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WO2009030153A1 - Rôles de micro-arn dans le diagnostic et le traitement du lupus érythémateux systémique - Google Patents

Rôles de micro-arn dans le diagnostic et le traitement du lupus érythémateux systémique Download PDF

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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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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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides 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

La présente invention concerne le domaine biotechnique et porte sur les utilisations d'une série de micro-ARN pour la préparation de réactifs ou de trousses permettant de détecter le lupus érythémateux systémique (LES). L'invention concerne également des réactifs ou des trousses permettant de détecter les niveaux d'expression de ces micro-ARN et, par conséquent, de diagnostiquer le LES. Pour la première fois, l'invention concerne et démontre la corrélation étroite entre ces micro-ARN et le LES. Ces micro-ARN sont de nouvelles cibles médicamenteuses dans le cadre de la prophylaxie et du traitement du LES, et les régulations des niveaux d'expression desdits micro-ARN représentent de nouvelles mesures pour le traitement de la maladie.
PCT/CN2008/072159 2007-08-27 2008-08-27 Rôles de micro-arn dans le diagnostic et le traitement du lupus érythémateux systémique WO2009030153A1 (fr)

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CN113196059A (zh) * 2018-10-18 2021-07-30 俄克拉荷马医学研究基金会 表征疾病活动性的系统性红斑狼疮(sle)疾病活动性免疫指数的生物标志物

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CN113196059A (zh) * 2018-10-18 2021-07-30 俄克拉荷马医学研究基金会 表征疾病活动性的系统性红斑狼疮(sle)疾病活动性免疫指数的生物标志物

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