The Role Of Cks1and Cks2on HepG2in Cell Proliferation And Apoptosis

Objective：We observe the proliferative and apoptotic ability of humanhepatocellular carcinoma cell line HepG2by transfection technology toexplore the possible mechanisms that cell cycle regulator Cks1and Cks2gene was involved in the tumorigenicity. To provide experimental supportand theoretical basis for the clinical of hepatocellular carcinoma.Methods：1、Two pieces of siRNA targeting the Cks1or Cks2mRNA was designed andsynthesized chemically to be tansfected into HepG2cells.2、Cks1or Cks2overexpression vector was transfected into HepG2byliposome. Succeeding esblishing transfected cell lines.3、Cks1and Cks2mRNA and protein expression were detected by real-timePCR and western blot,respectively.4、In the CCK-8test and cell counting,the proliferation of experimentalgroup and control group were compared.5、Cell apoptosis and cell cycle were analyzed by flow cytometer.6、The protein levels of Akt、GSK-3β、Bcl-2were detected to observe theaffection of Cks1and Cks2specific RNA interference.Results:1、The effective downregulations of Cks1and Cks2were identified from24h to96h after the siRNA transfections.We succeeded in establishing Cks1and Cks2gene overexpression humanhepatocellular carcinoma cell lines using the stable transfectiontechnology.2、CCK-8test and cell counting assay showed that the proliferation intransfected groups were changed significantly compared withnon-transfected groups(p<0.01). Cks1and Cks2protein levels and themultiplication rate had positive correlation. 3、Flow cytometer assay showed that apoptosis rate of Cks knockdown groupwas increased observably (p<0.01), either in the early or late stage.Inthe overexpression group,the variation trend was on the contrary.4、Neither the knockdown nor overexpression groups performed significantchanges.5、Phosphorylated Akt and phosphorylated GSK-3βwere down-regulated afterRNA interferences,but Bcl-2didn’t show any visible changes in thisexperiment.Conclusion:All these features together suggest that Cks1and Cks2may promote theprogress of hepatocellular carcinoma,these two genes might be thecandidate tumor promotor.We discovered firstly that inactivating GSK-3β via Akt plays an importantrole in Cks1,2expressions in HCC. Our findings suggest that theAkt/GSK-3β pathway is associated with the proliferation, apoptosis,which may be related to Cks1and Cks2expressions.