WO2018170750A1 - Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci - Google Patents
Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci Download PDFInfo
- Publication number
- WO2018170750A1 WO2018170750A1 PCT/CN2017/077591 CN2017077591W WO2018170750A1 WO 2018170750 A1 WO2018170750 A1 WO 2018170750A1 CN 2017077591 W CN2017077591 W CN 2017077591W WO 2018170750 A1 WO2018170750 A1 WO 2018170750A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mirna
- mir
- tud
- rna
- tud rna
- Prior art date
Links
- 230000014509 gene expression Effects 0.000 title claims abstract description 23
- 108091037426 miR-152 stem-loop Proteins 0.000 claims abstract description 18
- 108091027034 miR-148a stem-loop Proteins 0.000 claims abstract description 17
- 108091088477 miR-29a stem-loop Proteins 0.000 claims abstract description 17
- 108091029716 miR-29a-1 stem-loop Proteins 0.000 claims abstract description 17
- 108091092089 miR-29a-2 stem-loop Proteins 0.000 claims abstract description 17
- 108091066559 miR-29a-3 stem-loop Proteins 0.000 claims abstract description 17
- 239000013598 vector Substances 0.000 claims abstract description 17
- 239000012634 fragment Substances 0.000 claims abstract description 12
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- 239000013600 plasmid vector Substances 0.000 claims abstract description 6
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 claims abstract 3
- 230000009368 gene silencing by RNA Effects 0.000 claims abstract 3
- 238000000034 method Methods 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000001914 filtration Methods 0.000 abstract 1
- 108091008146 restriction endonucleases Proteins 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 239000002679 microRNA Substances 0.000 description 27
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 22
- 108091070501 miRNA Proteins 0.000 description 17
- 108700011259 MicroRNAs Proteins 0.000 description 12
- 206010028980 Neoplasm Diseases 0.000 description 9
- 238000003556 assay Methods 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 206010005003 Bladder cancer Diseases 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 201000005112 urinary bladder cancer Diseases 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108010009540 DNA (Cytosine-5-)-Methyltransferase 1 Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 108091068997 Homo sapiens miR-152 stem-loop Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091030146 MiRBase Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000007989 benign glioma Diseases 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 208000029824 high grade glioma Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 208000030173 low grade glioma Diseases 0.000 description 1
- 201000011614 malignant glioma Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008558 metabolic pathway by substance Effects 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 108091028606 miR-1 stem-loop Proteins 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 201000011461 pre-eclampsia Diseases 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000001743 silencing effect Effects 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 208000029387 trophoblastic neoplasm Diseases 0.000 description 1
Definitions
- the present invention relates to a Tud RNA for inhibiting expression of small RNA-29a, 148a and 152 and its use, and belongs to the field of genetic engineering technology.
- MicroRNAs are a class of endogenous, non-coding RNAs found in eukaryotes, typically between 22 and 25 nt in size. miRNAs are widely distributed in plants, animals, and multicellular organisms, and can Play an important regulatory role, and in the study of human miRNAs, it is found that the expression of miRNA in normal tissues and tumor tissues is significantly different, some miRNAs are lowly expressed in tumor tissues, and some are highly expressed in tumor tissues. This suggests that miRNAs play a crucial role in tumorigenesis.
- miR-29a is a small RNA closely related to cell proliferation. It is involved in many diseases and can act as a tumor suppressor gene in a variety of tumors. It has the ability to grow and invade cells such as human gastric cancer and bladder cancer. Correlation, its expression level is an important reference for evaluating the benign and malignant glioma, and it is also associated with diseases such as atherosclerosis liver fibrosis, and has important potential application value for the treatment of various tumors; miR-148a is in recent years A more studied micr 0 RNA.
- miR-148a is closely related to exogenous substance metabolism, apoptosis, occurrence, development and epigenetics of various cancers; miR-152 is a multifunctional miRNA, and the study found that miR-152 Related to methylation, such as methyltransferase DNMT1 content and enzyme activity, miR-152 can be methylated by endometrial cancer DNA into a silent gene, and it is associated with the development of various cancers, it is A tumor suppressing microRNA associated with many diseases such as pre-eclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, and ovarian cancer.
- miR-29a, miR-148a and miR-152 peers can work synergistically with other drugs to provide new epigenetic ideas for the treatment of cancer.
- MiRNA functional studies often require miRNA silencing techniques, including anti-miR, antago miR, miRNA Sponge et al., these technologies are all transient transfection techniques, unable to maintain long-term stable silencing of the target miRNA, and the silencing effect is far from optimal.
- Tough Decoy RNA is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in a long-term, stable, and efficient manner.
