+

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

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
mir
mirna
tud
tud rna
expression
Prior art date
Application number
PCT/CN2017/077592
Other languages
English (en)
Chinese (zh)
Inventor
毛吉炎
Original Assignee
深圳市博奥康生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市博奥康生物科技有限公司 filed Critical 深圳市博奥康生物科技有限公司
Priority to PCT/CN2017/077592 priority Critical patent/WO2018170751A1/fr
Publication of WO2018170751A1 publication Critical patent/WO2018170751A1/fr

Links

Definitions

  • 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.

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

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 WO2018170751A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
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

Publications (1)

Publication Number Publication Date
WO2018170751A1 true WO2018170751A1 (fr) 2018-09-27

Family

ID=63586251

Family Applications (1)

Application Number Title Priority Date Filing Date
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

Country Status (1)

Country Link
WO (1) WO2018170751A1 (fr)

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 *

Similar Documents

Publication Publication Date Title
EP2925866B1 (fr) Arn circulaire destiné à l'inhibition de micro-arn
EP3778892A1 (fr) Nouveau petit arn activateur
CN108796086B (zh) 一种环状RNAcircBCBM1及其非诊断性荧光定量检测方法
TW201842923A (zh) 包含ｍｉｒ－３０２前驅體的組合物在製造用於肺癌治療之藥物上的用途
MX2010012542A (es) Metodos para evaluar el cancer colorrectal y composiciones para usar en el.
US20200165607A1 (en) Composition and method of using mir-302 precursors as anti-cancer drugs for treating human lung cancer
Sun et al. Construction of lentivirus-based inhibitor of hsa-microRNA-338-3p with specific secondary structure
WO2018170751A1 (fr) Arn tud destiné à antagoniser l'expression d'un micro-arn, et son application
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
CN111411155B (zh) lncRNA IGFL2-AS1作为结肠癌诊断标志物的应用
WO2018170753A1 (fr) Arn tud pour l'inactivation de miarn-29a, de miarn-152, et de miarn-185, et application de celui-ci
WO2018170750A1 (fr) Arn tud pour supprimer les expressions de miarn-29a, de miarn-148a, et de miarn-152, et application de celui-ci
WO2017214948A1 (fr) Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-148a humain
WO2018170752A1 (fr) Arn tud pour le knock-down de miarn-29a, miarn-148a, et miarn-424, et application associée
WO2017214949A1 (fr) Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression d'arnmi-29a
WO2017219166A1 (fr) Vecteur lentiviral pour l'inhibition simultanée de l'expression de deux miarn, et application associée
CN101831461A (zh) 一种人小RNA-148a表达载体及应用
WO2017214953A1 (fr) Construction et application d'un vecteur lentiviral pour l'inhibition spécifique de l'expression de l'arnmi-424 humain
WO2017214951A1 (fr) Construction et application d'un vecteur lentiviral pour l'inhibition de l'expression de l'arnmi-152 humain
WO2018170620A1 (fr) Vecteur d'expression de deux micro-arn mis sous silence
WO2017219168A1 (fr) Vecteur lentiviral destiné à inactiver l'expression de miarn-29a et de mir-152 et application associée
WO2017219169A1 (fr) Vecteur lentiviral pour l'inhibition de l'expression de miarn-29a et de mir-185, et son application
WO2023225647A2 (fr) Nouvelles cibles contre le cancer de l'ovaire
WO2018170621A1 (fr) Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application

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: 17901686

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: 17901686

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载