WO2019227260A1 - Mammalian virus-mediated mirna overexpression method - Google Patents

Abstract

A mammalian virus-mediated miRNA overexpression method. The overexpression of endogenous target miRNA in a tumor cell is implemented mainly by infecting the tumor cell 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-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.

Technical solutions

The object of the present invention is to provide a method for mediating miR-155 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 miR-155 precursor fragment and package it into a lentivirus;

b. Jurkat cells were infected by lentivirus, and cells overexpressing miR-155 were selected by puromycin.

Beneficial effect

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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows miR-155 expression levels of Jurkat 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-155 Construction of an overexpression lentiviral vector

According to the gene sequence of human miR-155 in Genbank (accession number: AP000223.1), 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-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 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-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 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 Jurkat cells.

Example 3: Lentivirus infection Jurkat cell

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 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-155 The expression level

Normal Jurkat cells (control group) and miR-155 over-expressing 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-155 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-155 in the cells of the experimental group is more than 109 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-155 gene.

Industrial applicability

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.

Claims (3)

1. A method for mediating miRNA overexpression based on a mammalian virus, wherein the mammalian virus carries a human-derived miR-155 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-derived miR-155 precursor fragment and packaging it into a lentivirus;

   b. Jurkat cells were infected by lentivirus, and cells overexpressing miR-155 were selected by puromycin.