WO2019227260A1 - Procédé de surexpression de miarn médiée par un virus de mammifère - Google Patents

Procédé de surexpression de miarn médiée par un virus de mammifère Download PDF

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Publication number
WO2019227260A1
WO2019227260A1 PCT/CN2018/088535 CN2018088535W WO2019227260A1 WO 2019227260 A1 WO2019227260 A1 WO 2019227260A1 CN 2018088535 W CN2018088535 W CN 2018088535W WO 2019227260 A1 WO2019227260 A1 WO 2019227260A1
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Prior art keywords
cells
mammalian virus
mir
mirna
overexpression
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PCT/CN2018/088535
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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/CN2018/088535 priority Critical patent/WO2019227260A1/fr
Publication of WO2019227260A1 publication Critical patent/WO2019227260A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors

Definitions

  • the invention relates to a mammalian virus-based method for mediating miRNA overexpression, and the method can be used to study the role played by miRNA in tumorigenesis and development.
  • MicroRNA is a class of endogenous non-coding genes widely found in animal and plant cells, with a size of about 21-25 nt. It is highly conserved in the evolution of different species and plays an important role in regulating post-transcriptional gene expression. After miRNA is transcribed by polymerase, it forms a primary nucleotide product and is cut by the endonuclease Drosha to form a hairpin precursor. After being transported into the cytoplasm and then cleaved by enzymes, mature miRNA is finally formed. The mature miRNA, in the form of a RISC-miRNA complex, regulates the expression of the target gene by binding to the corresponding region of the 3 'untranslated region of the target gene.
  • miRNA-155 is a tumor-associated miRNA that is currently hotly studied. EisPS and other researchers have found that miRNA-155 levels in lymphoma cells are more abundant than circulating B lymphocytes in B-cell lymphoma patients as early as 2005, suggesting that high-level expression of miRNA-155 is more likely to activate B cells. Diffuse large B-cell lymphoma, not only means poor prognosis, but more and more studies have shown that miR-155 plays a very important role among inflammation, immune system, and tumor, and miRNA-155 both mediates and regulates inflammation. Occurrence, in turn, regulates the growth of immune cells, selective release of cytokines, and is closely related to the differentiation and maturation of tumor cells.
  • miRNA-155 is one of the earliest miRNA molecules that has been shown to promote cancer. Studies have shown that in, for example, diffuse large B-cell lymphoma, adult T-cell leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, Burkitt lymphoma, and mantle cells In most hematological malignancies such as lymphoma and solid tumors such as lung cancer and breast cancer, miRNA-155 is highly expressed. miRNA-155 plays an important role in the pathogenesis of T-cell lymphoma, but its biological functions and specific pathogenic mechanisms have not been fully elucidated, and there is also a lack of lentivirus-mediated overexpression that can be used for miR-155 function research in the prior art. method.
  • the object of the present invention is to provide a method for mediating miR-155 overexpression based on mammalian viruses.
  • the present invention adopts the following technical steps:
  • Jurkat cells were infected by lentivirus, and cells overexpressing miR-155 were selected by puromycin.
  • the mammalian virus-based miR-155 overexpression method provided by the present invention can greatly increase the expression level of miR-155 in tumor cells, and provides a new technical means for studying the role of miR-155 in tumorigenesis and development.
  • Figure 1 shows miR-155 expression levels of Jurkat cells in the control and experimental groups.
  • Embodiment one miR-155 Construction of an overexpression lentiviral vector
  • Age I and EcoR I enzymes were used to double digest the plasmid containing the synthetic sequence and the pLKO.1-puro vector, respectively, and then recovered and purified.
  • the recovered miR-155 sequence was mixed 1: 6 with pLKO.1-puro vector, and then ligated with NEB T4 DNA ligase.
  • the ligated product was transformed into competent E. coli DH5 ⁇ , and then expanded and sequenced to screen out bacteria that completely matched the expected results. Then expand the culture, and use the endotoxin-free plasmid extraction kit to extract the recombinant plasmid in E. coli and name it pLKO-miR155.
  • Example 2 Packaging of lentivirus
  • 293T cells were cultured, and well-growth cells were inoculated into six wells. Each well had 1,000,000 cells.
  • Recombinant plasmids pLKO-miR155 and pCMV-dR8.91, pCMV-VSV-G were cotransformed with 1 ⁇ g each of the auxiliary plasmids using Lipofectamine 2000 After staining to 293T cells, the virus-containing supernatant medium was collected 48 hours later, and the virus solution was filtered through a 0.45 ⁇ m sieve to infect Jurkat cells.
  • Jurkat cells were seeded in a six-well plate with 1,000,000 cells per well, and the cell density was about 50% after 12 hours.
  • the virus solution obtained in Example 2 was taken, and the virus was diluted 10-fold with DMEM complete medium. Polybrene was added to the final concentration. 8 ⁇ g / mL.
  • Remove the medium in the six-well plate add virus-containing DMEM complete medium (containing 10% fetal calf serum), discard the virus-containing DMEM complete medium after 24 hours, and replace with fresh DMEM complete medium (containing 1 ⁇ g / mL puromycin) for cell selection.
  • the screening time was 7 days, and the solution was changed (containing 1 ⁇ g / mL puromycin) every other day to eliminate the effect of dead cells on surviving cells and maintain the screening pressure. After screening, a large number of surviving cells were cultured.
  • the mammalian virus-based miR-155 overexpression method provided by the present invention can greatly increase the expression level of miR-155 in tumor cells, and provides a new technical means for studying the role of miR-155 in tumorigenesis and development.

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  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un procédé de surexpression de miARN médiée par un virus de mammifère. La surexpression du miARN cible endogène dans une cellule tumorale est mise en oeuvre principalement par infection de la cellule tumorale par un virus de mammifère portant un fragment précurseur de miARN cible.
PCT/CN2018/088535 2018-05-26 2018-05-26 Procédé de surexpression de miarn médiée par un virus de mammifère WO2019227260A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/088535 WO2019227260A1 (fr) 2018-05-26 2018-05-26 Procédé de surexpression de miarn médiée par un virus de mammifère

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/088535 WO2019227260A1 (fr) 2018-05-26 2018-05-26 Procédé de surexpression de miarn médiée par un virus de mammifère

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WO2019227260A1 true WO2019227260A1 (fr) 2019-12-05

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PCT/CN2018/088535 WO2019227260A1 (fr) 2018-05-26 2018-05-26 Procédé de surexpression de miarn médiée par un virus de mammifère

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024047115A1 (fr) 2022-09-02 2024-03-07 Leibniz-Institut Für Immuntherapie (Lit) Utilisation thérapeutique du snp mir155 rs377265631

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103025384A (zh) * 2010-03-26 2013-04-03 俄亥俄州立大学 涉及miR-155对于错配修复和基因组稳定性的调节的材料和方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103025384A (zh) * 2010-03-26 2013-04-03 俄亥俄州立大学 涉及miR-155对于错配修复和基因组稳定性的调节的材料和方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SONG, LE ET AL.: "Construction of miR-155-5p Lentiviral Vector and Its Effect on Normal Gastric Epithelial Proliferation", ACTA UNIVERSITATIS MEDICINALIS ANHUI, vol. 4, no. 53, 30 April 2018 (2018-04-30), ISSN: 1000-1492 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024047115A1 (fr) 2022-09-02 2024-03-07 Leibniz-Institut Für Immuntherapie (Lit) Utilisation thérapeutique du snp mir155 rs377265631

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