用单尾 Spearman检验方法。  C: Correlation between miR-146a expression and SLEDAI scores in SLE patients. Based on the previous results, a one-tailed Spearman test was used here.
D: miR-146a表达与 SLE患者肾脏 SLEDAI积分之间的相关性。  D: Correlation between miR-146a expression and renal SLEDAI scores in SLE patients.
图 3显示了 miR-146a表达水平与干扰素积分之间呈现负相关(r = -0. 3073, P = 0. 0378)。  Figure 3 shows a negative correlation between miR-146a expression levels and interferon scores (r = -0. 3073, P = 0. 0378).
图 4显示了 miR-146a在 I型干扰素通路活化中的作用。  Figure 4 shows the role of miR-146a in the activation of the type I interferon pathway.
A: 过表达 miR-146a能够抑制 I型干扰素的产生, 其中 IFN α引物能够识别最多的 IFN a亚型。 B:首先转染 miR-146a抑制序列或者随机无关序列预处理 PBMC后,用 R837 (5ug/ml) 剌激, 两小时后检测两种不同处理方法 miR-146a表达水平差异。 A: Overexpression of miR-146a is able to inhibit the production of type I interferons, which are capable of recognizing the most IFN a subtype. B: The PBMC was firstly transfected with the miR-146a inhibitory sequence or the random unrelated sequence, and then stimulated with R837 (5ug/ml). Two hours later, the difference in expression levels of miR-146a between the two different treatment methods was detected.
C: I型干扰素(1000U/ml)孵育 293T/ISRE 6小时后, 转染入 miR_146a,结果显示 miR-146a能够抑制 I SRE报告基因活性, 数据以 mean士 SEM格式显示。  C: Type I interferon (1000 U/ml) was incubated with 293T/ISRE for 6 hours and transfected into miR_146a. The results showed that miR-146a inhibited the activity of I SRE reporter gene, and the data was displayed in the SEM format.
D: I型干扰素(1000U/ml)孵育 PBMC6小时后, 转染入 miR_146a, PBMC原代细胞中 miR-146a能够抑制 I型干扰素下游干扰素诱导基因的产生。  D: Type I interferon (1000 U/ml) was incubated with PBMC for 6 hours and transfected into miR_146a. MiR-146a in PBMC primary cells inhibited the production of interferon-inducible genes downstream of type I interferon.
图 5显示了 miR-146a的两个靶基因 IRF5和 STAT1的鉴定。  Figure 5 shows the identification of two target genes miR-146a, IRF5 and STAT1.
A: 生物学预测 miR-146a与 IRF5和 STAT13 ' UTR结合位点。  A: Biological prediction miR-146a binds to IRF5 and STAT13 'UTR binding sites.
B: 过转染空载体或者 miR-146a表达载体后, 比较两者 IRF5和 STAT 13 ' UTR报告 基因的活性, 数据以 mean士 SEM格式显示。  B: After over-transfection of the empty vector or miR-146a expression vector, the activity of both IRF5 and STAT 13 ' UTR reporter genes was compared, and the data were displayed in the mean SEM format.
C: 293T细胞中应用 Western免疫印迹的方法检测 miR_146a对 IRF5和 STAT1蛋白 水平的影响。以 GAPDH的表达作为参照。其中, 以空质粒转染组的 IRF5 / GAPDH和 STAT1 I GAPDH的比值表示为 1。  C: Western blotting was used in 293T cells to detect the effect of miR_146a on IRF5 and STAT1 protein levels. The expression of GAPDH was used as a reference. The ratio of IRF5 / GAPDH to STAT1 I GAPDH in the empty plasmid transfection group was expressed as 1.
图 6显示了将病人来源的 PBMC中人为增加 miR-146a表达水平后,干扰素诱导基因 表达水平下降。  Figure 6 shows the decrease in the expression level of interferon-inducible genes after artificially increasing the expression level of miR-146a in patient-derived PBMC.
图 7显示了在正常人来源的 PBMC中检测不同剌激剂对 miR-146a表达水平的影响。 剌激剂浓度分别是 LPS (10 ug/ml), R837 (5 ug/ml), CpG-A (5 uM)和 I型干扰素(1000 U/ml) , 剌激时间为 6小时。  Figure 7 shows the effect of different stimulants on the expression level of miR-146a in normal human-derived PBMCs. The stimulant concentrations were LPS (10 ug/ml), R837 (5 ug/ml), CpG-A (5 uM) and type I interferon (1000 U/ml), and the stimulating time was 6 hours.
图 8显示了 miR-146a负反馈调节 I型干扰素通路。 具体实施方式  Figure 8 shows the miR-146a negative feedback regulation of the type I interferon pathway. detailed description
本发明人经过广泛的研究, 首次发现和证明了具有通式(I)所示的序列的小核糖核 酸(miRNA)与系统性红斑狼疮(SLE)密切相关。 本发明人还进一步验证了所述 miRNA的作 用靶基因。 并且, 本发明人还发现所述的小核糖核酸可抑制 I型干扰素通路, 从而可用 于防治 I型干扰素通路异常活化相关疾病(如系统性红斑狼疮)。 在此基础上完成了本发 明。 本文中, 除非另行定义, 所述的 " miR-146 " 是指 miR-146a或 miR-146b。  The inventors have extensively studied and found for the first time that small ribonucleotides (miRNAs) having the sequence represented by the general formula (I) are closely related to systemic lupus erythematosus (SLE). The inventors have further verified the target gene of the miRNA. Furthermore, the inventors have also found that the small ribonucleic acid can inhibit the type I interferon pathway and can be used to prevent diseases associated with abnormal activation of the type I interferon pathway (e.g., systemic lupus erythematosus). The present invention has been completed on this basis. As used herein, unless otherwise defined, the term "miR-146" refers to miR-146a or miR-146b.
本发明人发现, 一种小核糖核酸可调控 I型干扰素通路。 所述的小核糖核酸具有以 下通式:  The inventors have found that a small ribonucleic acid can regulate the type I interferon pathway. The small ribonucleic acid has the following formula:
5 ' UGAGAACUGAAUUCC AURGGYU 3 ' (I);  5 ' UGAGAACUGAAUUCC AURGGYU 3 ' (I);
其中, R选自 A或 G; Y选自 C、 U、 或丁。  Wherein R is selected from A or G; Y is selected from C, U, or D.
通式(I)所示的小核糖核酸只是在 3 ' 端有两个碱基不同,而该小核糖核酸发挥作用 的关键序列位于 5 ' 端 2-8个碱基;并且,通式(I)所示的小核糖核酸具有相同的靶基因, 因此它们具有基本相同的调控 I型干扰素通路功能。 所述的小核糖核酸优选具有 SEQ ID NO : 2 (miR-146a)或 SEQ ID NO : 3 (miR_146b)所示的序列。 作为本发明的优选方式, 所述的小核糖核酸是具有 SEQ ID NO : 2所示核酸序列的 小核糖核酸 (miR_146a), 其在系统性红斑狼疮中表达非常低, 进一步的研究发现, 所述 的小核糖核酸的表达信号与系统性红斑狼疮的活动程度和肾脏受累程度负相关; 在系统 性红斑狼疮中, 所述的小核糖核酸的靶基因较正常对照组显著升高且与该小核糖核酸的 表达信号呈负相关。 The small ribonucleic acid represented by the general formula (I) differs only in two bases at the 3' end, and the key sequence in which the small ribonucleic acid functions is located at the 5' end 2-8 bases; and, the general formula (I) The small ribonucleic acids shown have the same target genes, so they have substantially the same function of regulating the type I interferon pathway. The small ribonucleic acid preferably has the sequence shown by SEQ ID NO: 2 (miR-146a) or SEQ ID NO: 3 (miR_146b). As a preferred mode of the present invention, the small ribonucleic acid is a small ribonucleic acid (miR_146a) having the nucleic acid sequence of SEQ ID NO: 2, which is expressed very low in systemic lupus erythematosus, and further studies have found that The expression signal of the small ribonucleic acid is inversely related to the degree of activity of the systemic lupus erythematosus and the degree of renal involvement; in systemic lupus erythematosus, the target gene of the small ribonucleic acid is significantly higher than the normal control group and the small ribose sugar The expression signal of the nucleic acid is negatively correlated.
本发明人还发现, 选自下组的小核糖核酸均在系统性红斑狼疮中表达非常低(与正 常人的水平相比, 降低 6倍以上):  The inventors have also found that small ribonucleic acids selected from the group consisting of very low expression in systemic lupus erythematosus (more than 6 times lower than normal human levels):
具有 SEQ ID NO: 4所示核酸序列的小核糖核酸 (miR-130b),  a small ribonucleic acid (miR-130b) having the nucleic acid sequence of SEQ ID NO: 4,
具有 SEQ ID NO: 5所示核酸序列的小核糖核酸 (miR99a),  a small ribonucleic acid (miR99a) having the nucleic acid sequence of SEQ ID NO: 5,
具有 SEQ ID NO: 6所示核酸序列的小核糖核酸 (miR-10a),  a small ribonucleic acid (miR-10a) having the nucleic acid sequence of SEQ ID NO: 6,
具有 SEQ ID NO: 7所示核酸序列的小核糖核酸 (miR-134),  a small ribonucleic acid (miR-134) having the nucleic acid sequence of SEQ ID NO: 7,
具有 SEQ ID NO: 8所示核酸序列的小核糖核酸 (miR-31), 或  a small ribonucleic acid (miR-31) having the nucleic acid sequence of SEQ ID NO: 8, or
具有 SEQ ID NO: 9所示核酸序列的小核糖核酸 (miR-95)。 基于本发明的上述新发现,所述的小核糖核酸有多方面的新用途。这些用途包括 (但 不限于):  A small ribonucleic acid (miR-95) having the nucleic acid sequence of SEQ ID NO: 9. Based on the above new findings of the present invention, the picornacle nucleic acid has many new uses. These uses include (but are not limited to):
(i) 将所述小核糖核酸直接作为药物治疗小核糖核酸水平下降所致的疾病, 如系统 性红斑狼疮;  (i) treating the disease caused by a decrease in the level of small RNA, such as systemic lupus erythematosus, directly as a drug;
(ϋ) 用于制备系统性红斑狼疮 (SLE) 临床检测的试剂或试剂盒;  (ϋ) A reagent or kit for the clinical testing of systemic lupus erythematosus (SLE);
(i i i) 用于筛选防治系统性红斑狼疮的物质;  (i i i) for screening for substances that prevent systemic lupus erythematosus;
(iv) 用于调节肿瘤坏死因子受体相关因子 6、 白介素 1受体相关激酶 1、 干扰素诱 导因子 5或信号转导和转录激活子 1的表达水平, 或预防或治疗肿瘤坏死因子受体相关 因子 6、 白介素 1受体相关激酶 1、 干扰素诱导因子 5或信号转导和转录激活子 1表达 或活性失调相关的疾病;  (iv) for regulating the expression levels of tumor necrosis factor receptor-associated factor 6, interleukin-1 receptor-associated kinase 1, interferon-inducible factor 5 or signal transduction and activator of transcription 1, or preventing or treating tumor necrosis factor receptor a related factor 6, an interleukin 1 receptor-associated kinase 1, an interferon-inducible factor 5 or a signal transduction and a transcriptional activator 1 expression or activity disorder associated with a disease;
(v) 用于进行系统性红斑狼疮的分型、 鉴别诊断、 和 /或易感性分析。  (v) For the classification, differential diagnosis, and/or susceptibility analysis of systemic lupus erythematosus.
(vi) 用于评估相关人群系统性红斑狼疮的患病风险, 早期监测早期预防;  (vi) for assessing the risk of systemic lupus erythematosus in the relevant population, early monitoring of early prevention;
(vi i) 用于评估相关人群的系统性红斑狼疮治疗药物、 药物疗效、 预后, 以及选择 合适的治疗方法。  (vi i) For the evaluation of systemic lupus erythematosus treatment, drug efficacy, prognosis, and selection of appropriate treatments for the relevant population.
