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WO2018170621A1 - Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application - Google Patents

Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application Download PDF

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Publication number
WO2018170621A1
WO2018170621A1 PCT/CN2017/077171 CN2017077171W WO2018170621A1 WO 2018170621 A1 WO2018170621 A1 WO 2018170621A1 CN 2017077171 W CN2017077171 W CN 2017077171W WO 2018170621 A1 WO2018170621 A1 WO 2018170621A1
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WO
WIPO (PCT)
Prior art keywords
mir
sequence
vector
tud
mirna
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/CN2017/077171
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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/CN2017/077171 priority Critical patent/WO2018170621A1/fr
Publication of WO2018170621A1 publication Critical patent/WO2018170621A1/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 more particularly to a vector for reducing the expression of miR-148a and miR-424 and uses 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 has been 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-424 is in recent years A miRNA discovered, which acts on a target gene in a variety of tumors and participates in a signal pathway of target gene regulation, thereby affecting the biological effects and development of tumor cells, exerting effects similar to oncogenes, tumor suppressor genes, or promoting Inhibit the invasion and metastasis of tumors.
  • 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-ot; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing.
  • 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 target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in 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-424 after transformation of 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 ddH20, 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-424, 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 bacteria were amplified and cultured and extracted with a small amount of endotoxin-free extraction kit. The extracted plasmid is the homologous interference pLKO- required for the invention. Plasmid for TuD-148a-424.
  • the homologous interference miR-148a and miR-424 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-424 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-424 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 ddH20, mixed at an equimolar ratio, treated at 95 ° C for 5 min, and allowed to cool to room temperature by allowing them to stand at room temperature.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-Tud-148a-424, 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 for the homologous interference pLKO-TuD-148a-424 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-424 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-424 cell line.
  • the homologous interference miR-148a and miR-424 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

L'invention concerne un vecteur d'expression réduisant un micro-R-148a et un micro-R-424 et son application. Le procédé de mise au point d'un vecteur d'expression inhibant le micro-R-148a et le micro-R-424 consiste à lier une cible avec l'ARN Tud de micro-R-424 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-140-424. Le vecteur d'expression inhibe l'action de has-micro-R-148a et de has-micro-R-424.
PCT/CN2017/077171 2017-03-18 2017-03-18 Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application Ceased WO2018170621A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077171 WO2018170621A1 (fr) 2017-03-18 2017-03-18 Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077171 WO2018170621A1 (fr) 2017-03-18 2017-03-18 Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application

Publications (1)

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WO2018170621A1 true WO2018170621A1 (fr) 2018-09-27

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PCT/CN2017/077171 Ceased WO2018170621A1 (fr) 2017-03-18 2017-03-18 Vecteur d'expression réduisant un micro-r-148a et un micro-r-424 et son application

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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

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