WO2019227258A1 - Mammalian virus-mediated mirna overexpression method - Google Patents

Abstract

A Mammalian virus-mediated miRNA overexpression method, which achieves overexpression of an endogenous target miRNA in tumor cells by mainly infecting the tumor cells with a mammalian virus carrying a target miRNA precursor fragment.

Description

Mammalian virus-based miRNA overexpression method Technical field

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.

Background technique

MicroRNA (miRNA) 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.

technical problem

miRNA-7 is a recently reported tumor-related miRNA. It is significantly under-expressed in lung cancer, breast cancer, and Schwann cell tumors, and may be closely related to the occurrence, development, and metastasis of tumors. Kong and other studies found that miRNA-7, as a possible tumor suppressor gene, was significantly down-regulated in mouse gastric tumors. The proliferation of gastric cancer cells was significantly inhibited after transfection of miRNA-7 precursors into gastric cancer cells. It can be speculated that miRNA-7 It plays an important role in the pathogenesis of gastric cancer, but its biological functions and specific pathogenic mechanisms have not been fully elucidated, and there is also a lack of lentivirus-mediated overexpression methods that can be used for miRNA-7 function research in the prior art.

Technical solutions

An object of the present invention is to provide a method for mediating miR-7 overexpression based on mammalian viruses.

In order to achieve the above objective, the present invention adopts the following technical steps:

a. Construct a recombinant lentiviral vector carrying a human-derived miR-7 precursor fragment and package it into a lentivirus;

b. MGC-803 cells were infected with lentivirus, and cells overexpressing miR-7 were selected by puromycin. .

Beneficial effect

The mammalian virus-based miR-7 overexpression method provided by the present invention can greatly increase the expression level of miR-7 in tumor cells, and provides a new technical means for studying the role of miR-7 in tumorigenesis and development.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the miR-7 expression levels of MGC-803 cells in the control and experimental groups.

Embodiments of the invention

The invention is further described below with reference to the drawings and specific embodiments.

Embodiment one: miR-7 Construction of an overexpression lentiviral vector

According to the gene sequence of human miR-7 in Genbank (accession number: AL354733.15), an Age I restriction site was added to the 5 'end, and an EcoR I restriction site was added to the 3' end. Shanghai Biotech was commissioned to synthesize the sequence in the manner of gene synthesis.

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-7 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 DH5α, 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-miR7.

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-miR7 and pCMV-dR8.91 and pCMV-VSV-G were cotransformed with 1 μg 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 μm sieve to infect MGC-803 cells.

Example 3: Lentivirus infection MGC-803 cell

MGC-803 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 times with DMEM complete medium, and then polybrene was added to The final concentration was 8 μg / 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 μg / mL puromycin) for cell selection. The screening time was 7 days, and the solution was changed (containing 1 μg / 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.

Example 4: Quantitative Fluorescence PCR Detection miR-7 The expression level

Normal MGC-803 cells (control group) and miR-7 overexpressing cells (experimental group) obtained after screening were inoculated into six-well plates. When the cell density reached 80% -90%, total RNA of each group of cells was extracted with RNeasy Mini Kit, and mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit, and stored at -20 ° C.

Take 1 μL of cDNA from each group of cells as a template, use U6 as an internal reference, and detect the relative expression level of miR-7 by real-time quantitative PCR. Set the reaction conditions: 95 ° C for 30s, 1 cycle; 60 ° C for 30s, 40 cycles; 95 ° C for 5s, 60 ℃ for 1min, 95 ℃ for 15s. The results are shown in Figure 1. It can be seen that the expression level of miR-7 in the cells of the experimental group is more than 341 times higher than that of the cells in the control group, indicating that the miRNA overexpression method provided by the present invention can be specific, continuous, efficient and stable. Promote high expression of miR-7 gene.

Industrial applicability

The mammalian virus-based miR-7 overexpression method provided by the present invention can greatly increase the expression level of miR-7 in tumor cells, and provides a new technical means for studying the role of miR-7 in tumorigenesis and development.

Claims (3)

1. A method for mediating miRNA overexpression based on a mammalian virus, wherein the mammalian virus carries a human miR-7 precursor fragment.
2. According to a mammalian virus-based method for mediating miRNA overexpression, the mammalian virus is a lentivirus.
3. According to a mammalian virus-based method for mediating miRNA overexpression, it is characterized by the following steps:

   a. Construction of a recombinant lentiviral vector carrying a human miR-7 precursor fragment and packaging it into a lentivirus;

   b. MGC-803 cells were infected with lentivirus, and cells overexpressing miR-7 were selected by puromycin.