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WO2018170619A1 - Vecteur d'expression inhibant le micro-arn double brin et son application - Google Patents

Vecteur d'expression inhibant le micro-arn double brin et son application Download PDF

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Publication number
WO2018170619A1
WO2018170619A1 PCT/CN2017/077169 CN2017077169W WO2018170619A1 WO 2018170619 A1 WO2018170619 A1 WO 2018170619A1 CN 2017077169 W CN2017077169 W CN 2017077169W WO 2018170619 A1 WO2018170619 A1 WO 2018170619A1
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WO
WIPO (PCT)
Prior art keywords
mir
sequence
tud
vector
mirna
Prior art date
Application number
PCT/CN2017/077169
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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/CN2017/077169 priority Critical patent/WO2018170619A1/fr
Publication of WO2018170619A1 publication Critical patent/WO2018170619A1/fr

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Definitions

  • the present invention relates to the field of molecular biology, and more particularly to a vector for inhibiting expression of a dual miRNA and an application thereof.
  • RNA is an important substance in living organisms and plays various functions in life activities. RNA can be classified into messenger RNA (mRNA) and non-coding RNA depending on whether the protein is encoded or not.
  • mRNA messenger RNA
  • non-coding RNA depending on whether the protein is encoded or not.
  • RNA non-coding RNA, ncRNA.
  • Small RNA small RNA
  • RNA is an important class of ncRNAs. miRNAs are endogenous small RNAs in organisms that are typically 20-24 nt in length. The miRNA is part of the pri-miRNA (primary RNA) and is initially expressed in the nucleus by RNA polymerase ⁇ . The mature miRNA acts as a guiding molecule, binds to the target gene mRNA according to the principle of base pairing, and directs the silencing complex (RISC) to degrade mRNA or hinder its translation, thereby exerting a negative regulation effect on the expression of the target gene.
  • RISC silencing complex
  • miR-148a is a microRNA that has been studied more in recent years. It is reported that miR-148a is closely related to the metabolism of exogenous substances, apoptosis, the occurrence, development and epigenetics of various cancers, so it is important to study the function of miR-148a; miR-152 is a kind of With multifunctional miRNAs, it was found that miR-152 is associated with methylation, such as methyltransferase DNMT1 content and enzyme activity, miR-152 can be methylated by endometrial cancer DNA into a silent gene, and its Associated with the development of a variety of cancers, it is a tumor suppressor microRNA that is associated with many diseases such as pre-eclampsia, trophoblastic tumors, bladder cancer, gastrointestinal cancer, and ovarian cancer. By controlling the expression of miR-148a and miR-152, peers and other drugs work together to provide new epigenetic ideas for the treatment of cancer.
  • 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 adsorb target miRNAs. Since the inserted RNA is double-stranded and has a secondary structure of a stem loop, it is resistant to intracellular nuclease degradation and can inhibit miRNA for a long-term, stable, and efficient manner.
  • the technical problem to be solved by the present invention is to provide a dual miRNA suppression expression vector which is simple in structure, low in cost and simple in operation.
  • a Tud RNA targeting a dual miRNA the nucleotide sequence of which is shown in SEQ ID NO: 1 in the Sequence Listing.
  • a dual miRNA-inhibiting expression vector comprising the sequence of the invention SEQ ID NO: 1.
  • the double miRNA-inhibiting expression vector can inhibit the expression of miR-148a and miR-152 by transforming Hela cells.
  • a method for constructing a dual miRNA suppression expression vector according to the present invention comprises the following steps:
  • the synthesized sequence is two complementary single-stranded DNAs.
  • the two single-stranded DNAs were dissolved in ddH 2 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and then allowed to cool to room temperature at room temperature.
