Study On The Function Of PPP3CB Gene In Human Cutaneous Squamous Cell Carcinoma Colo16 And A431 Cells

Background:Cutaneous Squamous Cell Carcinoma（c SCC）is a common malignant tumor with high malignancy and easy metastasis,and its etiology and pathogenesis are still unclear,so it is necessary to further investigate the etiology and pathogenesis of c SCC.Previously,our group found that the expression of PPP3CB gene was reduced in c SCC tissues compared with paraneoplastic tissues,so we speculated that it might be an oncogene.To further verify this,we proposed to explore the function of PPP3CB in human cutaneous squamous cell carcinoma Colo16 and A431 cells using gene overexpression technology.Objective:Study the effect of overexpression of PPP3CB gene on the biological behavior of Colo16 and A431 cells.Methods:1.Construction overexpression lentiviral vector of target gene,packaging and titer assay.2.Construct human cutaneous squamous cell carcinoma cell lines Colo16 and A431stable transfection cell lines using overexpression lentivirus.The cell lines were divided into three groups:Colo16-control and A431-control:blank group of untransfected target gene;Colo16-Lv-control and A431-Lv-control:negative control group of transfected empty vector;Colo16-Lv-PPP3CB and A431-Lv-PPP3CB:experimental group of transfected PPP3CB gene.3.The expression of PPP3CB mRNA in Colo16 and A431 cell lines was detected by qPCR.4.The expression of PPP3CB protein in Colo16 and A431 cell lines was detected by Western blot.5.The proliferation ability of Colo16 and A431 cells lines was measured by CCK-8kit.6.The migration ability of Colo16 and A431 cell lines was examined separately using cell scratch assay.7.The Transwell cell invasion assay was used to detect changes in the invasive ability of the three groups of Colo16 and A431 cells.Results:1.The titers of overexpression lentivirus:LV_-PPP3CB=3×10⁸TU/ML,LV-control=1×10⁹TU/ML.The optimal MOI value of lentivirus-transfected Colo16 cells was 60,and the optimal puromycin concentration was 2ug/ml;the optimal MOI value of lentivirus-transfected A431 cells was 120,and the optimal puromycin concentration was 3ug/ml.2.The expression of PPP3CB mRNA in Colo16 and A431 cell lines was detected by qPCR,the negative control group had no significant difference compared with the blank group（P>0.05）,and the experimental group was significantly higher than the blank group（Colo16:F=275.4,P<0.05;A431:F=344.4,P<0.05）.Western Blot detection of PPP3CB protein expression showed that the negative control group had no significant difference compared with the blank group（P>0.05）,and the experimental group was significantly higher than the blank group（Colo16:F=49.9,P<0.05;A431:F=74.6,P<0.05）.3.The results of CCK8 showed that there was no significant difference in the value-added ability of the negative control group compared with the blank group,and the value-added ability of the experimental group was significantly weaker than that of the blank group（Colo16:F time=4021.9,F group=341.5,F time×group=83.7,P<0.05;A431:F time=1020.8,F group=149.2,F time×group=47.2,P<0.05）;scratch test results showed that there was no significant difference in the migration ability of cells in the negative control group compared with the blank group.Compared with the blank group,the cell migration ability of the group was significantly weakened（Colo16:F=257.4,P<0.05;A431:F=1412.6,P<0.05）;Transwell results showed that the invasion ability of the negative control group had no significant difference compared with the blank group,and the experimental group The invasive ability was significantly weakened compared with the blank group（Colo16:F=253.0,P<0.05;A431:F=1070.3,P<0.05）.Conclusion:The PPP3CB gene can inhibit the proliferation,migration and invasion ability of Colo16 and A431 cells and may play the role of oncogene,but its function and mechanism of oncogene suppression still need further research.