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WO2018170622A1 - Procédé de régulation négative synchrone des expressions de miarn-152 et de miarn-185 - Google Patents

Procédé de régulation négative synchrone des expressions de miarn-152 et de miarn-185 Download PDF

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Publication number
WO2018170622A1
WO2018170622A1 PCT/CN2017/077172 CN2017077172W WO2018170622A1 WO 2018170622 A1 WO2018170622 A1 WO 2018170622A1 CN 2017077172 W CN2017077172 W CN 2017077172W WO 2018170622 A1 WO2018170622 A1 WO 2018170622A1
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WO
WIPO (PCT)
Prior art keywords
mir
expression
pri
vector
microrna
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PCT/CN2017/077172
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English (en)
Chinese (zh)
Inventor
毛吉炎
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深圳市博奥康生物科技有限公司
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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/CN2017/077172 priority Critical patent/WO2018170622A1/fr
Publication of WO2018170622A1 publication Critical patent/WO2018170622A1/fr

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  • the present invention relates to the field of life sciences, and more particularly to a method for simultaneously down-regulating the expression of microRNA-152 and microRNA-185.
  • 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 DNA methylation of endometrial cancer becomes a silent gene, and it is associated with the development of various cancers. It is a tumor suppressor microRNA, with preeclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer.
  • microRNA-185 (miR-185) is a 22nt miRNA, located in human chromosome 22ql l.21, in the occurrence and invasion of colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer and other tumors. It plays an important role as a tumor suppressor gene.
  • it is a methylation-related tumor suppressor miRNA that affects the methylation level of the whole genome by directly targeting the expression of DNMT1, thereby regulating the methylation of certain genes. Modification of the state, affecting gene expression.
  • the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer.
  • 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-152 and miR-185 expression was down-regulated simultaneously.
  • the object of the present invention is to provide a method for simultaneously down-regulating the expression of miR-152 and miR-185, which is The method is simple, easy to operate, and can easily and effectively adjust the effects of miR-152 and miR-185 expression to overcome the deficiencies of the prior art.
  • a method for the present invention a method for down-regulating expression of miR-152 and miR-185, and constructing eukaryotic expression targeting miR-152 and miR-185 on a eukaryotic vector pRI-GFP/Neo containing a HI promoter Vector pRI-152-185, which regulates the expression of miR-152 and miR-185 in eukaryotic cells; after transfecting the vector into eukaryotic cells in vitro, the expression of miR-152 and miR-185 was detected by real-time PCR Level.
  • the sequences of pRI-GFP/Neo and Tud-152-185 were double-digested with Xho I and Bgl II, and the recovered fragments were purified and ligated overnight at 4 °C. After ligation, 6 colonies were picked and inoculated. After 12 hours of LB medium containing 1 (Vg/ml kanamycin), the bacterial solution was extracted and sent to Shanghai Yingjun for sequencing. The correctly sequenced vector was the successfully constructed recombinant plasmid pRI-152-185.
  • the present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-152-185 based on a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-185; After the vector was transfected into eukaryotic cells in vitro, the expression of miR-152 and miR-185 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-152 expression, b. miR-185 expression.
  • Example of a method for downregulating expression of miR-152 and miR-185 [0011] 1 First search for the sequence of hsa-miR-152 and hsa-miR-185 in the miRBase database, according to Tou gh Decoy RNA (Tud
  • the design principle of RNA is to design the Tud RNA targeting hsa-miR-152 and hsa-miR-185, and finally add the Xho I and Bgl II restriction sites at the 5' and 3' ends of the Tud RNA to obtain the desired The sequence of Tud-152-185.
  • the nucleotide sequence ⁇ IJ of Tud-152-185 is shown as SEQ ID No. 1.
  • HepG2 cells were seeded in a 6-well plate at a density of 30,000/ml, and 10 ug of plasmid pRI-152-185 was transiently transfected with Lip ofectamine 2000, and cultured at 5 ⁇ 3 ⁇ 4C02, 37 °C; After 48 hours, total RNA was extracted from each group, and the expression of miR-152 and miR-185 was detected by Realtime PCR. The results showed that miR-152 and miR-185 were expressed in pRI-152-185 transfected group. Significant down-regulation (Figure 2) suggests that this vector can be used to down-regulate the expression of miR-152 and miR-185.
  • the present invention utilizes a molecular biology technique to construct a eukaryotic expression vector containing Tud-152-185 based on a eukaryotic vector containing a CMV promoter, thereby down-regulating the expression of miR-152 and miR-185; After the vector was transfected into eukaryotic cells in vitro, the expression of miR-152 and miR-185 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 un procédé de réduction des expressions de miARN-152 et de miARN-185, comprenant la construction d'un vecteur d'expression eucaryote pRI-152-185 ciblant miR-152 et miR-185 sur un vecteur eucaryote pRI-GFP/Neo qui contient un promoteur H1, permettant ainsi de réguler à la baisse les expressions de miR-152 et miR-185.
PCT/CN2017/077172 2017-03-18 2017-03-18 Procédé de régulation négative synchrone des expressions de miarn-152 et de miarn-185 WO2018170622A1 (fr)

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PCT/CN2017/077172 WO2018170622A1 (fr) 2017-03-18 2017-03-18 Procédé de régulation négative synchrone des expressions de miarn-152 et de miarn-185

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077172 WO2018170622A1 (fr) 2017-03-18 2017-03-18 Procédé de régulation négative synchrone des expressions de miarn-152 et de miarn-185

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WO2018170622A1 true WO2018170622A1 (fr) 2018-09-27

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Citations (4)

* 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
CN102725632A (zh) * 2009-08-28 2012-10-10 奥斯瑞根公司 肺病的miRNA生物标志物
CN103987858A (zh) * 2011-11-22 2014-08-13 英特芒尼公司 诊断和治疗特发性肺纤维化的方法

Patent Citations (4)

* 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
CN102725632A (zh) * 2009-08-28 2012-10-10 奥斯瑞根公司 肺病的miRNA生物标志物
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
CN103987858A (zh) * 2011-11-22 2014-08-13 英特芒尼公司 诊断和治疗特发性肺纤维化的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIAO, XINGHUA 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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