(viii) 直接作为药物防治 I型干扰素通路异常相关疾病, 如系统性红斑狼疮。  (viii) Directly as a drug for the prevention of type I interferon pathway-associated diseases, such as systemic lupus erythematosus.
(X) 作为一种靶点, 筛选调控 I型干扰素通路的物质, 所述物质例如被用于制备抑 制 I型干扰素通路异常激活相关疾病的药物。 在得知了所述的小核糖核酸的用途后, 可以采用本领域熟知的多种方法来筛选调控 (X) As a target, a substance which regulates the type I interferon pathway, which is used, for example, to prepare a drug for inhibiting a disease associated with abnormal activation of the type I interferon pathway, is selected. After knowing the use of the small ribonucleic acid, various methods well known in the art can be used for screening and regulation.
I型干扰素通路的物质。 A substance of the type I interferon pathway.
在本发明的一种优选方式中, 提供一种筛选方法, 所述的方法包括: 将候选物质与 含有通式 (I)所示小核糖核酸的体系接触; 观察候选物质对于通式 (I)所示小核糖核酸的表 达的影响; 若所述候选物质可提高(优选显著提高, 如提高 20%或更低; 更优选提高 40% 或更高) 通式 (I)所示小核糖核酸的表达, 则表明该候选物质是抑制 I型干扰素通路的潜 在物质; 反之, 则该候选物质是促进 I型干扰素通路的潜在物质。 In a preferred aspect of the present invention, a screening method is provided, the method comprising: contacting a candidate substance with a system containing a small ribonucleic acid represented by the general formula (I); and observing the candidate substance for the general formula (I) Effect of expression of the displayed small ribonucleic acid; if the candidate substance can be increased (preferably significantly increased, such as by 20% or less; more preferably by 40%) Or higher) Expression of the small ribonucleic acid represented by the general formula (I) indicates that the candidate substance is a latent substance that inhibits the type I interferon pathway; conversely, the candidate substance is a latent substance that promotes the type I interferon pathway.
更优选的, 可通过设置对照组来观察候选物质对于通式 (I)所示小核糖核酸的表达的 影响状况; 所述对照组是不添加所述候选物质的含有通式 (I)所示小核糖核酸的体系。  More preferably, the influence of the candidate substance on the expression of the small ribonucleic acid represented by the general formula (I) can be observed by setting a control group; the control group is not containing the candidate substance and contains the formula (I) A system of small RNAs.
由于通式 (I)所示小核糖核酸的激动剂或拮抗剂可调节通式 (I)所示小核糖核酸的表 达, 因此, 所述的通式 (I)所示小核糖核酸的激动剂或拮抗剂可通过对通式 (I)所示小核糖 核酸的影响来调节 I型干扰素通路。  Since the agonist or antagonist of the small ribonucleic acid represented by the general formula (I) can modulate the expression of the small ribonucleic acid represented by the general formula (I), the agonist of the small ribonucleic acid represented by the general formula (I) Or the antagonist can modulate the type I interferon pathway by affecting the small ribonucleic acid represented by the general formula (I).
所述的通式 (I)所示小核糖核酸的激动剂是指任何可维持通式 (I)所示小核糖核酸的 稳定性、 促进通式 (I)所示小核糖核酸表达、 延长通式 (I)所示小核糖核酸有效作用时间的 物质, 这些物质均可用于本发明, 作为可用于调控(特别是抑制)1型干扰素通路有用的 物质。  The agonist of the small ribonucleic acid represented by the general formula (I) means any one which can maintain the stability of the small ribonucleic acid represented by the general formula (I), promote the expression of the small ribonucleic acid represented by the general formula (I), and prolong the passage. The substance having a small ribonucleic acid effective time as shown in the formula (I), which can be used in the present invention, is useful as a substance useful for regulating (especially inhibiting) the type 1 interferon pathway.
所述的通式 (I)所示小核糖核酸的拮抗剂是指任何可降低通式 (I)所示小核糖核酸的 稳定性、 抑制通式 (I)所示小核糖核酸表达的物质, 这些物质均可用于本发明, 作为可用 于调控(特别是促进)1型干扰素通路有用的物质。 所述的拮抗剂例如是 miR-146a或 miR-146b或它们的类似物的反义寡核苷酸链; 所述的拮抗剂通过增强 I型干扰素通路, 可开发为一些肿瘤和感染性疾病的治疗药物。  The antagonist of the small ribonucleic acid represented by the general formula (I) is any substance which can reduce the stability of the small ribonucleic acid represented by the general formula (I) and inhibit the expression of the small ribonucleic acid represented by the general formula (I). These materials can be used in the present invention as a useful substance for regulating (especially promoting) type 1 interferon pathways. The antagonist is, for example, an antisense oligonucleotide chain of miR-146a or miR-146b or an analog thereof; the antagonist can be developed into some tumors and infectious diseases by enhancing the type I interferon pathway. Therapeutic drugs.
在得知了所述通式 (I)所示小核糖核酸的用途后, 可以采用本领域熟知的多种方法来 将所述的通式 (I)所示小核糖核酸或其编码基因、 或其药物组合物给药于需要治疗的受试 者(如 SLE患者)。 优选的, 可采用基因治疗的手段进行。 比如, 可直接将通式 (I)所示小 核糖核酸或其类似物通过诸如注射等方法给药于受试者; 或者, 可通过一定的途径将携 带通式 (I)所示小核糖核酸或其类似物的表达单位递送到靶点上, 并使之表达通式 (I)所示 小核糖核酸, 这些均是本领域技术人员所熟知的。  After the use of the small ribonucleic acid represented by the general formula (I) is known, the small ribonucleic acid represented by the general formula (I) or a gene encoding the same can be used by various methods well known in the art, or The pharmaceutical composition is administered to a subject in need of treatment (e.g., a patient with SLE). Preferably, it can be carried out by means of gene therapy. For example, the small ribonucleic acid represented by the general formula (I) or an analog thereof can be directly administered to a subject by a method such as injection; or, the small ribonucleic acid represented by the general formula (I) can be carried by a certain route. The expression unit of the analog or analog thereof is delivered to the target and expressed to express the small ribonucleic acid represented by the general formula (I), which are well known to those skilled in the art.
所述的小核糖核酸的类似物是指关键区域与通式 (I)所示序列相同 (即 SEQ ID NO: 1 中第 2-8位),且具有与通式 (I)所示序列相同或接近的对 I型干扰素通路调控作用的小核 糖核酸。 较佳的, 该类似物与 SEQ ID NO: 2所示的序列具有 70%以上的同源性, 更佳 的具有 85%以上的同源性, 最佳的具有 95%以上的同源性。  The analog of the small ribonucleic acid means that the key region is identical to the sequence of the formula (I) (ie, positions 2-8 in SEQ ID NO: 1) and has the same sequence as the formula (I). Or close proximity to the small RNA that regulates the type I interferon pathway. Preferably, the analog has 70% or more homology with the sequence of SEQ ID NO: 2, more preferably 85% or more homologous, and most preferably has 95% or more homology.
本发明的小核糖核酸或其激动剂或拮抗剂, 当在治疗上进行施用 (给药)时, 可提供 不同的效果。 通常, 可将这些物质配制于无毒的、 惰性的和药学上可接受的水性载体介 质中, 其中 pH通常约为 5-8, 较佳地 pH约为 6-8, 尽管 pH值可随被配制物质的性质以 及待治疗的病症而有所变化。 配制好的药物组合物可以通过常规途径进行给药, 其中包 括 (但并不限于): 肌注、 静脉、 或皮下给药。  The picorucleic acid of the present invention or an agonist or antagonist thereof, when administered therapeutically (administered), provides different effects. Generally, these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary. The formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intramuscular, intravenous, or subcutaneous administration.
所述的小核糖核酸或其激动剂或拮抗剂可直接用于疾病治疗, 例如, 用于系统性红 斑狼疮的治疗。 在使用本发明小核糖核酸或其激动剂时, 还可同时使用其他治疗系统性 红斑狼疮的药剂。  The small ribonucleic acid or an agonist or antagonist thereof can be directly used for the treatment of diseases, for example, for the treatment of systemic lupus erythematosus. When using the small ribonucleic acid of the present invention or an agonist thereof, other agents for treating systemic lupus erythematosus can be used at the same time.
本发明还提供了一种药物组合物, 它含有安全有效量的本发明的小核糖核酸或其激 动剂或拮抗剂以及药学上可接受的载体或赋形剂。 这类载体包括 (但并不限于): 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 及其组合。 药物制剂应与给药方式相匹配。 本发明 的药物组合物可以被制成针剂形式, 例如用生理盐水或含有葡萄糖和其他辅剂的水溶液 通过常规方法进行制备。 药物组合物如针剂、 溶液宜在无菌条件下制造。 活性成分的给 药量是治疗有效量, 例如每天约 0.1微克 /千克体重-约 10毫克 /千克体重。 The invention also provides a pharmaceutical composition comprising a safe and effective amount of a small ribonucleic acid of the invention, or an agonist or antagonist thereof, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched to the mode of administration. this invention The pharmaceutical composition can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably prepared under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 0.1 microgram per kilogram body weight to about 10 milligrams per kilogram body weight per day.
使用药物组合物时, 是将安全有效量的所述小核糖核酸或其激动剂施用于哺乳动 物, 其中该安全有效量通常至少约 0.1微克 /千克体重, 而且在大多数情况下不超过约 10 毫克 /千克体重, 较佳地该剂量是约 0.1微克 /千克体重-约 100微克 /千克体重。 当然, 具 体剂量还应考虑给药途径、 病人健康状况等因素, 这些都是熟练医师技能范围之内的。  When a pharmaceutical composition is used, a safe and effective amount of the picorucleic acid or an agonist thereof is administered to a mammal, wherein the safe and effective amount is usually at least about 0.1 microgram per kilogram of body weight, and in most cases no more than about 10 The mg/kg body weight, preferably the dose is from about 0.1 microgram per kilogram body weight to about 100 micrograms per kilogram body weight. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
通过给予受试者所述的小核糖核酸 (如 miR-146a或 miR- 146b)或其激动剂或拮抗剂, 可提高受试者中该小核糖核酸的含量、 表达, 从而预防或治疗该小核糖核酸降低相关的 疾病, 如干扰素通路异常激活相关疾病。 已有研究显示, I型干扰素通路的异常激活在 狼疮发病中发挥着关键的作用。  By administering to the subject the small ribonucleic acid (such as miR-146a or miR-146b) or an agonist or antagonist thereof, the content and expression of the small ribonucleic acid in the subject can be increased, thereby preventing or treating the small Ribonucleotide-related diseases, such as disorders associated with abnormal activation of the interferon pathway. Studies have shown that abnormal activation of the type I interferon pathway plays a key role in the pathogenesis of lupus.
本发明还涉及定量和定性检测所述小核糖核酸水平, 从而判断系统性红斑狼疮的发 生或发展的诊断试验方法。 这些试验是本领域所熟知的, 例如包括 (但不限于;): 实时荧 光定量 PCR, 聚类分析, 非参数 Mann- Whitney检验, Spearman相关性分析等。  The present invention also relates to a diagnostic test method for quantitatively and qualitatively detecting said small ribonucleic acid levels to determine the occurrence or development of systemic lupus erythematosus. These assays are well known in the art and include, for example, but not limited to: real-time fluorescent quantitative PCR, cluster analysis, non-parametric Mann-Whitney test, Spearman correlation analysis, and the like.
