The Effects Of MiR-593-5p Transplantation Tumor In The Nude Mouse And Its Possible Related Mechanism

Objective:To observegastric cancer cells’ability of invasion and metastasis in transplantation tumor model of nude mice after the gene miR-593-5p are overexpressed and explore the possible molecular mechanisms.Methods:To culture human gastric cancer cells MGC-803 in RPMI 1640 containing 10% fetal bovine serum and put them in 37℃ and 5% CO2 incubator. When cells’growth achieve logarithmic phase, to collect human gastric cancer cell MGC-803 and culture them in six orifice plate, about 3.5×104/hole. According to the slow virus transfection manual of operation, they can be divided into three groups-the experimental group which the restructuring slow virus particles (Lv-hsa-miR-593-5p-GFP) infects human MGC-803 gastric cancer cells, the negative control group which the no-load slow virus particles (Lv-GFP) infects human MGC-803 gastric cancer cells, the blank control group whichdoes nothing with. Screening two groups of infection virus cells in puro, and making two groups of cells became stable infection strains.Using the real-time fluorescent quantitative PCR detect miR-593-5p gene expression of different group.To collect stable cells infection groups and adjust cell for 1×106/ul. Each group cell is inoculatedin nude mice left upper limb armpit, each mice at least 200 ul,10 mice in every group. To normal breed, observe every mouse and measure the volume of transplanted tuors each 4 days. After 20 days, all nude mice were put to death and taken nude mouse transplantation tumor, liver and lung tissue.Taking nude mice tumor tissue, liver and lung tissues do HE staining, taking each nude mice tumor tissuedo immunohistochemical detection, detection of CD44 groups of cells.miR-593-5p of human gastric cancer cell MGC-803 overexpress. Takingthe experimental group and the negative control group do gene chip experiments, and we get the differentially expressed genes. Application of bioinformatics prediction software Targetscan, miRDB and microRNA.org predict the downstream Target genes of the gene miR-593-5p. Combining the results we protocol possible miR-593-5p downstream Target genes MST4.Using real-time fluorescent quantitative PCR and western blot detect MST4 gene and protein expression, using double luciferase validate MST4 Target genes.Results:It’s successful to use the recombinant lentivirus to infect human gastric cell MGC-803. Using real-time fluorescent quantitative PCR to detect the stable infected cell lines and the miR-593-5p increased obviously in the experimental group. In vivo nude mouse transplantation tumor model was constructed, the growth of the transplanted tumor RTV, experimental group slower than the negative control group and blank control group. In the 20th days, volume of transplanted tumors had significantly less than the negative control group and blank control group.Taking pathology observations confirmes the transplanted tumor tissue is indeed for malignant tumor. We observed the liver and lung malignant tumor tissues in the experimental group are significantly less than other two groups and number of metastatic malignant tumor in nude mice are also less than other two groups. In Immunohistochemical detection of the experimental group, the number of blood vessels in tumor tissue relatively are less than other two groups, the expression of CD44 protein fewer than the other two groups. Molecular mechanism research of miR-593-5p,after up-regulation genetic miR-593-5p gene chip results in its downstream genes, raised a total of 212 genes, cut a total of 263 genes.Targetscan, miRDB, microRNA.org-the three online prediction software, may filter common target genes. Combining the gene chips with the purpose of comprehensive analysis, we find gene MST4, TMEM135, TWSG1 seven genes. Finally we protocol MST4 for gene miR-593-5p directly downstream target genes. Real-time fluorescent quantitative PCR detect MST4 gene expression, and in the experimental group gene MST4 is down-regulated. Usingwestern blot test MST4 protein expression and it shows MST4 protein is down-regulated in the experimental group. Using dual luciferase experiment detects target genes MST4 and the results show miR-593-5p can’t combineMST4.Conclusions:In nude mice transplantation tumor model, miR-593-5p is up-regulated and the blood vessel growth of nude mouse transplantation tumor is reduced.In human gastric cancer cells MGC-803, the overexpression of gene miR-593-5p can cut MST4 gene expression and protein expression.miR-593-5pdoesn’t directly regulate gene MST4, but it is highly possible to indirectly regulate the downstream gene MST4 to inhibit the growth of cancer.