- the present invention provides a Tud RNA which inhibits the expression of small RNA-29a, 148a and 152, and has a nucleotide sequence ⁇ IJ as shown in SEQ ID NO: 1, mainly inhibiting human miR-29a, miR-148a. And application in miR-152 expression preparations.
- the present invention also provides a method for preparing a shRNA interference carrier, and the steps are as follows:
- the interference fragment obtained in the step (1) was ligated to a plasmid vector linearized by BamHI and Hindlll to obtain a Tud RNA vector.
- the interference fragment obtained in the step (1) was ligated to the p-Ge nesil 1.0 plasmid vector linearized by BamHI and Hindlll to obtain the Tud RNA vector p-Genesil-Tud-29a-148a-152.
- a method of preparing an RNA vector for use in inhibiting expression of human miR-29a, miR-148a and miR-152 expression preparations is provided.
- Tud RNA sequences were designed by comparative analysis of human miR-29a, miR-148a and miR-152 sequences.
- the homologous interference miR-29a, miR-148a and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA is relatively common with the currently used single-stranded miRNA sponge.
- the binding efficiency is higher, and the same target for three targets can better achieve the interference of three miRNAs and improve the efficiency of miRNA function research.
- FIG. 1 miRNA expression levels of each group of cells, wherein, a. miR-29a expression, b.
- pGenesill.O vector was purchased from Biovector Plasmid Vector Culture Cell Genetic Collection Center; Reverse Transcription Kit
- SYBR fluorescence real-time quantitative PCR kits were purchased from Takara Reagent Co., Ltd.; endotoxin plasmid extraction kits were purchased from Tiangen Biochemical.
- TuD RNA oligonucleosides targeting miR-29a, miR-148a and miR-152 were designed based on the sequence information of miR-29a, miR-148a and miR-152 provided in the TuD RNA design sequence and miRBase.
- the Tud-29a-148a-152 sequence and p-Genesill.0 were separately treated with BamHI and Hindlll enzymes, respectively, and electrophoresed and recovered, and then ligated with T4 DNA ligase at 4 ° C overnight, and then transformed into competent E. coli ToplO, Single colonies were cultured and sent to Shanghai Biotech for sequencing.
- the correctly sequenced pG eneS il-T U d-29a-14 8a-152 vector was constructed.
- the p-Genesil-Tud-29a-148a-152 vector was extracted using an endotoxin plasmid extraction kit.
- 16HBE cells were seeded in 6-well plates at 1000000 cells per well, and the cell density was approximately 60% after 18 h.
- the p-Genesil-Tud-29a-148a-152 vector was transduced with 16HBE cells using jetPrime transfection reagent. After 4 h of reaction, the medium was changed to fresh DMEM complete medium and culture was continued for 24 h.
- 16HBE cells and 16HBE cells transduced with p-Genesil-Tud-29a-148a-152 vector were used to extract miRNAs from these cells using the miRcute miRNA extraction and isolation kit, followed by S-Poly(T) hsa-miR- 29a qPCR-assay primer set, S-Poly(T) hsa-miR-148a qPCR-assay primer set and S-Poly(T) hsa-miR-152 qPCR-assay primer set kit (both purchased from Shenzhen Anran Bio) The miRNA is reverse transcribed and tailed to obtain the corresponding cDNA. Take 2 kinds of cells of cDNA 2
- the homologous interference miR-29a, miR-148a and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA is more efficient than the currently used single-stranded miRNA sponge. High, and the same target for three targets, can better achieve the interference of three miRNAs, improve the efficiency of miRNA function research.