一种检测待测组织或样品中是否存在所述小核糖核酸及其存在量的方法是利用实 时荧光定量 PCR进行检测, 它包括: 制备各 miRNA 的特异性探针 (所述探针优选携带 可检测信号), 制备各 miRNA 的特异性引物, 进行 PCR, 然后通过检测 PCR结束后的 可检测信号的强弱来判断所述小核糖核酸的水平。作为本发明的优选方式,基于 TaqMan 荧光技术, 即以 TaqMan荧光探针为基础进行检测。 TaqMan荧光探针为一种寡核苷酸, 其两端分别标记一个荧光发射基团(作为可检测信号)和一个荧光淬灭基团。 当探针完 整时, 荧光发射基团发射的荧光信号被淬灭基团吸收; PCR扩增时, Taq酶的 5'_3'外切 酶活性将探针酶切降解, 使荧光发射基团和荧光淬灭基团分离, 从而通过荧光检测系 统可接受到荧光信号, 即每扩增一条 DNA链, 就有一个荧光分子形成, 即荧光信号的累 计与 PCR产物形成的完全同步, 从而实现定性和定量。  A method for detecting the presence or absence of the small ribonucleic acid and the amount thereof in a tissue or sample to be tested is detected by real-time fluorescent quantitative PCR, which comprises: preparing a specific probe for each miRNA (the probe preferably carries a probe The detection signal), specific primers for each miRNA are prepared, PCR is performed, and the level of the small RNA is judged by detecting the intensity of the detectable signal after the end of the PCR. As a preferred mode of the present invention, detection is based on TaqMan fluorescence technology, i.e., based on a TaqMan fluorescent probe. The TaqMan fluorescent probe is an oligonucleotide having a fluorescent emitting group (as a detectable signal) and a fluorescence quenching group, respectively, at both ends. When the probe is intact, the fluorescent signal emitted by the fluorescent emitting group is absorbed by the quenching group; when PCR is amplified, the 5'_3' exonuclease activity of the Taq enzyme degrades the probe, and the fluorescent emitting group and The fluorescence quenching group is separated, so that the fluorescence signal can be accepted by the fluorescence detecting system, that is, every time one DNA strand is amplified, one fluorescent molecule is formed, that is, the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product, thereby achieving qualitative and Quantitative.
所述的小核糖核酸特异性的引物和 /或探针也包含在本发明内, 用于检测样品中是 否存在所述小核糖核酸及其存在量, 从而用于判断受试者是否患有系统性红斑狼疮或 患病风险。 所述的探针可固定在微阵列 (microarmy)或基因芯片上, 用于分析组织或样 品中 miRNA的差异表达分析和基因诊断。  The small ribonucleic acid-specific primers and/or probes are also included in the present invention for detecting the presence or absence of the small ribonucleic acid in a sample and for determining whether the subject has a system. Lupus erythematosus or risk of illness. The probes can be immobilized on a microarmy or gene chip for analysis of differential expression analysis and genetic diagnosis of miRNAs in tissues or samples.
本发明还包括可用于检测 miRNA的表达水平,进而诊断系统性红斑狼疮的试剂盒, 所述的试剂盒中可包括特异性扩增所述的 miRNA (选自以下组的至少一个: miR-146a、 miR-130b、 miR99a、 miR_10a、 miR_134、 miR_31、 miR-95) 的引物。 优选的, 它还含有 以下试剂: 与所述的 miRNA特异性结合的探针; 更优选所述探针携带可检测信号。 作 为本发明的优选方式, 所述的小核糖核酸是通式 (I)所示小核糖核酸。 作为本发明的优 选方式, 所述的试剂盒中还含有系统性红斑狼疮临床检测的说明书。 本发明的主要优点在于:  The invention also includes a kit for detecting the expression level of a miRNA, thereby diagnosing systemic lupus erythematosus, which kit may comprise specifically amplifying said miRNA (selected from at least one of the group consisting of: miR-146a Primers for miR-130b, miR99a, miR_10a, miR_134, miR_31, miR-95). Preferably, it further comprises: a probe that specifically binds to said miRNA; more preferably said probe carries a detectable signal. In a preferred embodiment of the present invention, the small ribonucleic acid is a small ribonucleic acid represented by the general formula (I). As a preferred embodiment of the present invention, the kit further contains instructions for clinical detection of systemic lupus erythematosus. The main advantages of the invention are:
(1) 首次揭示和证明了一类小核糖核酸与系统性红斑狼疮密切相关, 找到了可作 为 SLE临床诊断的重要生物标志物, 为该疾病的诊断或防治提供了新的靶点。 (1) Revealed and proved that a small class of small RNA is closely related to systemic lupus erythematosus, and found It is an important biomarker for the clinical diagnosis of SLE and provides a new target for the diagnosis or prevention of this disease.
(2) 首次揭示通式(I)所示小核糖核酸的过表达可抑制 I型干扰素通路的激活, 从 而为 I型干扰素通路异常激活相关疾病的防治提供了有效途径。  (2) It is revealed for the first time that overexpression of the small ribonucleic acid represented by the general formula (I) can inhibit the activation of the type I interferon pathway, thereby providing an effective way for the prevention and treatment of diseases associated with abnormal activation of the type I interferon pathway.
(3) 本发明人分析了大量的 SLE患者中小核糖核酸的水平, 并与正常人作了细致的 比较, 因此结果准确可靠。 材料和方法  (3) The inventors analyzed the levels of small RNA in a large number of SLE patients and made a detailed comparison with normal people, so the results were accurate and reliable. Materials and Method
1. 研究对象  Research object
选择 SLE患者共 52例, 均为上海仁济医院风湿免疫科门诊与病房病人(来自于上海 市以及中国其它多个省市), 诊断均符合美国风湿病学会 SLE分类标准推荐的 1 1项标准 中的至少 4 项, 其中男 4例, 女 48例, 平均年龄 33. 5岁(12-60岁), 将其中 47例病 人按 SLE疾病活动性积分(SLEDAI 2K)分为 SLE非活动组(0〜4分, 即 SLE稳定组) 18例 和 SLE活动组( 5分) 29例。 其中 26例符合 ARA狼疮肾炎标准。  A total of 52 patients with SLE were selected, including the outpatient and ward patients of Shanghai Renji Hospital, which were from Shanghai and many other provinces and cities in China. The diagnosis was in accordance with the 11 criteria recommended by the American College of Rheumatology SLE classification criteria. At least 4 of them, including 4 males and 48 females, with an average age of 33.5 years (12-60 years), 47 patients were divided into SLE inactive groups according to SLE disease activity scores (SLEDAI 2K). 0 to 4 points, ie SLE stable group) 18 cases and SLE activity group (5 points) 29 cases. Of these, 26 met the criteria for ARA lupus nephritis.
另外, 设置正常对照组 29例, 来自医院体检正常人群(来自于上海市以及中国其它 多个省市), 其中男 2例, 女 27例, 平均年龄 28. 5岁(19-50岁)。 设置 6例白塞病(BD) 患者 o  In addition, 29 patients in the normal control group were enrolled in the hospital (from Shanghai and other provinces and cities in China), including 2 males and 27 females, with an average age of 28.5 years (19-50 years). Set up 6 patients with Behcet's disease (BD) o
各组性别和年龄差异无统计学意义, 无现存感染, 研究组与对照组每人采外周静脉 ACD抗凝血 lml, 作为检测样品。 2. 引物和探针设计  There was no statistically significant difference in gender and age between the groups. There was no existing infection. The study group and the control group each received peripheral blood ACD anticoagulation lml as a test sample. 2. Primer and probe design
miRNA逆转录引物和 Real-time PCR反应探针直接从 ABI (Appl ied Biosystems公司, TaqMan® MicroRNA (miRNA) Assays)购买获得; 此外, 本领域人员可以方便地根据所述 的 miRNA序列来设计所述引物和探针。  miRNA reverse transcription primers and Real-time PCR reaction probes were purchased directly from ABI (Applied Biosystems, Inc., TaqMan® MicroRNA (miRNA) Assays); in addition, one skilled in the art can conveniently design the miRNA sequences according to the described miRNA sequences. Primers and probes.
靶基因和 I型干扰素下游基因定量引物的设计: 检索 NCBI数据库查到人类 IRAK1、 TRAF6、 Ly6E、 0AS1、 MX1基因 cDNA全长序列, 以 Ol igo 6. 71软件设计扩增模板的引物 如下:  Design of target gene and type I interferon downstream gene quantification primers: The full-length cDNA sequences of human IRAK1, TRAF6, Ly6E, 0AS1, and MX1 genes were searched in the NCBI database. The primers designed to amplify the template by Oligo 6.71 software are as follows:
IRAKI正义链 5 ' TGGACTTTGCTGGCTACTG 3 ' (SEQ ID NO: 10) ,  IRAKI justice chain 5 ' TGGACTTTGCTGGCTACTG 3 ' (SEQ ID NO: 10) ,
IRAKI反义链 5 ' CTGTCCTGATGTAGAAACTGAAT 3 ' (SEQ ID NO: 1 1);  IRAKI antisense strand 5 ' CTGTCCTGATGTAGAAACTGAAT 3 ' (SEQ ID NO: 1 1);
TRAF6正义链 5 ' TTGCACAAGATGGAACTGAG 3 ' (SEQ ID NO: 12) ,  TRAF6 sense strand 5 ' TTGCACAAGATGGAACTGAG 3 ' (SEQ ID NO: 12) ,
TRAF6反义链 5 ' ATTGATGCAGCACAGTTGTC 3 ' (SEQ ID NO: 13);  TRAF6 antisense strand 5 ' ATTGATGCAGCACAGTTGTC 3 ' (SEQ ID NO: 13);
Ly6E正义链 5 ' CTTACGGTCCAACATCAGAC 3 ' (SEQ ID NO: 14) ,  Ly6E sense strand 5 'CTTACGGTCCAACATCAGAC 3 ' (SEQ ID NO: 14) ,
Ly6E反义链 5 ' GCACACATCCCTACTGACAC 3 ' (SEQ ID NO: 15);  Ly6E antisense strand 5 ' GCACACATCCCTACTGACAC 3 ' (SEQ ID NO: 15);
0AS1正义链 5 ' GAAGGCAGCTCACGAAAC 3 ' (SEQ ID NO: 16),  0AS1 sense strand 5 ' GAAGGCAGCTCACGAAAC 3 ' (SEQ ID NO: 16),
0AS1反义链 5 ' TTCTTAAAGCATGGGTAATTC 3 ' (SEQ ID NO: 17);  0AS1 antisense strand 5 ' TTCTTAAAGCATGGGTAATTC 3 ' (SEQ ID NO: 17);
MX1正义链: 5 ' GGGTAGCCACTGGACTGA 3 ' (SEQ ID NO: 18),  MX1 sense chain: 5 ' GGGTAGCCACTGGACTGA 3 ' (SEQ ID NO: 18),
MX1反义链: 5 ' AGGTGGAGCGATTCTGAG 3 ' (SEQ ID NO: 19);  MX1 antisense strand: 5 ' AGGTGGAGCGATTCTGAG 3 ' (SEQ ID NO: 19);
IFN a正义链: 5 ' -TCCATGAGATGATCCAGCAG-3 ' (SEQ ID NO: 24), IFNa反义链: 5, -ATTTCTGCTCTGACAACCTCCC-3' (SEQ ID NO: 25); IFNP正义链: 5, - TCTAGCACTGGCTGGAATGAG- 3, (SEQ ID NO: 26), IFN a sense strand: 5 ' -TCCATGAGATGATCCAGCAG-3 ' (SEQ ID NO: 24), IFNa antisense strand: 5, -ATTTCTGCTCTGACAACCTCCC-3' (SEQ ID NO: 25); IFNP sense strand: 5, - TCTAGCACTGGCTGGAATGAG-3, (SEQ ID NO: 26),
IFNP反义链: 5' - GTTTCGGAGGTAACCTGTAAG- 3' (SEQ ID NO: 27)。  IFNP antisense strand: 5'-GTTTCGGAGGTAACCTGTAAG-3' (SEQ ID NO: 27).
