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WO2018170620A1 - Vecteur d'expression de deux micro-arn mis sous silence - Google Patents

Vecteur d'expression de deux micro-arn mis sous silence Download PDF

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Publication number
WO2018170620A1
WO2018170620A1 PCT/CN2017/077170 CN2017077170W WO2018170620A1 WO 2018170620 A1 WO2018170620 A1 WO 2018170620A1 CN 2017077170 W CN2017077170 W CN 2017077170W WO 2018170620 A1 WO2018170620 A1 WO 2018170620A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
vector
sequence
mirna
tud
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/CN2017/077170
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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/CN2017/077170 priority Critical patent/WO2018170620A1/fr
Publication of WO2018170620A1 publication Critical patent/WO2018170620A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of molecular biology, and in particular to a vector for homologous knockdown of two miRNA expressions.
  • 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 exogenous substance metabolism, apoptosis, occurrence, development and epigenetics of various cancers, so it is important to study the function of miR-148a; miR-185 is a length
  • DNMT1 tumor suppressor miRNAs
  • DNMT1 tumor suppressor DNMT1
  • miR-148a and miR-185 the synergy with other drugs can 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 time, stably and efficiently.
  • 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-185 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-185, 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 small amount of extraction kit without endotoxin plasmid. The extracted plasmid was the plasmid of the homologous interference pLKO-TuD-148a-185 required for the present invention.
  • the homologous interference miR-148a and miR-185 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-185 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.
  • the TuD RNA oligonucleotide sequence targeting miR-148a and miR-185 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-185, 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, and the extracted plasmid was the plasmid of the homologous interference pLKO-TuD-148a-185 required for 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-185 was transduced into 16HBE cells with Lipfectamine 2000, and cultured for 48 h. Thereafter, the cells were cultured for 3 days in DMEM medium containing 1.0 g/ml of puromycin, and the obtained cell line was designated as TuD-148a-185 cell line.
  • the homologous interference miR-148a and miR-185 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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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Le vecteur d'expression de deux micro-ARN mis sous silence lie une cible avec l'ARN Tud de micro-R-185 et de micro-R148a sur un vecteur de clonage pLKO.1-puro afin d'obtenir un vecteur d'expression inhibant le micro-ARN double brin pLKO-Tud-148a-185, lequel vecteur d'expression de deux micro-ARN mis sous silence présente une action inhibant has-micro-R-148a et has-micro-R-185.
PCT/CN2017/077170 2017-03-18 2017-03-18 Vecteur d'expression de deux micro-arn mis sous silence Ceased WO2018170620A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077170 WO2018170620A1 (fr) 2017-03-18 2017-03-18 Vecteur d'expression de deux micro-arn mis sous silence

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077170 WO2018170620A1 (fr) 2017-03-18 2017-03-18 Vecteur d'expression de deux micro-arn mis sous silence

Publications (1)

Publication Number Publication Date
WO2018170620A1 true WO2018170620A1 (fr) 2018-09-27

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Family Applications (1)

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PCT/CN2017/077170 Ceased WO2018170620A1 (fr) 2017-03-18 2017-03-18 Vecteur d'expression de deux micro-arn mis sous silence

Country Status (1)

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WO (1) WO2018170620A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (1)

* Cited by examiner, † Cited by third party
Title
TAKESHI HARAGUCHI: "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), XP055538974, ISSN: 0305-1048 *

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