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
L'invention concerne un ARN leurre résistant (ARN Tud) pour supprimer les expressions de miARN-29a, de miARN-148a et de miARN-152. Une séquence nucléotidique de l'ARN Tud est représentée par la SEQ ID NO. 1. La présente invention concerne également un vecteur d'interférence d'ARN Tud pour supprimer les expressions de miARN-29a, de miARN-148a et de miARN-152. Une méthode de préparation du vecteur d'interférence d'ARN Tud comprend : la synthèse, selon les séquences des miR-29a, miR-148a, et miR-152a humains comparées et analysées, d'un fragment ayant des sites de coupure des enzymes de restriction BamHI et HindIII, connectant le fragment sur un vecteur plasmidique soumis à une linéarisation par BamHI et HindIII, et une filtration pour obtenir un vecteur d'interférence efficace.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CN2017/077591 WO2018170750A1 (fr) | 2017-03-21 | 2017-03-21 | Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CN2017/077591 WO2018170750A1 (fr) | 2017-03-21 | 2017-03-21 | Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018170750A1 true WO2018170750A1 (fr) | 2018-09-27 |
Family
ID=63586240
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2017/077591 WO2018170750A1 (fr) | 2017-03-21 | 2017-03-21 | Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2018170750A1 (fr) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010138263A2 (fr) * | 2009-05-28 | 2010-12-02 | University Of Massachusetts | Nouveaux virus adéno-associés (aav) et leurs utilisations |
WO2015164786A1 (fr) * | 2014-04-25 | 2015-10-29 | University Of Massachusetts | Vecteurs de virus adéno-associés recombinants utiles pour réduire une immunité contre des produits transgéniques |
-
2017
- 2017-03-21 WO PCT/CN2017/077591 patent/WO2018170750A1/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010138263A2 (fr) * | 2009-05-28 | 2010-12-02 | University Of Massachusetts | Nouveaux virus adéno-associés (aav) et leurs utilisations |
WO2015164786A1 (fr) * | 2014-04-25 | 2015-10-29 | University Of Massachusetts | Vecteurs de virus adéno-associés recombinants utiles pour réduire une immunité contre des produits transgéniques |
Non-Patent Citations (2)
Title |
---|
LINWENSI ZHU ET AL.: "The Structure and Clinical Roles of MicroRNA in Colorectal Cancer", GASTROENTEROLOGY RESEARCH AND PRACTICE, vol. 2016, 31 December 2016 (2016-12-31), pages 1 - 6, XP055540703, ISSN: 1687-630X * |
XIE, XING ET AL.: "Construction of a Human Bronchial Epithelial Hsa-miR-148a-3p Knockdown Cell Line", CARCINOGENESIS, TERATOGENESIS & MUTAGENESIS, vol. 26, no. 3, 14 May 2014 (2014-05-14), pages 204 - 208, 212, XP055447857, ISSN: 1004-616X, DOI: doi:10.3969/j.issn.1004-616x.2014.03.010 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2021263124A2 (fr) | Éléments génétiques commandant la traduction d'arn circulaire et procédés d'utilisation | |
JP7432521B2 (ja) | 新規小分子活性化rna | |
CN106032532A (zh) | 一种小激活rna及其制备方法和应用 | |
CN107058360A (zh) | 一种基于快速克隆技术的环状rna表达载体构建方法及其应用 | |
Moreno-Mateos et al. | Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance | |
CN108342386B (zh) | 一种多聚寡核酸分子及其在多靶标干扰中的应用 | |
Sun et al. | Construction of lentivirus-based inhibitor of hsa-microRNA-338-3p with specific secondary structure | |
WO2018170750A1 (fr) | Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci | |
WO2018165929A1 (fr) | Vecteur d'expression inhibant le micro-arn double brin, son procédé de mise au point et son application | |
WO2017214952A1 (fr) | Construction et application d'un vecteur lentiviral pour l'inhibition spécifique de l'expression de l'arnmi-185 humain | |
WO2018170751A1 (fr) | Arn tud destiné à antagoniser l'expression d'un micro-arn, et son application | |
WO2017214948A1 (fr) | Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-148a humain | |
WO2018170753A1 (fr) | Arn tud pour l'inactivation de miarn-29a, de miarn-152, et de miarn-185, et application de celui-ci | |
WO2018170752A1 (fr) | Arn tud pour le knock-down de miarn-29a, miarn-148a, et miarn-424, et application associée | |
WO2017219166A1 (fr) | Vecteur lentiviral pour l'inhibition simultanée de l'expression de deux miarn, et application associée | |
WO2017214951A1 (fr) | Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-152 humain | |
WO2017214949A1 (fr) | Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression d'arnmi-29a | |
WO2018170619A1 (fr) | Vecteur d'expression inhibant le micro-arn double brin et son application | |
WO2017219168A1 (fr) | Vecteur lentiviral destiné à inactiver l'expression de miarn-29a et de mir-152 et application associée | |
CN1927877A (zh) | 抑制人Ezrin基因表达的siRNA及其表达载体 | |
WO2017214953A1 (fr) | Construction et application d'un vecteur lentiviral pour l'inhibition spécifique de l'expression de l'arnmi-424 humain | |
WO2018170623A1 (fr) | Méthode de réduction des expressions de miarn-152 et de miarn-424 | |
Florescu et al. | Study of MIR-200c and MIR-9 methylation on patients with breast cancer | |
WO2017214950A1 (fr) | Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-140 humain | |
WO2018170620A1 (fr) | Vecteur d'expression de deux micro-arn mis sous silence |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17901992 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 17901992 Country of ref document: EP Kind code of ref document: A1 |