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WO2018170751A1 - Arn tud destiné à antagoniser l'expression d'un micro-arn, et son application - Google Patents

Arn tud destiné à antagoniser l'expression d'un micro-arn, et son application Download PDF

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Publication number
WO2018170751A1
WO2018170751A1 PCT/CN2017/077592 CN2017077592W WO2018170751A1 WO 2018170751 A1 WO2018170751 A1 WO 2018170751A1 CN 2017077592 W CN2017077592 W CN 2017077592W WO 2018170751 A1 WO2018170751 A1 WO 2018170751A1
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WIPO (PCT)
Prior art keywords
mir
mirna
tud
tud rna
expression
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PCT/CN2017/077592
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English (en)
Chinese (zh)
Inventor
毛吉炎
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Shenzhen Biocan Technologies Co ltd
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Shenzhen Biocan Technologies Co ltd
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Priority to PCT/CN2017/077592 priority Critical patent/WO2018170751A1/fr
Publication of WO2018170751A1 publication Critical patent/WO2018170751A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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  • the present invention relates to a Tud RNA which antagonizes the expression inhibition of miR-29a, miR-148a and miR-185 and an application thereof, 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-185 is a 22 nt miRNA, located on human chromosome 22ql L.21, plays an important role as a tumor suppressor gene in the development and invasion of tumors such as colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, etc.
  • DNMT1 methylation-related tumor suppressor miRNA that can be directly Targeting the expression of DNMT1 affects the methylation level of the whole genome, which in turn regulates the methylation status of certain genes and affects gene expression.
  • MiRNA functional studies often require miRNA silencing techniques, including anti-miR, antago miR, miRNA
  • 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 antagonizes miRNA expression, the nucleotide sequence of which is shown in SEQ ID NO: 1, and is mainly used for inhibiting expression products of human miR-29a, miR-148a and miR-185.
  • 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-185.
  • a method of preparing an RNA vector for use in inhibiting expression of human miR-29a, miR-148a and miR-185 expression products is provided.
  • Tud RNA sequences were designed by comparative analysis of human miR-29a, miR-148a and miR-185 sequences.
  • the homologous interference miR-29a, miR-148a and miR-185 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.
  • the TuD RNA design sequence and the sequence of miR-29a, miR-148a and miR-185 provided in miRBase In the column information, the TuD RNA oligonucleotide sequence ⁇ ijTud-29a-148a-185 targeting miR-29a, miR-148a and miR-185 was designed, and its sequence ⁇ ij is shown in SEQ ID NO 1.
  • the raw workers are synthesized by means of gene synthesis.
  • the Tud-29a-148a-185 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-185 vector was constructed.
  • the p-Genesil-Tud-29a-148a-185 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 about 60% after 18 h.
  • the p-Genesil-Tud-29a-148a-185 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-185 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-185 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 expression levels of miR-29a, miR-148a and miR-185 were detected by real-time PCR, and the experiment was repeated 3 times with 3 parallel samples per well, with snord 44 as an internal reference.
  • the results are shown in Figure 1. It can be seen that the expression level of miR-29a in TuD-29a-148a-185 cells is 52 ⁇ 3 ⁇ 4 lower than that in 16HBE cells, and the expression level of miR-1 48a is 57% lower than that in 16HBE cells, miR- The expression level of 185 was 56 ⁇ 3 ⁇ 4 lower than that of 16HBE cells. The difference was statistically significant (/? ⁇ 0.01), indicating that the TuD-29a-148a-185 cell line was successfully constructed.
  • the homology of the present invention interferes with miR-29a, miR-148a and miR-185 TuD RNA sequences with stem loops
  • the structure is not easily degraded.
  • the double-stranded Tud RNA has higher binding efficiency than the commonly used single-stranded miRNA sponge, and the same target can better interfere with the three miRNAs and enhance the miRNA function. The efficiency of the research.

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Abstract

L'invention concerne un ARN Tud destiné à antagoniser une expression d'un micro-ARN. Une séquence nucléotidique de l'ARN Tud est représentée par SEQ ID NO. 1. L'invention concerne également un vecteur d'interférence ARN Tud destiné à supprimer les expressions du micro-ARN-29a, du micro-ARN-148a et du micro-ARN-185. Le vecteur d'interférence ARN Tud est construit par le procédé suivant : la synthèse, selon les séquences du micro-ARN-29a, du micro-ARN-148a et du micro-ARN-185 humains comparées et analysées, un fragment comportant des sites de coupe d'enzyme de restriction BamHI et HindIII, reliant le fragment à un vecteur plasmidique soumis à une linéarisation BamHI et HindIII, et le filtrage en vue d'obtenir le vecteur d'interférence.
PCT/CN2017/077592 2017-03-21 2017-03-21 Arn tud destiné à antagoniser l'expression d'un micro-arn, et son application Ceased WO2018170751A1 (fr)

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PCT/CN2017/077592 WO2018170751A1 (fr) 2017-03-21 2017-03-21 Arn tud destiné à antagoniser l'expression d'un micro-arn, et son application

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PCT/CN2017/077592 WO2018170751A1 (fr) 2017-03-21 2017-03-21 Arn tud destiné à antagoniser l'expression d'un micro-arn, et son application

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Citations (2)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
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 (3)

* Cited by examiner, † Cited by third party
Title
LI, QUANQIU ET AL.: "Research Progress on the Relationship between miR-185 and Tumor", JOURNAL OF MODERN MEDICINE & HEALTH, vol. 31, no. 3, 15 February 2015 (2015-02-15), pages 380 - 382, ISSN: 1009-5519 *
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 - 7, XP055538564, 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 *

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