引物设计好后通过 BLAST分析(www. ncbi. nlm. nih. gov/BLAST )避免扩增的目的片 段序列存在非特异性, 由上海生工公司合成。  After the primers were designed, the target fragment sequence of the amplification was prevented from being non-specific by BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) and synthesized by Shanghai Biotech Co., Ltd.
内参照基因一-核糖体蛋白 L13a(RPL13A)的定量引物来源于 Quantitative PCR Primer Database (QPPD)网站所报导序列, 也为上海生工公司合成:  The quantitative reference primer for the internal reference gene-ribosomal protein L13a (RPL13A) was derived from the sequence reported on the Quantitative PCR Primer Database (QPPD) website, and was also synthesized by Shanghai Shenggong Company:
RPL13A正义链 5' CCTGGAGGAGAAGAGGAAAGAGA 3' (SEQ ID NO: 20),  RPL13A sense strand 5' CCTGGAGGAGAAGAGGAAAGAGA 3' (SEQ ID NO: 20),
RPL13A反义链 5' TTGAGGACCTCTGTGTATTTGTCAA 3' (SEQ ID NO: 21)。  RPL13A antisense strand 5' TTGAGGACCTCTGTGTATTTGTCAA 3' (SEQ ID NO: 21).
3. RNA抽提和逆转录 3. RNA extraction and reverse transcription
应用 Trizol酚氯仿法抽提 (Invitrogen公司产品)血液样品的总 RNA, 得到的 RNA 用毛细管电泳 (NanoDrop Specthophotometer) 鉴定其完整性, 紫外分光光度计测定其 浓度。  The total RNA of the blood sample (Invitrogen product) was extracted by Trizol phenol chloroform method, and the obtained RNA was identified by capillary electrophoresis (NanoDrop Specthophotometer), and its concentration was determined by an ultraviolet spectrophotometer.
一部分总 RNA用作 oligo dT逆转录, 使用 Superscript II逆转录酶试剂盒 A portion of total RNA was used as oligo dT reverse transcription, using the Superscript II reverse transcriptase kit
(Invitrogen 公司产品)逆转录为 cDNA, 首先加 RNA 8ul、 oligo dT lul、 dNTP 4ul混 匀离心后 65°C 5min, 快速置于冰上再加入 5 X FS 缓冲液 4 ul、 0.1M DTT 2 ul, 混匀 离心后 42°C 2min, 最后加入 1 ul的 SSRT II混匀离心后于 42°C 50min, 70。C 15min 条件下反应。 (Invitrogen products) reverse transcription into cDNA, first add RNA 8ul, oligo dT lul, dNTP 4ul mixed and centrifuged at 65 ° C for 5min, quickly placed on ice and then added 5 X FS buffer 4 ul, 0.1M DTT 2 ul Mix and centrifuge at 42 ° C for 2 min, finally add 1 ul of SSRT II and mix and centrifuge at 42 ° C for 50 min, 70. Reaction under C 15 min conditions.
另一部分总 RNA用作 miRNA特异性引物逆转录, 加样体系为:  Another part of total RNA was used as a miRNA-specific primer for reverse transcription. The loading system was:
dNTP 0.03ul, MMLV 0.2ul, 10X缓冲液 0· 3ul, RNase 抑制剂 0.02ul, dd¾0 (无 RNase) 0.45ul, 引物 lul, RNA lul。  dNTP 0.03ul, MMLV 0.2ul, 10X buffer 0· 3ul, RNase inhibitor 0.02ul, dd3⁄40 (no RNase) 0.45ul, primer lul, RNA lul.
逆转录反应条件为 16°C 30min, 42 °C 30min, 85 °C 5min。 4. 实时荧光定量 PCR (Real-time PCR)  The reverse transcription reaction conditions were 16 ° C for 30 min, 42 ° C for 30 min, and 85 ° C for 5 min. 4. Real-time PCR (Real-time PCR)
在 ABI Prism 7900测序仪 (Applied Biosystems公司) 上进行实时荧光定量 PCR 操作。  Real-time PCR was performed on an ABI Prism 7900 sequencer (Applied Biosystems).
针对每一个所检测的 miRNA, 用 miRNA Taqman方法的加样体系为 Master Mix 2ul、 特异性探针 lul和模板 lul, 每个样本做两负孔, 负孔之间 CT值差异均控制在 0.5个 CT之内, 每块板上均设有板间对照(Control)。 反应条件为 50°C 2min, 95 °C lOmin; 之后 95°C 15s, 60 °C lmin共进行 40个循环。  For each miRNA detected, the loading system using miRNA Taqman method was Master Mix 2ul, specific probe lul and template lul. Two negative wells were made for each sample, and the difference of CT values between negative holes was controlled at 0.5. Within the CT, there is an inter-plate control on each board. The reaction conditions were 50 ° C for 2 min, 95 ° C lOmin; then 95 ° C for 15 s, 60 ° C lmin for a total of 40 cycles.
靶基因 SYBR Green定量加样体系为 SYBR Green Master Mix 2.5ul、 R0X 0. lu 引物(正义链 0. lul, 反义链 0. lul)、 dd¾0 1.2ul和 cDNA模板 lul, 同样每个样本做 两负孔,负孔之间 CT值差异控制在 0.5个 CT之内,每块板上设置有板间对照(Control)。 反应条件为 95°C 15s; 之后 95°C 5s, 60 °C 30s共进行 40个循环; 之后 95°C 15s, 60 V 15s, 95 °C 15s。 5. IFN积分计算 The target gene SYBR Green quantitative loading system is SYBR Green Master Mix 2.5ul, R0X 0. lu primer (sense strand 0. lul, antisense strand 0. lul), dd3⁄40 1.2ul and cDNA template lul, also do two for each sample The difference between the negative and negative CT values is controlled within 0.5 CT, and each plate is provided with an inter-plate control. The reaction conditions were 95 ° C for 15 s; then a total of 40 cycles were carried out at 95 ° C for 5 s and 60 ° C for 30 s; then 95 ° C for 15 s, 60 V for 15 s, and 95 ° C for 15 s. 5. IFN integral calculation
IFN积分计算根据已有的相关研究报道 [Xuebing Feng, Hui Wu, Bevra H. Hahn, and Betty P. Tsao, et al Association of Increased Interferon-Inducible Gene Expression With Disease Activity and Lupus Nephritis in Patients With Systemic Lupus Erythematosus.  IFN integral calculations are based on existing related research reports [Xuebing Feng, Hui Wu, Bevra H. Hahn, and Betty P. Tsao, et al Association of Increased Interferon-Inducible Gene Expression With Disease Activity and Lupus Nephritis in Patients With Systemic Lupus Erythematosus .
Arthritis Rheum 2006 Sep;54(9):2951-62.] , 本发明人选用 Ly6E、 0AS1、 MX1三个基因来 计算 IFN积分。 首先利用正常对照组各个基因表达水平的平均值和标准差来标化每个样 本相应基因的表达水平, 然后将每个样本的标化值相加即可得 IFN积分。 公式如下: Arthritis Rheum 2006 Sep; 54(9): 2951-62.], the inventors selected three genes, Ly6E, 0AS1, and MX1, to calculate IFN scores. First, the average level and standard deviation of the expression levels of each gene in the normal control group were used to standardize the expression levels of the corresponding genes of each sample, and then the normalized values of each sample were added to obtain IFN scores. The formula is as follows:
^ Ge e iSLF ―' ' ' . C¾i^ ^ Ge ei SLF ―''' . C3⁄4i^
:: ¾ '' i = Ly6E、 0AS1、 MX1三个基因中的一个基因,  :: 3⁄4 '' i = one of the three genes Ly6E, 0AS1, MX1,
Gene iSLE = 每个 SLE患者的基因表达水平, Gene i SLE = gene expression level in each SLE patient,
Gene ictr = 正常对照组的基因表达水平。 Gene i ctr = gene expression level in the normal control group.
SLE患者中 IFN积分的平均值为 65. 3, 最小值和最大值分别为 _0. 45和 412. 6; 正 常对照组中三个值分别是为 0、 -1. 88和 6. 55。 6. 数据分析  The mean value of IFN score in SLE patients was 65. 3, the minimum and maximum values were _0. 45 and 412. 6 respectively; the three values in the normal control group were 0, -1.88 and 6.55, respectively. 6. Data analysis
分析各孔 CT值, 选取 CT值小于 33并且复孔间重复性好的样本, 计算出用内参标 化后的 2(A A CT)值,即代表该样本该基因初始拷贝数的数量。第一批表达数据首先运用 SAM 2. 20软件分析, 然后将表达有差异的数据导入 HCE 3. 0软件作聚类分析。 后一批扩大样 本数据用 Graph Pad 4. 03软件进行分析, 两组独立样本数据比较应用非参数 The CT values of each well were analyzed, and the samples with CT values less than 33 and good repeatability between the two wells were selected, and the 2 ( -AA CT ) value after standardization with internal parameters was calculated, which is the number of initial copy numbers of the gene. The first batch of expression data was first analyzed using SAM 2. 20 software, and then the data with different expressions were imported into HCE 3. 0 software for cluster analysis. The latter batch of expanded sample data was analyzed with Graph Pad 4. 03 software, and two sets of independent sample data were compared for non-parameters.
Mann-Whitney检验, 相关分析应用 Spearman相关性检验。 值小于 0. 05代表有统计学 意义。 Mann-Whitney test, correlation analysis applied Spearman correlation test. A value less than 0.05 indicates statistical significance.
7. miR-146a表达载体的构建 7. Construction of miR-146a expression vector
以采用常规方法获得的基因组 DNA为模板, 运用 PCR方法扩增出长度约为 280bp的 miR-146a前体片段, 引物如下:  Using the genomic DNA obtained by the conventional method as a template, a miR-146a precursor fragment of about 280 bp in length was amplified by PCR, and the primers were as follows:
上游引物 5 ' GTGAGATCTGCATTGGATTTACC 3 ' (SEQ ID NO: 22);  Upstream primer 5 ' GTGAGATCTGCATTGGATTTACC 3 ' (SEQ ID NO: 22);
下游引物: 5 ' GACCTCGAGACTCTGCCTTCTGT 3 ' (SEQ ID NO: 23)。  Downstream primer: 5 'GACCTCGAGACTCTGCCTTCTGT 3 ' (SEQ ID NO: 23).
利用 Bgl l l/Xho I进行酶切, 然后将其插入经过同样酶切的 pSUPER basic载体 (Ol igoEngine)内。 通过常规测序方法确定插入片段的完整性和准确性, 获得正确的 miR-146a表达载体。  The enzyme was digested with Bgl l l/Xho I and inserted into the pSUPER basic vector (OligoEngine) which was similarly digested. The integrity and accuracy of the insert was determined by conventional sequencing methods to obtain the correct miR-146a expression vector.
