The Role Of SGK3 In Hepatocarcinoma Cell BEL-7402 And It Leads To Enhance The Anti-apoptosis Ability Of BEL-7402 Growing In Steroid Hormone Stripped Fetal Bovine Serum

Object: To build vector pCDH-SGK3 expressing SGK3 sequence and to transfect it into hepatocellular carcinoma cell lines BEL-7402 and SMMC-7721 which stably expressing SGK3 protein, to know how SGK3 takes part in regulation in hepatocellular carcinoma cells signaling transduction and the effect of anti-apoptosis ability to survive cultured in steroid hormone free serum. Methods:(1)SGK3 gene fragment was amplified by PCR, and then linked to vector pCDH to construct lentivirus vector pCDH-SGK3, the vector and its empty vector pCDH-NC set as control were transfected into cell 293 T respectively with their packaging carriers.Lentivirus-SGK3 and Lentivirus-NC were produced. Hepatocellular carcinoma cells BEL-7402 and SMMC-7721 stably expressing SGK3 were made by infecting with produced lentiviruses and filtering with Puromycin.(2) the growth of cells expressing SGK3 with its control was tested by CCK-8, the expression level of SGK3 and its regulation of downstream GSK3β, S6 K was tested by Western Blot.(3) The growth of BEL-7402 cells overexpressing SGK3 and the change of C-PARP and pS6 K were tested respectively by CCK-8 and Western Blot in cells by when cells were cultured in the DMEM contained 10% FBS or 10% steroid hormone stripped serum, it showed that SGK3 enhanced the anti-apoptosis ability of hepatocellular carcinoma cell line BEL-7402 to growth in steroid hormone stripped serum via regulation of its downstream molecules. Results:(1) vector carrying SGK3 coding sequence was successfully constructed, BEL-7402 and SMMC-7721 cells that stably overexpressing SGK3 protein with empty vector were built.(2) Overexpressing SGK3 could enhance the growth of cell BEL-7402 tested by CCK-8 but the change of SMMC-7721 growth seemed no significant,the phosphorylation of GSK3β and the phosphorylation of pS6 K under different concentrations of FBS revealed how SGK3 effected the downstream signaling in hepatocellular carcinoma cells.(3) The expression ofandrogen receptor was detected by Western Blot and the growth of cell BEL-7402 were inhibited cultured in steroid hormone stripped FBS. Cells overexpressing SGK3 showed strong ability of anti-apoptosis cultured in the DMEM with 10% steroid hormone stripped FBS with S6 activated. Conclusion: Overexpression of SGK3 in hepatocellular carcinoma cell could enhance its growth but it does not fit for all hepatocarcinoma cells.(2) SGK3 could phosphorylate GSK3β and conditional phosphorylate S6 in low level of serum revealed SGK3 took party in PI3K/AKT signaling pathway, but may not be a predominate one.(3) SGK3 could enhance the ability of anti-apoptosis cultured in the DMEM with 10% steroid hormone stripped FBS via regulated of downstream molecules.