Construction Of Sh Rna Lentiviral Expression Vector Of ITGA5 Gene And Its Effect On Human Bel-7404 Hepatoma Cells

Objective To construct integrin α5(ITGA5)gene sh RNA lentiviral vector and ITGA5 gene stable transfection Bel-7404 human hepatoma carcinoma cell line,to detect the biological function of ITGA5 gene about the proliferation,apoptosis,invasion,metastasis in Bel-7404 hepatoma cells.Methods 1.Design specific sh RNA target of ITGA5 gene,construct sh RNA lentiviral vector of ITGA5 gene and infect Hepatocellular carcinoma Bel-7404 cell,construct the cell line of ITGA5 gene stable silencing.Viral vectors containing non-specific sequences were used as negative controls and untransfected cell was used as a blank control.The m RNA level of ITGA5 in Bel-7404 cells was detected by q PCR,and the expression of integrin α5 protein was detected by Western Blot,to identify the effect of ITGA5 gene silencing.2.Platelet colony formation assay,flow cytometry,Transwell and cell scratch were used to detect the proliferation,apoptosis,invasion and migration of Bel-7404 cells after ITGA5 gene silencing.Result 1 The sh RNA-ITGA5 lentiviral vector was successfully constructed and infiltrated Bel-7404 cells.ITGA5 gene silencing cell line was established.q PCR and Western blot indicated that ITGA5 m RNA and protein expression levels in the silencing cell line were significantly lower than those of blank and negative control groups.Plate colony formation assay results showed that the cloning rate of the gene silencing group was lower than that of the blank control group and the negative control group,the difference was statistically significant(P < 0.05);Transwell results showed that number of cells through the membrane of the gene silencing cells line less than the blank and negative control,different result have statistically significant(P < 0.05).The results of apoptosis detection by flow cytometry showed that the gene silencing group had more apoptosis than the blank and negative control group,different result have statistically significant(P < 0.05).Conclusions 1.Successfully constructed the sh RNA lentivirus expression vector of ITGA5 gene and the hepatoma cell line of ITGA5 gene stable silencing.2.ITGA5 gene may promote hepatoma metastasis by affecting the proliferation,invasion,migration and apoptosis of hepatoma cells.