Study On The Mechanism Of MG132-induced Apotosis And Autophagy In Hepatocellular Carcinorna Cells

BACKGROUNDHepatocellular carcinoma(HCC)is a malignant digestive system tumor,that has no clear clinical pathological definition,and is a serious threat to human health[1-3].The recurrence rate and mortality rate of HCC are high.Less than 15 % of patients can live 5 years,while it was 10.1 % in China [4].In HCC patients,the men are more than the women.There are several treatments for HCC.At present,the most effective treatment of hepatocellular carcinoma are surgical resection and the use of chemotherapy drugs such as Sorafenib [5].But,in the course of surgical resection treatment,patients suffer great physical harm,and the general chemotherapy drugs are helpful to some patients,it still cannot save advanced hepatocellular carcinoma patients.Therefore,it is necessary to find better and more effective drugs for the treatment of HCC.HCC cells have the characteristics of immortal proliferation,and proteasome inhibitors(PIs)can inhibit the infinite proliferation of tumor cells by inhibiting the cycle of tumor cells.In particular,PIs can select high-grade malignant cells to exert their toxic effects first.So choosing PIs to treat HCC is a good way.MG132 is one of PIs,and is a kind of natural PIs.It has been proved that MG132 can exert toxic effects on HCC cells [6].The toxic effects of MG132 on HCC cells involve multiple pathways,such as p53,MAPK and AKT etc.,as well as various genes such as TRAIL [7-9].However,the more comprehensive regulation mechanism of MG132 in the treatment of HCC cells is not very clear.In order to treat HCC earlier and better,it is necessary to conduct more in-depth and more comprehensive discussions on the regulatory mechanisms of MG132 in the treatment of HCC.SIRT2 is deacetylase,and it can interact with some E3 ubiquitin ligases.SIRT2 is involved in cell mitosis,and it can affect cell proliferation through the cell cycle.In addition,SIRT2 is highly expressed in HCC tissues and cells,and is positively correlated with the degree of malignancy of HCC.There is evidence that lentivirus-mediated silencing of SIRT2 can inhibit the proliferation of HCC cells[10],indicating that down-regulation of SIRT2 will contribute to the treatment of HCC.However,whether MG132 will cause down-regulation of SIRT2 in the treatment of HCC has not been studied.Therefore,inorder to understand the role of SIRT2 in the treatment of HCC clearly,it is necessary to develop the study of MG132 and SIRT2 in SMMC-7721.PURPOSEOur purpose is to investigate the mechanismof proteasome inhibitor MG132 on the proliferation,apoptosis and autophagy in hepatocellular carcinoma cell line SMMC-7721,preliminary discussion the role of SIRT2 in the treatment of hepatocellular carcinoma by MG132.And through the study of the function of MG132 in SMMC-7721,it provides more theoretical basis for the treatment of hepatocellular carcinoma and will contribute to the development of new drugs for the treatment of hepatocellular carcinoma eventually.METHOD1.Hepatocellular carcinoma cell line SMMC-7721 was selected as the research object,SMMC-7721 cells were treated with MG132 at 2.5 μM,5 μM,10 μM,20 μM,40 μM and 80 μM for 24 h,these cells viability were assayed by MTT.2.SMMC-7721 cells were treated with MG132 at 20 μM for 24 h respectively.hoechst33258 staining was used to assay the apoptotic bodies;western blot was used to assay the expression of cleaved-caspase3;MDC staining was used to assay small autophagy body;western blot was used to assay the changes of LC3 I and LC3 II.3.HCC cells were treated with 20 μM MG132 for 6 h,12 h and 24 h,western blot was used to assay the changes of SIRT2,LC3 I,LC3II,ATG7,STAT3 and P-STAT3.4.SIRT2 overexpression SMMC-7721 cell line was constructed.5.Normal cells,cells transfected with PCDNA3.1-HA plasmid and with SIRT2-PCDNA3.1-HA plasmid were treated with 20 μM MG132 for 6 h,12 h,18 h and 24 h,the inhibition rates were assayed by MTT.6.Cells transfected with PCDNA3.1-HA plasmid and with SIRT2-PCDNA3.1-HA plasmid were treated with 20 μM MG132 for 24 h,the changes of LC3 I and LC3 II were assayed by western blot;the changes of autophagy were assayed by MDC staining;the apoptotic rate was assayed by hoechst33258 and flow cytometry.RESULT1.The results of MTT showed that the proliferation of SMMC-7721 was inhibited significantly with the increase concentration of MG132.2.The results of MDC staining、hoechst33258 staining and western blot showed that apoptosis and autophagy were observed when treated with MG132 for 24 h.3.Normal cells were treated with 20 μM MG132 for 12 h and 24 h,SIRT2 was down-regulation;the autophagy-related protein ATG7 was up-regulation;protein LC3 II was up-regulation;and P-STAT3 was down-regulation.4.Cells were treated with 20 μM MG132 for 6 h,12 h,18 h and 24 h,the inhibition rat of normal cells and cells transfected with PCDNA3.1-HA plasmid were higher than cells transfected with SIRT2-PCDNA3.1-HA plasmid.5.Cells were treated with 20 μM MG132 for 24 h,the reasults of western blot,MDC staining,flow cytometry and hoechst33258 staining showed that the apoptosis degree and autophagy degree of cells transfected with PCDNA3.1-HA plasmid were more obvious than cells transfected with SIRT2-PCDNA3.1-HA plasmid.CONCLUSION1.MG132 can inhibit effectively the proliferation of SMMC-7721 in a concentration-dependent manner,MG132 can induce apoptosis and autophagy in SMMC-7721.2.MG132 inhibits SMMC-7721 proliferation,induce apoptosis and autophagy in SMMC-7721 by redecining SIRT2.