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WO2018170624A1 - Méthode de réduction des expressions de mir-185 et de mir-424 - Google Patents

Méthode de réduction des expressions de mir-185 et de mir-424 Download PDF

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Publication number
WO2018170624A1
WO2018170624A1 PCT/CN2017/077174 CN2017077174W WO2018170624A1 WO 2018170624 A1 WO2018170624 A1 WO 2018170624A1 CN 2017077174 W CN2017077174 W CN 2017077174W WO 2018170624 A1 WO2018170624 A1 WO 2018170624A1
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WO
WIPO (PCT)
Prior art keywords
mir
expression
pri
vector
microrna
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/CN2017/077174
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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/CN2017/077174 priority Critical patent/WO2018170624A1/fr
Publication of WO2018170624A1 publication Critical patent/WO2018170624A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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  • the present invention relates to the field of life sciences, and more particularly to a method of reducing the expression of microRNA-185 and microRNA-424.
  • MicroRNA-185 (miR-185) is a 22 nt miRNA located in human chromosome 22ql l.
  • DNMT1 methylation-related tumor suppressor miRNA that can be directly targeted.
  • the expression of DNMT1 affects the methylation level of the whole genome, which in turn regulates the methylation status of certain genes, affecting gene expression; microRNA
  • miR-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, thereby affecting the biological effects and development of tumor cells.
  • 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 silencing is the presentation of artificially synthesized oligonucleotide small molecules into cells, and endogenous miRNAs.
  • a heteroduplex allows the miRNA to reduce the inhibition of the target gene to achieve regulation of gene function.
  • Silencing of miRNAs is currently difficult to achieve.
  • Commonly used methods for silencing miRNA mainly include anti-miR, antagomiR, miRNA sponge, etc. These methods have short effects such as short inter-turn, poor stability, complex construction, high requirements on instrument conditions, and complicated technical operations, which are difficult to achieve, simple, and effective. The effect of miR-185 and miR-424 expression was reduced.
  • the object of the present invention is to provide a method for reducing the expression of miR-185 and miR-424, which is simple in design, easy to operate, and can easily and effectively down-regulate the expression of miR-185 and miR-424, Overcoming the deficiencies of the prior art.
  • a method for the present invention a method for down-regulating expression of miR-185 and miR-424, and constructing eukaryotic expression targeting miR-185 and miR-424 on a eukaryotic vector pRI-GFP/Neo containing a HI promoter Vector pRI-185-424, which regulates the expression of miR-185 and miR-424 in eukaryotic cells; after transfecting the vector into eukaryotic cells in vitro, the expression of miR-185 and miR-424 was detected by real-time PCR Level.
  • the sequences of pRI-GFP/Neo and Tud-185-424 were double-digested with Xho I and Bgl II, and the recovered fragments were purified and ligated overnight at 4 ° C. After ligation, single colonies were picked and inoculated. In LB medium containing l (Vg/ml kanamycin, 12 hours later, the bacterial solution was extracted and sent to Shanghai Yingjun for sequencing. The correctly sequenced vector was the successfully constructed recombinant plasmid pRI-185-424.
  • the present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-185-424 based on a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-185 and miR-424; After the vector was transfected into eukaryotic cells in vitro, the expression of miR-185 and miR-424 was detected by a real-time PCR instrument.
  • the invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.
  • FIG. 1 is a schematic diagram showing the structure of the pRI-GFP/Neo vector
  • FIG. 2 is a quantitative PCR method for detecting the expression level of miRNA, wherein a. miR-185 expression, b. miR-424 expression.
  • Tud RNA targeting hsa-miR-185 and hsa-miR-424 was designed according to the design principle of Tough Decoy RNA (Tud RNA).
  • Tud RNA Tough Decoy RNA
  • Tud-185-424 The enzyme was digested to obtain the desired Tud-185-424 sequence.
  • the nucleotide sequence of Tud-185-424 is shown as SE Q ID No 1.
  • 16HBE cells were seeded in a 6-well plate at a density of 30,000/ml, and 10
  • Plasmid pRI-185-424 was transfected with Lipofectamine 2000 and continuously cultured at 5% CO 2 at 37 ° C. After 48 sputum, total RNA was extracted from each group, using Realtime.
  • miR-185 and miR-424 were detected by PCR, and the results showed that the expression levels of m iR-185 and miR-424 were significantly down-regulated in the pRI-185-424 transfection group (Fig. 2), suggesting that the vector can be used for The expression of miR-185 and miR-424 was reduced.
  • the present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-185-424 based on a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-185 and miR-424; After the vector was transfected into eukaryotic cells in vitro, the expression of miR-185 and miR-424 was detected by a real-time PCR instrument.
  • the invention is not only suitable for transfection of cells, but also for the production of whole horizontal transgenic animals.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une méthode de réduction des expressions de miR-185 et de miR-424. La méthode comprend la construction d'un vecteur d'expression eucaryote pRI-185-424 ciblant miR-185 et miR-424 sur un vecteur eucaryote pRI-GFP/Neo qui contient un promoteur H1, régulant ainsi négativement les expressions de miR-185 et de miR-424.
PCT/CN2017/077174 2017-03-18 2017-03-18 Méthode de réduction des expressions de mir-185 et de mir-424 Ceased WO2018170624A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077174 WO2018170624A1 (fr) 2017-03-18 2017-03-18 Méthode de réduction des expressions de mir-185 et de mir-424

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077174 WO2018170624A1 (fr) 2017-03-18 2017-03-18 Méthode de réduction des expressions de mir-185 et de mir-424

Publications (1)

Publication Number Publication Date
WO2018170624A1 true WO2018170624A1 (fr) 2018-09-27

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PCT/CN2017/077174 Ceased WO2018170624A1 (fr) 2017-03-18 2017-03-18 Méthode de réduction des expressions de mir-185 et de mir-424

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WO (1) WO2018170624A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101368213A (zh) * 2008-10-13 2009-02-18 南京大学 血清微小核糖核酸试剂盒及其在乙肝早期诊断中的应用
CN101755208A (zh) * 2007-07-25 2010-06-23 路易斯维尔大学研究基金会公司 作为诊断标记物的外来体相关微rna
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101755208A (zh) * 2007-07-25 2010-06-23 路易斯维尔大学研究基金会公司 作为诊断标记物的外来体相关微rna
CN101368213A (zh) * 2008-10-13 2009-02-18 南京大学 血清微小核糖核酸试剂盒及其在乙肝早期诊断中的应用
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XING-HUA LIAO ET AL.: "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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