Effects Of RASSF6 Gene On Biological Behavior Of Gastric Cancer Cells

Objective:To study the effects of RASSF6 gene on proliferation,migration,cell cycle,apoptosis and chemosensitivity of gastric cancer cell line BGC823 to oxaliplatin.Methods:In this study,human gastric cancer BGC823 cells were cultured in vitro and lentivirus transduction gene expression regulation technology was used to construct lentivirus negative control?Nc?,RASSF6 gene interference?Ni?and RASSF6 gene overexpression?OP?.Normal cultured cells served as control group?Control?.After 72 hours,the transduction efficiency was observed by fluorescence microscopy.RASSF6 mRNA and protein expression in each group were detected by real-time quantitative polymerasechain reaction?qRT-PCR?and Western Blot.Cell proliferation and migration were tested by CCK-8 and cell scratch assay.Cell cycle distribution and apoptosis were detected by flow cytometry.To research chemotherapy sensitive of oxaliplatin,cells were divided into normal control+oxaliplatin treatment group?L-OHP group?,lentiviral negative control+oxaliplatin treatment group?Nc+L-OHP group?,RASSF6 gene silencing+oxaliplatin treatment group?Ni+L-OHP group?,RASSF6 gene overexpression+oxaliplatin treatment group?OP+L-OHP group?and BGC823cells in normal culture were used as normal control group?Control group?.Cell apoptosis in each group was analyzed by flow cytometry.The relative expression levels of apoptosis-related proteins?Bcl-2,Bax and Caspase-3?in each group were examined by Western Blot.Results:1.After transduction of gastric cancer BGC823 cells with RASSF6 gene silencing,overexpressed lentiviral vector and lentiviral empty vector for 72h,the abundance of fluorescence expression was more than 80%.2.The results of qRT-PCR showed that there were no significant differences in the expression levels of RASSF6 mRNA between Nc and control group?P>0.05?.RASSF6 mRNA expression levels were decreased in the Ni group and increased in the OP group?P<0.05?.The results of Western Blot were consistent with those of qRT-PCR.3.The absorbance value?OD value?of each group at 24h,48h and 72h was analyzed by CCK-8 cell proliferation test.Compared with the control group,there was no difference in Nc group?P>0.05?.Cell proliferation were increased in the Ni group and decreased in the OP group?P<0.05?.4.There was no difference between the Nc group and the control group in terms of cell scratch area ratio?P>0.05?.The cell scratch area ratio was decreased in the Ni group and increased significantly in the OP group?P<0.05?.5.There were no significant differences in cell cycle distributions between the Nc group and the control group?P>0.05?.In the Ni group,the proportions of G0/G1 cells were decreased,and the proportions of G2/G1 cells were increased.In the OP group,the proportions of G0/G1 cells were increased,and the proportions of G2/S cells were decreased?P<0.05?.6.The apoptosis of cells in each group was examined by flow cytometry.The results showed that there were no significant differences in the apoptosis rates between the Nc group and the control group?P>0.05?The apoptosis rate in the Ni group was decreased,while in the OP group was significantly increased?P<0.05?.7.After 48h treatment of oxaliplatin on gastric cancer BGC823 cells,IC₅₀ was 13.33?g/ml,and IC₂₀ was 5?g/ml.Subsequently,5?g/ml was selected as the experimental concentration of oxaliplatin.8.The apoptosis rates of four groups treated with L-OHP were higher than the control group?P<0.05?.There were no differences in apoptosis rates between the L-OHP group and Nc+L-OHP group?P>0.05?.The apoptosis rates of Ni+L-OHP group were decreased,and the apoptosis rates of OP+L-OHP group were increased?P<0.05?.9.Western Blot analysis of Bcl-2,Bax and Caspase-3 protein expression in each group showed that compared with the control group,the relative expression levels of three proteins in the four groups were all increased?P<0.05?.The protein expression levels of Bcl-2,Bax and Caspase-3 were little difference between L-OHP and Nc+L-OHP group?P>0.05?.The relative expression levels of Bcl-2 protein in the Ni+L-OHP group were increased,the relative expression levels of Bax and Caspase-3 protein in the OP+L-OHP group were higher than L-OHP group?P<0.05?.Conclusion:Overexpression of RASSF6 gene inhibited the proliferation and migration of gastric cancer BGC823 cells,blocked the cell cycle in G0/G1 phase,and promoted apoptosis.Up-regulation of RASSF6 gene can increase the sensitivity of oxaliplatin to chemotherapy by promoting the apoptosis of BGC823 cells.RASSF6 gene may be a tumor suppressor gene for gastric cancer,and it may be a new target for the targeted therapy of gastric cancer.