将干扰素诱导因子 5 ( IRF5 ) 和信号转导和转录激活子 1 ( STAT 1 ) 基因 3 ' 端非翻 译区 (UTR) 内预测可与 miR-146a互补结合的一段序列克隆接入 pMIR-REPORT 载体 Interferon-inducible factor 5 ( IRF5 ) and a 3 ' untranslated region (UTR) of the signal transduction and activator of transcription 1 ( STAT 1 ) gene are predicted to bind to a sequence of clones that bind to miR-146a to pMIR-REPORT Carrier
( Ambion公司) 萤火虫荧光素酶下游的 Sac I /Hind III 酶切位点内, 构建 3 ' UTR荧光 报告载体。 引物序列如下: ( Ambion) A 3 ' UTR fluorescent reporter vector was constructed within the Sac I /Hind III restriction site downstream of firefly luciferase. The primer sequences are as follows:
IRF5正向弓 I物: 5_ ' GTCGAGCTCTCTTGTGTATATTC-3 ' (SEQ ID NO: 28) , IRF5反向弓 I物: 5, - GAGAAGCTTGGAGTGTGCAGAGAT- 3, (SEQ ID NO: 29); STAT1正向弓 I物: 5 ' -GTGGAGCTCTTTACTGTTTGTTATGG-3 ' (SEQ ID NO: 30), STAT1反向弓 |物: 5 ' -ACGAAGCTTAATAGACTAAATACCAC-3 ' (SEQ ID NO: 31)。 IRF5 forward bow I: 5_ ' GTCGAGCTCTCTTGTGTATATTC-3 ' (SEQ ID NO: 28) , IRF5 reciprocal I: 5, - GAGAAGCTTGGAGTGTGCAGAGAT-3, (SEQ ID NO: 29); STAT1 forward arch I: 5 '-GTGGAGCTCTTTACTGTTTGTTATGG-3 ' (SEQ ID NO: 30), STAT1 recurve | : 5 ' -ACGAAGCTTAATAGACTAAATACCAC-3 ' (SEQ ID NO: 31).
所有载体均通过测序方法确定其插入片段的完整性和准确性。表达载体扩增提取采 用 EndoFree Plasmid Maxi kit (Qiagen公司)方法。 转染以后 miR_146a是否成功过表 达采用 RT-PCR方法检测。  All vectors were determined by sequencing methods for the integrity and accuracy of their inserts. The expression vector was amplified and extracted using the EndoFree Plasmid Maxi kit (Qiagen) method. After transfection, whether miR_146a was successfully expressed or not was detected by RT-PCR.
8 . 细胞培养, 转染和剌激 8. Cell culture, transfection and stimulation
293T禾 P SMMC-7721细胞培养于加了 10%FBS的 DMEM营养液中, 293T/ISRE (稳转 ISRE 报告基因的 293T细胞)则培养于加了 10%FBS和 20μ /πι1的潮霉素 Β的 DMEM营养液中, 这三种细胞系采用脂质体 2000 (lnvitrogen公司)转染方法。 通过密度梯度离心方法 (Cedarlane公司)从人外周血中分选得到外周血单个核细胞(PBMC), 然后在加了 10%FBS 的 RPMI 1640中培养 2小时, 运用电转仪(Amaxa公司, programme T-16)将 1. 5μ 空载 体或者 miR-146表达载体分别电转入 3 X 106的 PBMC中, 否则电转入 3μ miRIDIAN™ (有 miR-146a抑制剂)或一段随机无关序列(Dharmacon公司)。 剌激细胞时, 首先在转染后 18或者 24小时之后细胞换液, 然后将细胞分别培养于单独新鲜培养基中和加入 I型干 扰素(PBL Interferonsource公司)或各种 TLR配体(Invivogen)的新鲜培养基中。 293T and P SMMC-7721 cells were cultured in DMEM supplemented with 10% FBS, and 293T/ISRE (293T cells stably transfected with ISRE) were cultured in hygromycin with 10% FBS and 20μ /πι1. In the DMEM nutrient solution, these three cell lines were transfected with liposome 2000 (l nv itrog en ). Peripheral blood mononuclear cells (PBMC) were sorted from human peripheral blood by density gradient centrifugation (Cedarlane), and then cultured for 2 hours in RPMI 1640 supplemented with 10% FBS using an electrorotator (Amaxa, programme T -16) The 1. 5μ empty vector or miR-146 expression vector was separately transferred into 3×10 6 PBMC, otherwise electroporated into 3 μM RIDIANTM (with miR-146a inhibitor) or a random unrelated sequence (Dharmacon Inc.) ). When stimulating cells, first change the cells 18 or 24 hours after transfection, then culture the cells separately in fresh medium and add type I interferon (PBL Interferonsource) or various TLR ligands (Invivogen). In fresh medium.
9 . 荧光报告系统 9. Fluorescence reporting system
首先把 SMMC-7721细胞系铺于 96孔板,将 20ng 3 ' UTR荧光报告载体, 10ng pRL-TK 载体(Promega公司)和 270ng空载体(pSUPER basic)或者 miR_146a表达载体混匀后转染 入每孔 S匪 C-7721细胞, 转染后细胞培养 24小时, 裂解后即可按照双荧光报告系统 (Promega公司)说明上机(TR717, Appl ied Biosystems公司)检测, 对于每个孔而言可 以得到一个 ISRE与 Reni l la的荧光信号比值。 293T/ISRE细胞系步骤同上, 不同的是转 入 300ng空载体或 miR-146a表达载体。 每次实验均为四复孔, 每个实验重复三次。  First, the SMMC-7721 cell line was plated in a 96-well plate, and 20 ng of 3' UTR fluorescent reporter vector, 10 ng of pRL-TK vector (Promega) and 270 ng of empty vector (pSUPER basic) or miR_146a expression vector were mixed and transfected into each. Well S匪C-7721 cells were cultured for 24 hours after transfection. After lysis, they were assayed according to the dual fluorescence reporter system (Promega) (TR717, Applied Biosystems). For each well, The ratio of the fluorescence signal of an ISRE to Reni l la. The 293T/ISRE cell line was subjected to the same procedure as above, except that 300 ng of empty vector or miR-146a expression vector was transferred. Each experiment was a quadruple well and each experiment was repeated three times.
10. Western 免疫印迹法 10. Western immunoblotting
首先将 293T细胞培养在 6孔板上, 然后分别在每孔细胞内转染入 3 g miR-146a表 达载体, 转染 24小时后, 裂解细胞抽取蛋白。 上清加入 SDS-PAGE胶中进行电泳, 用直 接抗体进行免疫印迹, 通过 Luminol/Enhancer试剂(Pierce公司)检测。 IRF5和 GAPDH 抗体分别由 Abeam公司和 Chen icon公司购买获得, SATAl和 HRP标记的二抗来源于 Santa Cruz公司。 蛋白质相对表达量通过 Quantity One 4. 52软件(Bio-Rad公司)计算分析获 得。 实施例 1 一组 miRNA在 SLE患者和正常对照组之间表达水平存在差异  The 293T cells were first cultured in a 6-well plate, and then transfected into 3 g of miR-146a expression vector in each well. After transfection for 24 hours, the cells were lysed by lysis. The supernatant was subjected to electrophoresis by adding SDS-PAGE gel, and immunoblotted with a direct antibody, and detected by Luminol/Enhancer reagent (Pierce). IRF5 and GAPDH antibodies were purchased from Abeam and Chen icon, respectively, and SATAl and HRP-labeled secondary antibodies were sourced from Santa Cruz. The relative expression of protein was obtained by calculation and analysis by Quantity One 4. 52 software (Bio-Rad). Example 1 Difference in expression levels between a group of miRNAs in SLE patients and normal controls
为探讨 miRNA在 SLE方面发挥的作用, 本发明人应用 Real-time PCR方法对 SLE患 者和正常对照组外周血 156个成熟 miRNA表达水平差异进行了研究。 结果发现, 有 42个 miRNA表达水平在 SLE患者中明显低于正常人, 其中包括 miR-146a在内的 7个 miRNA表达水平降低高达 6倍以上, 见图 1A和表 1。 To investigate the role of miRNAs in SLE, the inventors used Real-time PCR to study the difference in expression levels of 156 mature miRNAs in peripheral blood of SLE patients and normal controls. The results showed that 42 miRNA expression levels were significantly lower in SLE patients than in normal subjects, and the expression levels of 7 miRNAs including miR-146a were reduced by more than 6-fold, as shown in Figure 1A and Table 1.
表 1  Table 1
Figure imgf000014_0001
接着, 本发明人对 47例 SLE患者, 6例 BD患者和 21例正常对照运用同样的方法 检测 miR-146a的表达水平。 miR_146a在 SLE患者中表达水平显著低于正常对照
Figure imgf000014_0001
Next, the inventors used the same method to detect the expression level of miR-146a in 47 patients with SLE, 6 patients with BD, and 21 normal controls. The expression level of miR_146a in SLE patients was significantly lower than that of normal controls.
(尸〈0. 0001), 而 BD患者相比正常人无明显变化, 见图 1B。 (The corpse <0. 0001), and BD patients have no significant changes compared with normal people, see Figure 1B.
根据病人具体情况和个体化治疗原则, 所有病人用药种类和剂量不尽相同, 进一步 本发明人分析了用药剂量与 miR-146a表达之间的关系。 挑选了 29例激素用量较大的活 动性病人, 分析激素用量与 miR-146a之间的相关性, 发现激素水平对 miR_146a表达并 无影响(/ ). 1442)。 根据是否同时服用非特异性免疫抑制剂将病人分成两组, 两组之间 miR-146a表达同样不存在统计学差异(/ ). 7149)。 综上表明在 SLE病人中 miR_146a表 达水平降低系原发性的, 与用药情况无关。 实施例 2 miR-146表达信号与疾病活动程度和肾脏受累程度负相关  According to the patient's specific conditions and individualized treatment principles, the types and doses of all patients were different, and the inventors analyzed the relationship between the dose and the expression of miR-146a. Twenty-nine active patients with high hormonal doses were selected to analyze the correlation between hormone dosage and miR-146a, and it was found that hormone levels had no effect on miR_146a expression (/). 1442). Patients were divided into two groups according to whether they were taking non-specific immunosuppressive agents at the same time. There was also no statistical difference in miR-146a expression between the two groups (/). 7149). In summary, the decrease in miR_146a expression level in SLE patients is primary, and has nothing to do with medication. Example 2 miR-146 expression signal is inversely related to the degree of disease activity and renal involvement
根据本发明人前面数据结果, 进一步研究了 miR-146a表达水平与疾病的相关性。 结果显示, miR-146a在 SLE患者中显著低于正常对照组, 非参数 Mann-Whitney检 验显示差异有统计学意义(/^值均〈0. 0001), 见图 2A。  Based on the results of the previous data of the present inventors, the correlation between the expression level of miR-146a and disease was further studied. The results showed that miR-146a was significantly lower in the SLE patients than in the normal control group, and the nonparametric Mann-Whitney test showed statistically significant differences (/^ values were <0. 0001), as shown in Figure 2A.
miR-146a水平在 SLE患者活动组(SLEDAI积分 5)显著低于稳定组(SLEDAI积分 0〜 4分), 非参数 Mann-Whitney检验显示差异有统计学意义(7 ). 0080), 见图 2A。  The miR-146a level was significantly lower in the SLE patient activity group (SLEDAI score 5) than in the stable group (SLEDAI score 0 to 4 points), and the nonparametric Mann-Whitney test showed a statistically significant difference (7). 0080), see Figure 2A. .
在此基础之上进一步应用 Spearman单尾相关性检验发现 SLEDAI积分和 miR_146a 两者之间存在负相关(r=_0. 2882, 7 ). 0247), 见图 2C。  Based on this, the Spearman one-tailed correlation test is used to find that there is a negative correlation between SLEDAI integral and miR_146a (r=_0. 2882, 7). 0247), see Figure 2C.
排除其他肾脏疾患影响后本发明人根据 24小时尿蛋白水平将 SLE分成尿蛋白阳性 组(24小时尿蛋白大于 0. 5g)和尿蛋白阴性组(24小时尿蛋白小于 0. 5g )两组, 分析后 发现 miR-146a水平在两组之间同样存在明显差异(7 ). 0271), 见图 2B。  After excluding the effects of other kidney diseases, the inventors divided the SLE into a urine protein-positive group (24-hour urine protein greater than 0.5 g) and a urine protein-negative group (24-hour urine protein less than 0.5 g) according to the 24-hour urine protein level. After analysis, it was found that the level of miR-146a was also significantly different between the two groups (7). 0271), see Figure 2B.
