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 PDFInfo
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- 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
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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.
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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.
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| 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 |
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| Application Number | Priority Date | Filing Date | Title |
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| PCT/CN2017/077173 WO2018170623A1 (fr) | 2017-03-18 | 2017-03-18 | Méthode de réduction des expressions de miarn-152 et de miarn-424 |
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| WO2018170623A1 true WO2018170623A1 (fr) | 2018-09-27 |
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Citations (5)
| 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 |
-
2017
- 2017-03-18 WO PCT/CN2017/077173 patent/WO2018170623A1/fr active Application Filing
Patent Citations (5)
| 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)
| 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 * |
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