Influence Of Overexpression Of SEPT9Gene On Proliferation And Invasion And Metastasis Of Human Hepatoma HepG2Cell

Objectives: By building SEPT9gene eukaryoticexpression vector to transfect human hepatoma cell line HepG2with inertmetastatic potential,explore the influence of overexpression of SEPT9gene on proliferation,transference,invasion and metastasis of hepatomacell lines HepG2, and investigate the role of SEPT9in the occurrence anddevelopment of hepatoma. It will provide experimental basis for theSEPT9to become a new target for hepatoma gene therapy and a possiblemarker of hepatic carcinoma metastasis. Methods: HepG2cells wereroutinely cultured and plated in6-well cell culture plates till The growthstate of the cells is in good condition and achieve exponential growthphase,and use constructed eukaryotic expression vector pIRES2/EGFP-SEPT9to transfect the HepG2cells after24hours. Set up threegroups:the overexpressing group(transfected vectors pIRES2/EGFP-SEPT9)、the empty plasmid group (transfection vectors pIRES2/EGFP)and the blank group (normally cultured without any specialreagents). Match transfection complexes with liposomes LipofectamineTM200010μl and DNA4μg per well,add them to each well HepG2cells fortransfection.①After48h,according to the ratio of green fluorescence cellsunder the fluorescence microscope,transfection efficiency could be judged.②After72h,use western blot method to detect the expression ofSEPT9protein: to extract total cellular protein with cell lysates,and theconcentration of protein were detected by BCA assay,and use GAPDHprotein as the control to determine the interference efficiency bySDS-PAGE,and use semi-dry assay to transfer the total protein to themembrane,close with skim milk,primary and second antibodyincubation hybridization,the SEPT9antibody, chemiluminescencesolution development and auto exposure with UV gel imaging analysissystem The results were analyzed by Image J2X software,detectexpression of SEPT9protein in each group relative to the internalreference protein GAPDH.③Use0h,24h,48h,72h as time points,CCK-8assay to detect influence of SEPT9gene overexpression on cellgrowth and proliferation.④Cell scratch test: to detect the migrationathletic ability of HepG2cells in0h,24h,48h,72h.⑤Transwellexperiment: to detect the ability change of invasion and metastasis inHepG2cell in each group.Results:①Observe the HepG2cells underfluorescence microscope48h after transfection, the transfection efficiencyarrive to70%according to the ratio of SEPT9cells with a greenfluorescent.②The results of analysis of the western blot showed that thebrightness of SEPT9protein bands in the overexpressing group wassignificantly stronger than that in the negative control group and theblank group,and it also showed that the relative expression of SEPT9 protein in the blank group、 the empty plasmid group and theoverexpressing group were0.7078±0.018、0.7136±0.012、1.0526±0.020.The data were analyzed by Paired samplest test andanalysis of variance,and the relative expression of SEPT9protein in theoverexpressing group was48%higher than the other two groups,thedifference had statistical significance(P<0.05).③The results of CCK-8assay showed that compare to the other two groups, HepG2cells in theoverexpressing group had faster proliferation rate in24~72h aftertransfection, the difference had statistical significance(P<0.05),and theproliferation rate in48h reached21.742%,72h reached30.126%aftertransfection.④Cell scratch experiment showed that compared with theother twogroups,the scratch width of the overexpressing group wassignificantly narrowed in the period of24~72h and appeal obvioustendency of heal. After the measurement and calculation of the migrationdistance of each period analyzed: cell migration in the overexpressinggroup had statistical significance(P<0.05).⑤Cell Transwell experiment:Both the Invasion assay and migration assay results showed the numberof cell wells in the overexpressing group were significantly higher thanother two groups(P<0.05). Conclusion:①T heconstructed eukaryoticexpression vector pIRES2/EGFP-SEPT9of SEPT9gene may increasethe expression of SEPT9protein in inert metastatic potential hepaticcarcinoma HepG2cells.②The overexpressing SEPT9gene can significantly enhance proliferation of HepG2cells.③The overexpressingSEPT9gene can significantly enhance the ability of migration,invasionand metastasis in HepG2cells.