单纯根据肾脏累及的四项指标(血尿、脓尿、蛋白尿和管型尿)计算得到肾脏(Renal) 积分, 本发明人也发现 miR-146a水平与肾脏积分之间存在负性相关(r= -0. 3815, 7^0. 0081), 见图 2D。 实施例 3 miR-146a水平与 I型干扰素通路过度活化相关 The renal (Renal) score was calculated based on the four indicators of the kidney (hematuria, pyuria, proteinuria, and tubular urine). The inventors also found a negative correlation between miR-146a levels and kidney scores (r= - 0. 3815, 7^0. 0081), see Figure 2D. Example 3 Levels of miR-146a are associated with excessive activation of type I interferon pathway
已有研究结果显示 IRAKI和 TRAF6活化后可通过多种信号通路传导导致下游 I型干 扰素的产生, 而且 I型干扰素通路在狼疮发病中发挥着关键性作用。 本发明人通过检测 I型干扰素下游三个代表性基因的 mRNA表达水平来反映病人体内 I型干扰素通路的活化 程度, 并探索 SLE中 miR-146a表达水平降低与 I型干扰素通路的关系。 Spearman双尾 相关性检验结果显示, miR-146a表达水平与 IFN积分水平之间存在负性相关(r = -0.3073, P = 0.0378), 见图 3。 实施例 4 miR-146a可负反馈调节 I型干扰素通路  Studies have shown that IRAKI and TRAF6 can be transmitted through multiple signaling pathways leading to the production of downstream type I interferon, and that type I interferon pathway plays a key role in the pathogenesis of lupus. The present inventors detected the activation level of type I interferon pathway in patients by detecting the mRNA expression levels of three representative genes downstream of type I interferon, and explored the relationship between the decrease of miR-146a expression level and the type I interferon pathway in SLE. . Spearman's two-tailed correlation test showed a negative correlation between miR-146a expression and IFN scores (r = -0.3073, P = 0.0378), as shown in Figure 3. Example 4 miR-146a Negative Feedback Regulation Type I Interferon Pathway
为了进一步研究 miR-146a表达与 I型干扰素通路过度活化之间的相关性, 本发明 人对 miR-146a对 I型干扰素的产生及干扰素通路下游活化的影响作了进一步研究。 把 miR-146a电转入 1名正常人来源的 PBMC中,然后用 TLR7的配体 R837 (购自 invivogen) 剌激, 检测 I型干扰素的 mRNA水平。  To further investigate the correlation between miR-146a expression and over-activation of type I interferon pathway, the inventors further investigated the effects of miR-146a on the production of type I interferon and downstream activation of the interferon pathway. miR-146a was electroporated into a normal human-derived PBMC and then stimulated with TLR7 ligand R837 (purchased from invivogen) to detect type I interferon mRNA levels.
结果发现, 过表达 miR-146a能够明显抑制 IFNa和 的表达(见图 4 A)。 预先 处理将体内 miR-146a抑制后, 可发现 I型干扰素表达明显上调(见图 4 B), 结果提示, miR-146a能够负反馈调节 I型干扰素的产生。  It was found that overexpression of miR-146a significantly inhibited the expression of IFNa and (see Figure 4A). Pretreatment with in vivo inhibition of miR-146a revealed a significant up-regulation of type I interferon expression (see Figure 4B). The results suggest that miR-146a can negatively regulate the production of type I interferon.
I型干扰素被诱导产生后, 可与细胞膜表面干扰素受体 (如 IFNAR1或 IFNAR2) 结 合使下游 STAT磷酸化。活化的 STAT1和 STAT2,与 DNA结合蛋白干扰素诱导因子 9(IRF9) 共同形成干扰素剌激基因因子 3(ISGF3), ISGF3可结合于 ISRE产生活化信号, 剌激下 游干扰素诱导基因的产生 [Platanias IX. Mechanisms of type-I- and  After type I interferon is induced, it binds to cell membrane surface interferon receptors (such as IFNAR1 or IFNAR2) to phosphorylate downstream STAT. Activated STAT1 and STAT2, together with the DNA-binding protein interferon-inducible factor 9 (IRF9), form interferon-stimulating gene factor 3 (ISGF3), which binds to ISRE to generate an activation signal that stimulates downstream interferon-inducible gene production [ Platanias IX. Mechanisms of type-I- and
type-I I-interf eron-mediated signalling. Nat Rev Immunol 2005; 5: 375-86]。 因 此, 本发明人通过测定 ISRE的荧光活性来估计 miR-146a对 I型干扰素下游信号传导的 影响。在用 I型干扰素剌激的 293T/ISRE细胞系中过表达 miR-146a后能够明显抑制 ISRE 报告基因活性(见图 4 C),提示 miR-146a能够直接调控干扰素受体下游活化信号的传导。 type-I I-interf eron-mediated signalling. Nat Rev Immunol 2005; 5: 375-86]. Therefore, the inventors estimated the effect of miR-146a on the downstream signaling of type I interferon by measuring the fluorescence activity of ISRE. Overexpression of miR-146a in a 293T/ISRE cell line stimulated with type I interferon significantly inhibited ISRE reporter gene activity (see Figure 4 C), suggesting that miR-146a directly regulates the activation signal downstream of the interferon receptor. Conduction.
为了进一步阐明 miR-146a对干扰素诱导基因表达的影响, 本发明人将来源于另外 一个正常人的 PBMC按照上面同样步骤处理, 过表达 miR-146a后同样能够抑制干扰素诱 导基因的表达(见图 4 D)。 综上 miR-146a同时能够有效地调节 I型干扰素下游的激活。 实施例 5 miR-146a通过多个关键信号分子来调控 I型干扰素通路  To further elucidate the effect of miR-146a on interferon-inducible gene expression, the inventors treated PBMC derived from another normal human according to the same procedure as above, and overexpression of miR-146a also inhibited the expression of interferon-inducible genes (see Figure 4 D). In summary, miR-146a can effectively regulate the activation of type I interferon downstream. Example 5 miR-146a regulates type I interferon pathway through multiple key signaling molecules
上面的研究结果提示 miR-146a是 I型干扰素通路的一个负反馈调控因子, 为了探 索其作用发挥的分子机制, 本发明人运用生物信息学分析 miR-146a潜在的靶基因。 已 有的很多软件分析结果都显示 miRNA主要作用于基因的阳性调节基序及其下游的信号转 导分子网络。 因此, 本发明人将 I型干扰素通路上关键的信号蛋白与 miR-146a序列综 合预测, 寻找其潜在的结合位点。 使用 miRBase  The above results suggest that miR-146a is a negative feedback regulator of the type I interferon pathway. In order to explore the molecular mechanism of its action, the inventors used bioinformatics to analyze the potential target genes of miR-146a. A number of software analyses have shown that miRNAs act primarily on the positive regulatory motif of the gene and its downstream network of signal transduction molecules. Therefore, the inventors predicted the key signaling proteins of the type I interferon pathway and the miR-146a sequence to find potential binding sites. Use miRBase
(http://microrna. sanger. ac. uk/targets/v5/) 禾口 TargetScan (http://microrna. sanger. ac. uk/targets/v5/) and PortTargetScan
(http:〃 www. targetscan. org/)软件。 结果发现, 在已经确定的两个靶基因 IRAKI和 TRAF6以外, IRF5禾 P STAT13' UTR同样存在 miR_146a的结合位点, 见图 5 A。 已知这几 个蛋白质都是 I型干扰素通路上的相关分子。 在多个种族中均发现 IRF5基因上的一个 单倍型可以影响 IRF5多个剪切体的表达从而增加 SLE发病易感性, 且 SLE病人和狼疮 鼠模型中也同时检测到基础状态下及剌激后状态下 STAT1表达水平皆明显升高。 (http: 〃 www. targetscan. org/) software. As a result, it was found that, in addition to the two target genes IRAKI and TRAF6, the binding sites of miR_146a were also present in IRF5 and P STAT13' UTR, as shown in Fig. 5A. Known here Each protein is a related molecule on the type I interferon pathway. A haplotype on the IRF5 gene was found in multiple races to affect the expression of multiple ISF5 splicing and increase the susceptibility to SLE, and both SLE patients and lupus mouse models also detected basal state and stimuli. The expression level of STAT1 was significantly increased in the post-state.
进一步体外实验验证了本发明人这一预测, 在过转染 miR-146a的 SMMC-7721细胞 系中, IRF5和 STAT1 3 ' UTR报告基因的活性明显受到抑制, 见图 5 B, 提示 miR_146a 可通过互补结合于预测序列抑制这两个基因的表达。 在 293T细胞系过表达 miR-146a后 使用 Western免疫印迹检测, 发现蛋白水平上 IRF5和 STAT 1的表达均有明显下降, 见 图 5 C。 因此, miR-146a可通过调节 I型干扰素通路上的关键信号分子来调控固有免疫 应答。 实施例 6 人为升高 SLE患者中 miR-146a表达水平可部分逆转 I型干扰素活化水平 miR-146a通过调节 I型干扰素通路上的关键信号分子来调控固有免疫应答, 那么 人为升高 SLE患者体内 miR-146a表达水平是否会逆转 I型干扰素通路过度活化的程度。 将 miR-146a电转入来源于一例 SLE患者的 PBMC, miR-146a水平升高能够降低部分干扰 素诱导基因的水平, 见图 6。 计算干扰素积分, 也发现过转染 miR-146a载体的干扰素 积分(67. 87)低于过转空载体的干扰素积分(153. 59), 提示人为升高 SLE患者体内 miR-146a表达水平可作为潜在的治疗手段。 实施例 7 药物筛选  Further in vitro experiments confirmed the inventors' prediction that in the SMMC-7721 cell line transfected with miR-146a, the activity of the IRF5 and STAT1 3 ' UTR reporter genes was significantly inhibited, as shown in Figure 5 B, suggesting that miR_146a can pass Complementary binding to the predicted sequence inhibits the expression of both genes. Western blot analysis of the 293T cell line overexpressing miR-146a revealed a significant decrease in the expression of IRF5 and STAT 1 at the protein level, see Figure 5 C. Thus, miR-146a regulates the innate immune response by modulating key signaling molecules on the type I interferon pathway. Example 6 Human elevation of miR-146a expression levels in SLE patients can partially reverse type I interferon activation levels miR-146a regulates innate immune responses by modulating key signaling molecules on the type I interferon pathway, then artificially elevated SLE patients Whether the level of miR-146a expression in vivo reverses the extent of over-activation of the type I interferon pathway. Transferring miR-146a to PBMC from a patient with SLE, elevated levels of miR-146a reduced the level of some interferon-inducible genes, as shown in Figure 6. Computation of interferon scores also revealed that the interferon score (67.87) transfected with the miR-146a vector was lower than the interferon score of the transposon vector (153.59), suggesting that human expression of miR-146a is elevated in SLE patients. Levels can be used as a potential treatment. Example 7 Drug Screening
以 HEK 293T为受试对象, 检测候选物质剌激前后的 HEK 293T中 miR-146的表达状 况; 检测方法如前述 "材料和方法" 中第 4项、 第 8项和实施例 4中所述。  Using HEK 293T as a test subject, the expression of miR-146 in HEK 293T before and after the challenge of the candidate substance was detected; the detection method is as described in Items 4, 8 and 4 of the above-mentioned "Materials and Methods".
测试组: 添加候选物质的重组的 HEK 293T (制备方法见 "材料和方法" 中第 8项) 培养物;  Test group: Recombinant HEK 293T with candidate substance added (for preparation method, see item 8 in Materials and Methods) Culture;
对照组: 不添加候选物质的重组的 HEK 293T (制备方法见 "材料和方法" 中第 8 项)培养物。  Control group: Recombinant HEK 293T (see Section 8 of Materials and Methods) cultures without the addition of candidate substances.
