| Part 1 BackgroundHepatocellular Carcinoma(HCC)is the most common primary liver cancer and one of the major medical problems in the world.There were significant gender differences in the occurrence and development of HCC.The related research found that gender differences in HCC are closely related to sex hormones,inflammatory factors,etc.However,hormone therapy for HCC has not acquired perfect effects.Therefore,the mechanism of gender difference of HCC still needs to be further studied.Objective1.To explore the effect of estradiol on the proliferation of hepatocellular carcinoma cells.2.To explore the mechanism of estradiol inhibiting the proliferation of hepatocellular carcinoma cells.Methods1.CCK-8 assay were used to detect the proliferation of hepatocellular carcinoma cell treated with different concentrations of estradiol for 24 h or at the experimental concentration of estradiol for 0,6,12,24,48 h.Colony formation assay was used to detect the Colony forming ability of hepatocellular carcinoma cell treated with different concentrations of estradiol.2.miRNA mimics and inhibitor were transfected with Lipofectamine 2000 to up-regulate or down-regulate miR-125 b.3.After corresponding experimental treatment,Western Blot was used to detect the expression of related proteins,and Real-time fluorescent quantitative PCR was used to detect the expressions of miR-125 b and U6.Results1.SMMC-7721 and Hep-G2 cells were inhibited when the concentration of estradiol was 0.1?M or above,which was concentration dependent.SMMC-7721 and Hep-G2 cells were inhibited apparently at the concentration of estradiol(0.1?M)for 0,6,12,24,48 h,which was time-dependent.The expression of p-AKT was downregulated markedly after estradiol treatment,but no significant change of the expression of T-AKT.Furthermore,the expression of proliferation and apoptosis related proteins such as Bcl-2,Cyclin D1 decreased.After treatment with estradiol(0.1 ?M)and AKT inhibitor(MK2206 1 ?M),the proliferation of cells was significantly inhibited and p-AKT levels were significantly reduced.When estradiol(0.1 ?M)was added with activator(SC79 0.1 ?M),the inhibition disappearred and p-AKT level increased,which was not significantly different from normal group.2.It was found that estradiol up-regulated the expression of miR-125 b by Realtime fluorescent quantitative PCR.The proliferation of HCC cells was inhibited and AKT phosphorylation levels were reduced by transfection of miR-125 b mimics.If transfecting with miR-125 b inhibitor at the same time,the inhibition by estradiol was reversed.ConclusionsEstradiol inhibits AKT phosphorylation by up-regulating miR-125 b to inhibit proliferation of hepatocellular carcinoma cells.Part 2 BackgroundLiver transplantation(LT)is the best treatment for HCC with liver failure.However,recurrence and metastasis are important factors affecting the prognosis after LT.Rapamycin(mTOR inhibitor)is one of the commonly used immunosuppressive agents after LT for HCC,which plays both immunosuppressive and anticarcinogenic role.However,related studies found that mTOR targeted therapy did not achieve the expected results,because mTOR inhibitors such as rapamycin can negative feedback regulate the phosphorylation of AKT.Objective1.To explore the effect of rapamycin on the proliferation of hepatocellular carcinoma cells.2.To explore the mechanism of estradiol-enhanced rapamycin inhibiting the proliferation of hepatocellular carcinoma cells.Methods1.Colony formation assay was used to detect the Colony forming ability of hepatocellular carcinoma cell treated with rapamycin at different concentrations.CCK-8 assay were used to detect the proliferation of hepatocellular carcinoma cell treated with estradiol,rapamycin or both.Sphere-formation assays was used to observe the pellet-forming ability of hepatocellular carcinoma cell treated with estradiol,rapamycin or both.2.miRNA mimics and inhibitor were transfected with Lipofectamine 2000 to up-regulate or down-regulate miR-125 b.After corresponding experimental treatment,Western Blot was used to detect the expression of related proteins.Results1.Rapamycin inhibits the proliferation of SMMC-7721 and Hep-G2 cells in concentration-dependence.The proliferation of cells was inhibited more significantly by combined treatment of rapamycin and estradiol.Through sphere-formation assays,it was found that rapamycin or estradiol inhibited the pellet-forming ability of SMMC-7721 and Hep-G2,and the combination therapy group was more significant decrease than alone therapy group.2.The negative feedback AKT phosphorylation level was increased after rapamycin treatment.When rapamycin was combined with estradiol,it was found that estradiol could partially inhibit the increase of AKT phosphorylation.The miR-125 b mimics transfected with rapamycin also reduced the negative feedback AKT phosphorylation of rapamycin.ConclusionsEstradiol is synergistic with rapamycin by inhibiting AKT phosphorylation to inhibit proliferation of hepatocellular carcinoma cells. |
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