[go: up one dir, main page]

WO2018170623A1 - Méthode de réduction des expressions de miarn-152 et de miarn-424 - Google Patents

Méthode de réduction des expressions de miarn-152 et de miarn-424 Download PDF

Info

Publication number
WO2018170623A1
WO2018170623A1 PCT/CN2017/077173 CN2017077173W WO2018170623A1 WO 2018170623 A1 WO2018170623 A1 WO 2018170623A1 CN 2017077173 W CN2017077173 W CN 2017077173W WO 2018170623 A1 WO2018170623 A1 WO 2018170623A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
pri
expression
microrna
vector
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2017/077173
Other languages
English (en)
Chinese (zh)
Inventor
毛吉炎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Biocan Technologies Co Ltd
Original Assignee
Shenzhen Biocan Technologies Co Ltd
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 Shenzhen Biocan Technologies Co Ltd filed Critical Shenzhen Biocan Technologies Co Ltd
Priority to PCT/CN2017/077173 priority Critical patent/WO2018170623A1/fr
Publication of WO2018170623A1 publication Critical patent/WO2018170623A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Definitions

  • the present invention relates to the field of life sciences, and more particularly to a method of reducing the expression of microRNA-152 and microRNA-424.
  • MicroRNA-152 (miR-152) is a multifunctional miRNA. It was found that miR-152 is related to methylation, such as methyltransferase DNMT1 content and enzyme activity, miR-152 can be endometrium. Cancer DNA methylation becomes a silent gene, and it is associated with the development of various cancers. It is a tumor suppressing microRNA, and many diseases such as preeclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer, etc. Related; microRNA-424 (miR-424) is a miRNA discovered in recent years, which affects the biological effects and development of tumor cells by acting on target genes in various tumors and participating in the signal pathway of target gene regulation.
  • miR-424 is a multifunctional miRNA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF- ⁇ ; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing.
  • miRNA function research is generally achieved by miRNA overexpression and silencing.
  • miRNA silencing is the presentation of artificially synthesized oligonucleotide small molecules into cells, forming heteroduplexes with endogenous miRNAs, and reducing miRNAs to target genes. Inhibition to achieve regulation of gene function. Silencing of miRNAs, especially long-term stable silencing, is currently more difficult to achieve.
  • Commonly used methods for silencing miRNA mainly include anti-miR, antagomiR, miRNA sponge, etc. These methods have short acting time, poor stability, complex construction, high instrumental requirements and complicated technical operation, which is difficult to achieve convenient, simple and effective. The effect of miR-152 and miR-424 expression was reduced.
  • the object of the present invention is to provide a method for reducing the expression of miR-152 and miR-424, which is simple in design, easy to operate, and can easily and effectively down-regulate the expression of miR-152 and miR-424 to overcome the existing The lack of technology.
  • the present invention is a method for down-regulating the expression of miR-152 and miR-424, and constructing a eukaryotic expression vector pRI- targeting miR-152 and miR-424 on the eukaryotic vector pRI-GFP/Neo containing the H1 promoter. 152-424, the expression of miR-152 and miR-424 in eukaryotic cells was down-regulated; after transfecting the vector into eukaryotic cells in vitro, the expression levels of miR-152 and miR-424 were detected by a real-time PCR instrument.
  • pRI-152-424 specifically, the sequence design of Tud-152-424 was performed by software; the backbone vectors pRI-GFP/Neo and Tud-152-424 sequences were double-digested with Xho I and Bgl II. The recovered fragments were purified and then ligated overnight at 4 °C; after transformation, 6 colonies were picked and inoculated into LB medium containing 10 ⁇ g/ml kanamycin. After 12 hours, the bacterial liquid was extracted and sent to Shanghai Yingjun. Sequencing. The correctly constructed vector was the successfully constructed recombinant plasmid pRI-152-424.
  • the present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-152-424 on the basis of a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-424; After transfecting eukaryotic cells in vitro, the expression of miR-152 and miR-424 was detected by real-time PCR.
  • the invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.
  • Figure 1 is a schematic diagram showing the structure of the pRI-GFP/Neo vector
  • Figure 2 is a quantitative PCR detection of miRNA expression levels, wherein a. miR-152 expression, b. miR-424 expression.
  • Tud RNA targeting hsa-miR-152 and hsa-miR-424 was designed according to the design principle of Tough Decoy RNA (Tud RNA).
  • Tud RNA targeting hsa-miR-152 and hsa-miR-424 was designed according to the design principle of Tough Decoy RNA (Tud RNA).
  • Xho I and Bgl II restriction sites were added to the 5' and 3' ends of the Tud RNA to obtain the desired Tud-152-424 sequence.
  • the nucleotide sequence of Tud-152-424 is shown in SEQ ID No. 1.
  • the recombinant plasmid pRI-152-424 was extracted using an endotoxin-free plasmid extraction kit (Tiangen Biochemical).
  • the present invention utilizes molecular biology techniques to construct a eukaryotic expression vector containing Tud-152-424 on the basis of a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-424; After transfecting eukaryotic cells in vitro, the expression of miR-152 and miR-424 was detected by real-time PCR.
  • the invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une méthode de réduction des expressions de miARN-152 et de miARN-424, comprenant la construction d'un vecteur d'expression eucaryote pRI-152-424 ciblant miR-152 et miR-424 sur un vecteur eucaryote pRI-GFP/Neo qui contient un promoteur H1, ce qui permet de réguler négativement les expressions de miR-152 et de miR-424.
PCT/CN2017/077173 2017-03-18 2017-03-18 Méthode de réduction des expressions de miarn-152 et de miarn-424 Ceased WO2018170623A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077173 WO2018170623A1 (fr) 2017-03-18 2017-03-18 Méthode de réduction des expressions de miarn-152 et de miarn-424