观察测试组和对照组的 miR-146的表达状况, 如果测试组中 miR-146的表达上升, 则说明该候选物质是对于防治 I型干扰素通路异常激活相关疾病有用的物质。 实施例 8 miR-146a的拮抗剂可用来增强 I型干扰素通路  The expression status of miR-146 in the test group and the control group was observed, and if the expression of miR-146 in the test group was increased, it indicates that the candidate substance is a substance useful for preventing a disease associated with abnormal activation of the type I interferon pathway. Example 8 Antagonists of miR-146a can be used to enhance the type I interferon pathway
上面的研究结果表明 miR-146a能抑制 I型干扰素通路。而降低 miR_146a的水平或 用 miR-146a的反义寡核苷酸链及其类似物抑制 miR_146a的作用, 将会增强 I型干扰素 通路。 见图 4B, 利用 miR-146a的反义寡核苷酸抑制 miR_146a, IFN a和 的表达 明显高于对照组。 讨论  The above results indicate that miR-146a can inhibit the type I interferon pathway. Decreasing the level of miR_146a or inhibiting the action of miR_146a with the antisense oligonucleotide chain of miR-146a and its analogs would enhance the type I interferon pathway. As shown in Figure 4B, the antisense oligonucleotides of miR-146a inhibited miR_146a, and the expression of IFN a was significantly higher than that of the control group. Discussion
本发明首先检测 SLE患者中 miRNA的表达差异, 分析差异表达的 miR_146a在 I型 干扰素通路活化中的左右, 对自身免疫性疾病的发病机理做了进一步的扩展。 miR-146a 表达与疾病活动之间的明显相关也提示 miR-146a可作为 SLE的一个新的生物标志物。 本发明在样本筛选阶段排除了感染因素的影响, 其次将 miR-146a表达水平与不同 用药(包括激素和免疫抑制剂)剂量之间做相应统计分析后所得均未发现差异, 故排除了 药物因素对结果的干扰, 表明结果差异确实是因为疾病本身情况所致。 The invention firstly detects the difference of miRNA expression in SLE patients, and analyzes the differential expression of miR_146a in the activation of type I interferon pathway, and further expands the pathogenesis of autoimmune diseases. miR-146a The apparent correlation between expression and disease activity also suggests that miR-146a can be used as a new biomarker for SLE. The invention excludes the influence of infectious factors in the sample screening stage, and secondly, the difference between the expression level of miR-146a and the dose of different drugs (including hormones and immunosuppressive agents) is not found, so the drug factor is excluded. The interference with the results indicates that the difference in results is indeed due to the disease itself.
miR-146 a在病人中水平显著低于正常对照, 且其水平与反映病情活动程度和肾脏 损害情况的 SLEDAI积分和 Renal积分呈负性相关,提示 miR-146 a可以作为判断病情活 动程度和肾脏损害严重度的一个生物标记物, 为早期便捷地对 SLE进行诊断, 评估疾病 活动程度和肾脏受累严重情况提供了良好的依据。  The level of miR-146 a was significantly lower in patients than in normal controls, and its level was negatively correlated with SLEDAI score and Renal score reflecting the degree of disease activity and renal damage, suggesting that miR-146 a can be used as a measure of disease activity and kidney. A biomarker of severity of damage provides a good basis for early and convenient diagnosis of SLE, assessing the extent of disease activity and the severity of kidney involvement.
已知干扰素通路在 SLE发病中发挥重要作用,干扰素通路异常激活是 SLE的一个主 要分子表型 [Pasciml V等, Banchereau J Systemi c lupus erythematosus: al l roads lead to type I interferons. Curr Op in Immunol. 2006 Dec ; 18 (6): 676-82. Epub 2006 Oct 2]。 TRAF6/IRAK1作为 TLR通路下游的接头蛋白, 在诱导 I型 IFN的产生中起着关 键的作用 [Uematsu S, Sato S Interleukin-1 receptor-assoc iated kinase-1 plays an essent ial role for Tol l-l ike receptor (TLR) 7- and TLR9-mediated  The interferon pathway is known to play an important role in the pathogenesis of SLE. Abnormal activation of the interferon pathway is a major molecular phenotype of SLE [Pasciml V et al., Banchereau J Systemi c lupus erythematosus: al l roads lead to type I interferons. Curr Op in Immunol. 2006 Dec; 18 (6): 676-82. Epub 2006 Oct 2]. TRAF6/IRAK1 acts as a linker protein downstream of the TLR pathway and plays a key role in the induction of type I IFN production [Uematsu S, Sato S Interleukin-1 receptor-assocated kinase-1 plays an essent ial role for Tol ll receptor (TLR) 7- and TLR9-mediated
interferon- {alpha} induction. J Exp Med. 2005 Mar 21 ; 201 (6) : 915-23. Epub 2005 Mar 14.禾口 Kawai T, Sato S等, Interf eron-alpha induct ion through Tol l-l ike receptors involves a direct interaction of IRF7 wi th MyD88 and TRAF6. Nat Immunol. 2004 0ct ; 5 (10) : 1061-8. Epub 2004 Sep 7]。 本发明人的研究结果表明 miRNA参与 SLE 疾病的发生发展, miRNA表达信号可作为 SLE临床诊断的一个重要生物标志物; miR_146a 可作为一个新的药物干预靶点, 特异性干预 miR-146a的表达水平可发展为新的治疗手 段, 这种有针对性和特异性的干预有望高效低副作用地对疾病进行治疗。 Interferon- {alpha} induction. J Exp Med. 2005 Mar 21 ; 201 (6) : 915-23. Epub 2005 Mar 14. and Kawai T, Sato S et al, Interf eron-alpha induct ion through Tol ll ike receptors a direct interaction of IRF7 wi th MyD88 and TRAF6. Nat Immunol. 2004 0ct ; 5 (10) : 1061-8. Epub 2004 Sep 7]. The results of the present inventors indicate that miRNA is involved in the development of SLE disease, and miRNA expression signal can be used as an important biomarker for clinical diagnosis of SLE; miR_146a can be used as a new drug intervention target to specifically interfere with the expression level of miR-146a. It can be developed as a new treatment, and this targeted and specific intervention is expected to treat the disease with high efficiency and low side effects.
TLR通过与其相应的配体结合在自身免疫反应中发挥着重要的作用, 在 SLE患者的 浆细胞样树突状细胞( P DC)中, RNA结合核蛋白和自身 DNA与相应的自身抗体结合形成 的免疫复合物可通过结合于 TLR7或者 TLR9促进 I型干扰素的大量产生, 这个反应需要 包括 MyD88, IRAKI , TRAF6 , IRF5和 IRF7在内的信号接头蛋白和转录蛋白的次序激活 来实现。 目前研究发现包含 RNA的自身免疫复合物主要通过结合于细胞内涵体膜上组成 性表达的 TLR7和 TLR9 , 使下游 IRF5激活, 在 I型干扰素产生中发挥重要的作用。 干扰 素一旦产生, 便可结合于干扰素受体, 通过 STAT蛋白的活化最终导致干扰素诱导基因 基因的转录产生。 生理状态下, 免疫细胞可自发地通过多种机制来负反馈调节 TLR信号 传导通路从而保证免疫系统处于一个相对平衡的状态, 这些机制包括细胞内组成性表达 或者诱导表达负调节因子(如 MyD88s, IRAKM, S0CS1),负反馈调节的缺陷造成正向信号的 过度传导最终引起人体疾病。 这些观点已经在很多研究中得到证实, 比如哮喘病人中遗 传学研究发现 IRAKM基因存在非活性损伤, 同时在 S0CS1基因缺陷狼疮鼠模型中可发现 类似于系统性自身免疫病的临床表型。 现在的研究结果发现 miRNA也属于负调节因子中 的一类, 如在人单核细胞 THP-1细胞系中发现 miR-146可通过形成负反馈调节通路来调 节 TLR4信号传导。 在原代细胞中, 本发明人也发现 R837 (TLR7配体)、 CpG (TLR9配体)、 I型干扰素和 LPS (TLR4配体)能够剌激正常人来源的 PBMC中 miR_146a表达升高(见图 7 )。 这说明 miR-146a确实参与了固有免疫应答的复杂的调节网络。 虽然以往的研究中 曾显示 THP-1细胞系中 TLR7和 TLR9并不能剌激 miR_146a表达, 但可能是 TLR表达模 式的不同和剌激反应强度不同所致。 因为已有研究证明在体外简单培养基中单一细胞的 反应没有办法重复人外周血内激素与细胞因子多重相互作用下的实验结果。 TLR plays an important role in autoimmune response by binding to its corresponding ligand. In plasma-derived dendritic cells (P DC) of SLE patients, RNA-binding nucleoprotein and self-DNA bind to the corresponding autoantibodies. The immune complex can promote the large-scale production of type I interferon by binding to TLR7 or TLR9, which requires the activation of the sequence of signal adaptor proteins and transcription proteins including MyD88, IRAKI, TRAF6, IRF5 and IRF7. The current study found that autoimmune complexes containing RNA mainly activate TRF7 and TLR9, which are constitutively expressed on the endosomal membrane of cells, to activate downstream IRF5 and play an important role in the production of type I interferon. Once interferon is produced, it can bind to the interferon receptor, and the activation of the STAT protein ultimately leads to the transcription of the interferon-inducible gene. Under physiological conditions, immune cells can spontaneously regulate the TLR signaling pathway through a variety of mechanisms to ensure that the immune system is in a relatively balanced state, including intracellular constitutive expression or induction of negative regulators (such as MyD88s, IRAKM, S0CS1), a defect in negative feedback regulation causes excessive conduction of the positive signal and ultimately causes human disease. These observations have been confirmed in many studies. For example, genetic studies in asthma patients have found that the IRAKM gene has inactive damage, and a clinical phenotype similar to systemic autoimmune disease can be found in the S0CS1 gene-deficient lupus mouse model. The current study found that miRNAs also belong to a class of negative regulators. For example, in human monocyte THP-1 cell line, miR-146 was found to regulate TLR4 signaling by forming a negative feedback regulatory pathway. In primary cells, the inventors also found that R837 (TLR7 ligand), CpG (TLR9 ligand), type I interferon and LPS (TLR4 ligand) are capable of stimulating the expression of miR_146a in normal human-derived PBMC (see Figure 7). This suggests that miR-146a does participate in a complex regulatory network of innate immune responses. Although previous studies have shown that TLR7 and TLR9 in THP-1 cell lines do not stimulate miR_146a expression, it may be due to differences in TLR expression patterns and different stimuli. Because studies have shown that single cell reactions in simple medium in vitro have no way to replicate the experimental results of multiple interactions between human peripheral blood hormones and cytokines.
