WO2019227262A1

WO2019227262A1 - Method for mediating mirna overexpression based on mammalian virus

Info

Publication number: WO2019227262A1
Application number: PCT/CN2018/088537
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2018-05-26
Filing date: 2018-05-26
Publication date: 2019-12-05

Abstract

Provided is a method for mediating miRNA overexpression based on a mammalian virus. The method achieves the overexpression of an endogenous target miRNA in tumor cells mainly by infecting the tumor cells with the 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

Osteosarcoma is the primary clinical malignant bone tumor with the highest incidence rate. It usually occurs in the dry end of the femur and the upper end of the cavity. It can also occur in fatty bones, fat bones, skeletal bones, and spine. According to authoritative epidemiological data from the US Department of Health's SEER organization, the incidence of osteosarcoma has two peaks in adolescents and the elderly, and the incidence rate in the 0-24 age group is 4.4 / 1 million, second only to lymphoma. The peak incidence of adolescents is around 12 years old, mainly with primary osteosarcoma; the second peak incidence age is around 75 years old, mainly with secondary, the primary disease includes Paget's disease and other bone diseases, the male to female ratio is 1.22 :1. Osteosarcoma has a high degree of malignancy, about 80% Metastasis can occur in ~ 90% of patients. It may have broken through the bone cortex and the medullary cavity early in the course of the disease, and local infiltration followed by hematogenous metastasis. The most common disease is metastasis to the lungs. A few can metastasize to the brain, prostate, and kidneys. organ.

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

miR-664 is a miRNA that has been reported less frequently in rat models of spontaneous diabetes, patients with atrial fibrillation of rheumatoid heart disease and animal models, and patients with pituitary prolactin adenoma. ､ Autism spectrum disorder miR-664 is also abnormally expressed. 胃 In gastric cancer, miR-664 expression is lower in tumor cells and adjacent tissues than in tumor-free gastric tissues, especially in gastric cancer cells; in acute T lymphocytic leukemia Medium-highly expressed miR-664 promotes tumor cell invasion and metastasis by inhibiting the expression of PLP2 at the protein level, but miR-664 has a low expression state in malignant skin melanoma and can activate PLP2 expression and promote tumor proliferation. Metastasis MiR-664 is over-expressed in liver cancer and inhibits the expression of methionine adenosyltransferase 1A (MAT1A). The knockout of miR-664, miR-485-3p and miR-496 genes can induce the re-expression of MAT1A and slow the tumor Proliferation cells promote cell regulation and reduce tumor invasion and metastasis. The above studies suggest that miR-664 plays an important role in invasion, metastasis and apoptosis of other malignant tumors. It plays the role of proto-oncogene or tumor suppressor gene in tumors from different tissues, which may be future gene therapy. Potential boot sites for malignant tumors, but there has not been any related research on miR-664's abnormal expression in osteosarcoma and its biological function, and there is also a lack of lentivirus-mediated lentivirus-based functional studies in the prior art Over-expression method.

Technical solutions

An object of the present invention is to provide a method for miR-664-based 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-664 precursor fragment and package it into a lentivirus;

b. Lentivirus infected MG-63 cells and screened for over-expressing miR-664 by puromycin.

Beneficial effect

The mammalian virus-based miR-664 overexpression method provided by the present invention can greatly increase the expression level of miR-664 in tumor cells, and provides a new technical means for studying the role of miR-664 in tumorigenesis and development.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows miR-664 expression levels of MG-63 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.

实施例一Example one ：: miR-664miR-664 过表达慢病毒载体的构建Construction of an overexpression lentiviral vector

According to the gene sequence of human miR-664 (accession number: AL445435.11) in Genbank, 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-664 sequence was mixed with the pLKO.1-puro vector 1: 6, 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-miR664.

实施例二：慢病毒的包装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-miR664 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 MG-63 cells.

实施例三Example three ：: 慢病毒感染Lentivirus infection MG-63MG-63 细胞cell

Inoculate MG-63 cells in a six-well plate with 1,000,000 cells per well. The cell density is about 50% after 12 hours. Take the virus solution obtained in Example 2. Dilute the virus 10 times with DMEM complete medium, and add polybrene to The final concentration was 8 μg / mL. Remove the medium from 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.

实施例四：荧光定量Example 4: Quantitative Fluorescence PCRPCR 检测Detection miR-664miR-664 表达水平The expression level

Normal MG-63 cells (control group) and miR-664 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 and use U6 as an internal reference to detect the relative expression level of miR-664 by real-time quantitative PCR. Set the reaction conditions: 95 ° C 30s, 1 cycle; 60 ° C 30s 40 cycles; 95 ° C 5s, 60 ℃ for 1min, 95 ℃ for 15s. The results are shown in FIG. 1. It can be seen that the expression level of miR-664 in the cells of the experimental group is more than 179 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-664 gene.

Industrial applicability

Claims

A mammalian virus-based method for miRNA overexpression, characterized in that the mammalian virus carries a human-derived miR-664 precursor fragment.
According to a mammalian virus-based method for mediating miRNA overexpression, the mammalian virus is a lentivirus.
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-664 precursor fragment and packaging it into a lentivirus;

b. Lentivirus infected MG-63 cells, and screened for over-expressing miR-664 by puromycin.