  • the digested vector was recovered using the MinElute Reaction Cleanup Kit, and the TuD obtained in the previous step was further treated with T4 DNA ligase.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-148a-152, and finally the ligation product was transformed into competent E. coli Stbl3 and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Single colonies were picked and sequenced. The bacteria with the correct sequencing were expanded and extracted with a small amount of extraction kit without endotoxin plasmid. The extracted plasmid was the plasmid for the homologous interference pLKO-TuD-148a-152 required by the present invention.
  • the homologous interference miR-148a and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.
  • Figure 1 shows the miRNA expression levels of 16HBE cells and TuD-148a-152 cells, wherein, a.
  • the lentiviral plasmid pLKO.l-puro vector used in the present invention was purchased from Addgene; the human bronchial epithelial cells (16HBE cell strain) used in the present invention were purchased from ATCC, USA.
  • TuD RNA oligonucleotide sequence targeting miR-148a and miR-152 was designed, and its sequence is SEQ ID.
  • the synthesized sequence is two complementary single stranded DNAs.
  • the two single-stranded DNAs were dissolved in ddH 2 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and allowed to stand at room temperature to allow them to naturally cool to room temperature.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-Tud-148a-152, and finally the ligation product was transformed into competent E. coli Stbl3 and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Single colonies were picked and sequenced. The correct sequencing bacteria were expanded and extracted with a non-endotoxin plasmid miniprep kit. The extracted plasmid was the plasmid of the same interference interference pLKO-TuD-148a-152 required by the present invention.
  • 16HBE cells were seeded in 6-well plates, 1000000 cells per well, and the cell density was about 60% after 18 hours.
  • the plasmid pLKO-TuD-148a-152 was transduced into 16HBE cells with Lipfectamine 2000, and culture was continued for 48 h. Thereafter, the cells were cultured for 3 days in DMEM medium containing 1.0 g/ml puromycin, and the cell line obtained by the screening was named TuD-148a-152 cell line.
  • the homologous interference miR-148a and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un vecteur d'expression inhibant le micro-ARN double brin et son application. Le vecteur d'expression selon l'invention lie une cible avec l'ARN Tud de micro-R-152 et de micro-R148a sur un vecteur de clonage pLKO.1-puro, le vecteur d'expression inhibant le micro-ARN double brin pLKO-Tud-148a-152 ainsi obtenu présente une action inhibant has-micro-R-148a et has-micro-R-152.
PCT/CN2017/077169 2017-03-18 2017-03-18 Vecteur d'expression inhibant le micro-arn double brin et son application WO2018170619A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077169 WO2018170619A1 (fr) 2017-03-18 2017-03-18 Vecteur d'expression inhibant le micro-arn double brin et son application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077169 WO2018170619A1 (fr) 2017-03-18 2017-03-18 Vecteur d'expression inhibant le micro-arn double brin et son application

Publications (1)

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WO2018170619A1 true WO2018170619A1 (fr) 2018-09-27

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101988060A (zh) * 2009-07-30 2011-03-23 江苏命码生物科技有限公司 结直肠癌检测标记物及其检测方法、试剂盒和生物芯片
CN103623425A (zh) * 2012-08-27 2014-03-12 苏州圣诺生物医药技术有限公司 应用双靶标拮抗寡核酸抑制新血管增生疾病的药物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101988060A (zh) * 2009-07-30 2011-03-23 江苏命码生物科技有限公司 结直肠癌检测标记物及其检测方法、试剂盒和生物芯片
CN103623425A (zh) * 2012-08-27 2014-03-12 苏州圣诺生物医药技术有限公司 应用双靶标拮抗寡核酸抑制新血管增生疾病的药物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HARAGUCHI, T. ET AL.: "Vectors Expressing Efficient RNA Decoys Achieve the Long-term Suppression of Specific MicroRNA Activity in Mammalian Cells", NUCLEIC ACIDS RESEARCH, vol. 37, no. 6, 17 February 2009 (2009-02-17), pages e43, XP055538716, ISSN: 0305-1048 *
XIE, XING ET AL.: "Construction of a Human Bronchial Epithelial Has-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 *

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