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077173 WO2018170623A1 (fr) 2017-03-18 2017-03-18 Méthode de réduction des expressions de miarn-152 et de miarn-424

Publications (1)

Publication Number Publication Date
WO2018170623A1 true WO2018170623A1 (fr) 2018-09-27

Family

ID=63584003

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/077173 Ceased WO2018170623A1 (fr) 2017-03-18 2017-03-18 Méthode de réduction des expressions de miarn-152 et de miarn-424

Country Status (1)

Country Link
WO (1) WO2018170623A1 (fr)

Citations (5)

* 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
CN102218144A (zh) * 2010-04-13 2011-10-19 江苏命码生物科技有限公司 一种调节生物体内微小核糖核酸含量的方法及其用途
CN103920164A (zh) * 2014-05-05 2014-07-16 山东大学 MiR-424-5p在抑制转移性肝癌中的应用
CN105779638A (zh) * 2016-05-21 2016-07-20 崔学俊 用于直肠癌诊断的miRNA生物标志物及检测试剂盒
CN105950730A (zh) * 2016-05-21 2016-09-21 崔学俊 用于甲状腺癌诊断的miRNA生物标志物及检测试剂盒

Patent Citations (5)

* 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
CN102218144A (zh) * 2010-04-13 2011-10-19 江苏命码生物科技有限公司 一种调节生物体内微小核糖核酸含量的方法及其用途
CN103920164A (zh) * 2014-05-05 2014-07-16 山东大学 MiR-424-5p在抑制转移性肝癌中的应用
CN105779638A (zh) * 2016-05-21 2016-07-20 崔学俊 用于直肠癌诊断的miRNA生物标志物及检测试剂盒
CN105950730A (zh) * 2016-05-21 2016-09-21 崔学俊 用于甲状腺癌诊断的miRNA生物标志物及检测试剂盒

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XING-HUA LIAO: "Human cytomegalovirus immediate early protein 2 enhances myocardin-mediated survival of rat aortic smooth muscle cells", VIRUS RESEARCH, vol. 192, 23 August 2014 (2014-08-23), pages 85 - 91, XP029071011, ISSN: 1872-7492 *

Similar Documents

Publication Publication Date Title
Fathi Dizaji Strategies to target long non-coding RNAs in cancer treatment: progress and challenges
CN110959042B (zh) 一种新型小激活rna
Liang et al. RETRACTED ARTICLE: Systematic analyses reveal long non-coding RNA (PTAF)-mediated promotion of EMT and invasion-metastasis in serous ovarian cancer
Connelly et al. Spatiotemporal control of microRNA function using light-activated antagomirs
Luo et al. microRNA133a targets Foxl2 and promotes differentiation of C2C12 into myogenic progenitor cells
Elbarbary et al. Human cytosolic tRNase ZL can downregulate gene expression through miRNA
Prakash et al. RNA interference by 2′, 5′-linked nucleic acid duplexes in mammalian cells
JP2021520220A (ja) ｐ２１遺伝子の発現を活性化させる方法
WO2018165929A1 (fr) Vecteur d'expression inhibant le micro-arn double brin, son procédé de mise au point et son application
WO2018170623A1 (fr) Méthode de réduction des expressions de miarn-152 et de miarn-424
Liu et al. Identification of microRNA–RNA interactions using tethered RNAs and streptavidin aptamers
CN103103189B (zh) 过表达单一MicroRNA成熟体序列的新方法
CN102433352A (zh) 一种基于microRNA结构的肝细胞选择性多靶点干扰质粒载体的构建方法及其功能验证
WO2018170622A1 (fr) Procédé de régulation négative synchrone des expressions de miarn-152 et de miarn-185
EP2955228A1 (fr) Petit arn interférant pour l'inhibition de l'expression intracellulaire de la protéine ribosomale s3
WO2018170624A1 (fr) Méthode de réduction des expressions de mir-185 et de mir-424
CN110564743A (zh) 一种六盘山黄牛circR-UQCC1基因及其过表达载体、构建方法和应用
WO2018170621A1 (fr) Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application
CN101892236B (zh) 靶向znf268基因的rna干扰表达载体构建及应用
CN106591311B (zh) 核酸及其用途
WO2018170620A1 (fr) Vecteur d'expression de deux micro-arn mis sous silence
WO2018170619A1 (fr) Vecteur d'expression inhibant le micro-arn double brin et son application
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
RU2022116800A (ru) Генетическая конструкция, содержащая последовательности химерных направляющих РНК для делеции гена SMN1 человека в культурах клеток человека

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: 17901672

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: 17901672

Country of ref document: EP

Kind code of ref document: A1