在本发明中,发现 miR-146a可通过 TLR7通路抑制下游 I型干扰素的产生及干扰素 下游的反应性。 为了探讨 miR-146a的作用机制, 本发明人采用生物信息学方法预测, 在原有的发现基础上, 预测得到两个新的靶基因: IRF5和 STAT1 , 所有这些基因分子都 是干扰素通路上下游关键的信号分子, 因此 miR-146a能够调节 I型干扰素通路中的多 个组成部分(见图 8 )。 也许 miR-146a对于某一个靶基因而言调节作用有限, 但是几个 靶基因的协同作用使 miR-146a的调节明显放大。这与 miR-181a的作用机制有类似之处, miR-181a通过对多种磷酸酶的温和调节, 有效地降低 T细胞对抗原识别的兴奋阈值, 这 是 siRNA的单靶点强大的调节作用所无法比拟的。 miR-146a的表达缺陷, 导致其靶基因 的大量蓄积, 可进一步促进 I型干扰素的大量产生及其下游信号的过度活化, 结果中显 示的 miR-146a表达与 I型干扰素之间的负相关也同样符合上面假设, 本研究揭示了 miRNA失调在自身免疫性疾病中发挥的作用。  In the present invention, it was found that miR-146a inhibits the production of downstream type I interferon and the reactivity downstream of interferon through the TLR7 pathway. In order to investigate the mechanism of action of miR-146a, the inventors used bioinformatics methods to predict, based on the original findings, predicted two new target genes: IRF5 and STAT1, all of which are upstream and downstream of the interferon pathway. A key signaling molecule, therefore miR-146a is able to modulate multiple components of the type I interferon pathway (see Figure 8). Perhaps miR-146a has a limited regulatory effect on a target gene, but the synergy of several target genes significantly amplifies the regulation of miR-146a. This is similar to the mechanism of action of miR-181a. miR-181a effectively reduces the excitatory threshold of T cell recognition by antigen by mild regulation of multiple phosphatases, which is a powerful regulation of siRNA single target. incomparable. Defective expression of miR-146a leads to a large accumulation of its target gene, which can further promote the large-scale production of type I interferon and the excessive activation of downstream signals. The results show a negative relationship between miR-146a expression and type I interferon. The correlation is also consistent with the above hypothesis, and this study reveals the role of miRNA dysregulation in autoimmune diseases.
为探讨 SLE病人中 miR-146a下降原因,本发明人应用生物信息学分析发现 miR-146a 启动子区域有一个潜在甲基化位点(http:〃 cpgislands. usc. edu/)。 该 CpG岛的位置与一 个预测的 STAT1结合位点和一个确证的 NF K B结合位点明显重叠  To investigate the cause of miR-146a decline in SLE patients, the inventors applied bioinformatics analysis to find a potential methylation site in the miR-146a promoter region (http: cp cpgislands. usc. edu/). The location of the CpG island overlaps significantly with a predicted STAT1 binding site and a corroborating NF K B binding site.
(http: / / ecrbrowser. dcode. org/)。 (http: / / ecrbrowser. dcode. org/).
因为 I型干扰素在 SLE发病机制中举足轻重的地位,导致 TLR和 IFN的拮抗剂成为 靶点治疗 SLE的热门, 然而由于其对固有免疫和适应性免疫的潜在威胁使人们不得不谨 慎对待。 而不同于以往的有或无的调节特点, miRNA主要是在数量上对靶基因进行调节。 因此人为调节体内 miRNA的表达水平有可能发展成为对抗 SLE的新生手段。 本研究结果 也显示当人为增加 SLE病人 PBMC中 miR-146a表达水平时, 反应干扰素通路活化程度的 干扰素诱导基因的表达均明显下调。 综上提示 miR-146a可作为新的 SLE治疗的靶点。  Because type I interferons play a pivotal role in the pathogenesis of SLE, TLR and IFN antagonists are targets for the treatment of SLE, but they have to be treated with caution because of their potential threat to innate and adaptive immunity. Unlike previous regulatory features with or without miRNAs, miRNAs regulate the target genes quantitatively. Therefore, artificial regulation of miRNA expression levels in vivo may develop into a new means of combating SLE. The results of this study also showed that when the expression level of miR-146a in PBMC of SLE patients was artificially increased, the expression of interferon-inducible genes in response to the activation of interferon pathway was significantly down-regulated. In summary, miR-146a can be used as a target for new SLE treatment.
综上所述, SLE患者中 miR-146a表达缺陷与疾病生物学及临床表型密切相关。 本 发明人的结果提示 miR-146a可作为 SLE—个新的生物标志物, 人为改变患者体内 miR-146a表达水平可发展为新的治疗手段。 在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引 用作为参考那样。 此外应理解, 在阅读了本发明的上述讲授内容之后, 本领域技术人员 可以对本发明作各种改动或修改, 这些等价形式同样落于本申请所附权利要求书所限定 的范围。  In summary, miR-146a expression defects in SLE patients are closely related to disease biology and clinical phenotype. The inventors' results suggest that miR-146a can be used as a new biomarker for SLE, and artificially changing the expression level of miR-146a in patients can be developed into a new treatment. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entirety in the the the the the the the the the the In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the invention.

Claims

1. 一种小核糖核酸的用途, 其特征在于, 用于制备系统性红斑狼疮临床检测的试 剂或试剂盒; A use of a small ribonucleic acid, characterized by a reagent or kit for preparing a clinical test for systemic lupus erythematosus;
其中, 所述的小核糖核酸具有 SEQ ID NO: 2所示的序列。  Wherein the picorucleic acid has the sequence shown in SEQ ID NO: 2.
2. 如权利要求 1所述的用途, 其特征在于, 所述的小核糖核酸还包括选自下组的小 核糖核酸:  2. The use according to claim 1, wherein the small ribonucleic acid further comprises a small ribonucleic acid selected from the group consisting of:
具有 SEQ ID NO: 4所示核酸序列的小核糖核酸,  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 4,
具有 SEQ ID NO: 5所示核酸序列的小核糖核酸,  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 5,
具有 SEQ ID NO: 6所示核酸序列的小核糖核酸,  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 6,
具有 SEQ ID NO: 7所示核酸序列的小核糖核酸,  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 7,
具有 SEQ ID NO: 8所示核酸序列的小核糖核酸, 或  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 8, or
具有 SEQ ID NO: 9所示核酸序列的小核糖核酸。  A small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 9.
3. 一种可用于系统性红斑狼疮临床检测的试剂盒, 其特征在于, 所述的试剂盒包 含:  3. A kit for clinical detection of systemic lupus erythematosus, characterized in that said kit comprises:
容器; 和  Container; and
位于容器中的特异性地针对小核糖核酸或其前体的引物或探针,所述的小核糖核酸 具有 SEQ ID NO: 2所示的序列; 以及  a primer or probe specifically located in a container for a small ribonucleic acid or a precursor thereof, the small ribonucleic acid having the sequence set forth in SEQ ID NO: 2;
用于检测系统性红斑狼疮的说明书。  Instructions for detecting systemic lupus erythematosus.
4. 如权利要求 3所述的试剂盒, 其特征在于, 所述试剂盒中还包括特异性地针对选 自下组的小核糖核酸或其前体的引物或探针:  4. The kit according to claim 3, further comprising a primer or probe specifically targeting a small ribonucleic acid or a precursor thereof selected from the group consisting of:
具有 SEQ ID NO: 4所示核酸序列的小核糖核酸,  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 4,
具有 SEQ ID NO: 5所示核酸序列的小核糖核酸,  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 5,
具有 SEQ ID NO: 6所示核酸序列的小核糖核酸,  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 6,
具有 SEQ ID NO: 7所示核酸序列的小核糖核酸,  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 7,
具有 SEQ ID NO: 8所示核酸序列的小核糖核酸, 或  a small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 8, or
具有 SEQ ID NO: 9所示核酸序列的小核糖核酸。  A small ribonucleic acid having the nucleic acid sequence of SEQ ID NO: 9.
5. 一种小核糖核酸的用途, 其特征在于, 用于制备调控 I型干扰素通路的组合物; 或用于筛选调控 I型干扰素通路的物质,  5. Use of a small ribonucleic acid, characterized in that it is used to prepare a composition for regulating a type I interferon pathway; or for screening for a substance that regulates a type I interferon pathway,
其中, 所述的小核糖核酸具有通式(I)所示的序列:  Wherein the small ribonucleic acid has the sequence represented by the general formula (I):
5 ' UGAGAACUGAAUUCC AURGGYU 3 ' (I);  5 ' UGAGAACUGAAUUCC AURGGYU 3 ' (I);
其中, R选自 A或 G; Y选自 C、 U、 或丁。  Wherein R is selected from A or G; Y is selected from C, U, or D.
6. 如权利要求 5所述的用途, 其特征在于, 所述的组合物还用于: 防治 I型干扰 素通路异常活化相关疾病。  The use according to claim 5, wherein the composition is further for: preventing and treating a disease associated with abnormal activation of the type I interferon pathway.
7. 如权利要求 6所述的用途, 其特征在于, 所述的调控 I型干扰素通路选自: (a) 抑制肿瘤坏死因子受体相关因子 6的表达、 (b)抑制白介素 1受体相关激酶 1 的表达、 (c)抑制干扰素诱导因子 5的表达; (d)抑制信号转导和转录激活子 1的表达(e)抑制 IFN α的表达; (f)抑制 的表达; (g)抑制粘液病毒抗性因子 1表达; (h)抑制 5 ' 寡核 苷酸合成酶表达; 或(i)抑制淋巴细胞抗原 6表达。 7. The use according to claim 6, wherein the regulated type I interferon pathway is selected from the group consisting of: (a) inhibiting expression of a tumor necrosis factor receptor-associated factor 6, and (b) inhibiting an interleukin 1 receptor Expression of related kinase 1 , (c) inhibition of expression of interferon-inducible factor 5; (d) inhibition of signal transduction and expression of activator of transcription 1 (e) inhibition of IFN Expression of α; (f) expression of inhibition; (g) inhibition of mucinous virus resistance factor 1 expression; (h) inhibition of 5 'oligonucleotide synthetase expression; or (i) inhibition of lymphocyte antigen 6 expression.
8. 一种筛选调控 I型干扰素通路的潜在物质的方法, 其特征在于, 所述方法包括 步骤:  8. A method of screening for potential substances that modulate a type I interferon pathway, the method comprising the steps of:
(a) 将候选物质与含有小核糖核酸的体系接触, 所述的小核糖核酸具有通式(I)所 示的序列:  (a) contacting the candidate substance with a system containing a small ribonucleic acid having the sequence of the formula (I):
5 ' UGAGAACUGAAUUCC AURGGYU 3 ' (I);  5 ' UGAGAACUGAAUUCC AURGGYU 3 ' (I);
其中, R选自 A或 G; Y选自 C、 U、 或 T;  Wherein R is selected from A or G; Y is selected from C, U, or T;
(b) 观察候选物质对于小核糖核酸的表达的影响;  (b) observing the effect of candidate substances on the expression of small RNA;
其中, 若所述候选物质可提高小核糖核酸的表达, 则表明该候选物质是抑制 I型干 扰素通路的潜在物质; 若所述候选物质可降低小核糖核酸的表达, 则表明该候选物质是 促进 I型干扰素通路的潜在物质。  Wherein, if the candidate substance can increase the expression of the small ribonucleic acid, it indicates that the candidate substance is a latent substance that inhibits the type I interferon pathway; if the candidate substance can reduce the expression of the small ribonucleic acid, it indicates that the candidate substance is Potential substances that promote the type I interferon pathway.
9. 如权利要求 8所述的方法, 其特征在于,  9. The method of claim 8 wherein:
步骤(a)包括: 在测试组中, 将候选物质加入到含有小核糖核酸的体系中; 和 /或 步骤 (b)包括: 检测测试组的体系中小核糖核酸的表达, 并与对照组比较, 其中所 述的对照组是不添加所述候选物质的含有小核糖核酸的体系;  Step (a) comprises: adding a candidate substance to the system containing the small RNA in the test group; and/or step (b) comprises: detecting the expression of the small ribonucleic acid in the system of the test group, and comparing with the control group, The control group described therein is a small ribonucleic acid-containing system that does not add the candidate substance;
如果测试小核糖核酸的表达在统计学上高于对照组,就表明该候选物是抑制 I型干 扰素通路的物质; 如果测试小核糖核酸的表达在统计学上低于对照组, 就表明该候选物 是促进 I型干扰素通路的物质。  If the expression of the test ribonucleic acid is statistically higher than the control group, it indicates that the candidate is a substance that inhibits the type I interferon pathway; if the expression of the test ribonucleic acid is statistically lower than the control group, it indicates that The candidate is a substance that promotes the type I interferon pathway.
10. 如权利要求 8所述的方法, 其特征在于, 所述的体系是细胞体系。  10. The method of claim 8 wherein said system is a cellular system.
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