Mechanism Study Of SCLU Mediating Apoptosis Resistance In Hepatocellular Carcinoma Under Tunicamycin-induced

Objective:Hepatocellular carcinoma(HCC)is one of the most common cancer in the world which is fifth common.More than half of new cases and death cases occur in China every year.Clusterin gene is a single copy gene,and the functional protein sCLU plays an anti-apoptotic role in tumor by regulating multiple pathways.Through analyzing clinical data,it was concluded that sCLU protein was highly expressed in HCC,and the expression of sCLU significantly affected the survival rate of patients(p=0.035).The purpose of this study is to explore the role of sCLU protein in the occurrence and progression of HCC and its related molecular mechanism by combining bioinformatics investigation of clinical cases and cellular mechanism studies.By exploring the protective mechanism of HCC under various stress states,we can improve the sensitivity of HCC patients to targeted drugs or chemotherapeutic drugs,so as to prolong the long-term survival rate of patients.Methods:1.Bioinformatics technology was used to predict the relationship between sCLU and HCC.By using ONCOMINE、TCGA、GEPIA and other databases to analyze clinical samples,,we can predict the expression of sCLU protein in HCC tissues,and explore the impact of sCLU protein expression on the survival rate of HCC patients.2.Verify the expression of sCLU protein in HCC cell.The common HCC cell lines Huh-7,SK-Hep-1 and Hep3B were cultured.The high expression of sCLU in HCC cells was verified by Western blot and PCR.3.Construct sCLU overexpression and sCLU RNAi.sCLU overexpression and sCLU RNAi model were designed and transfected into hepatoma cells.The expression efficiency of sCLU regulation model was tested by fluorescence microscope and Western blot.4.Explore the relationship between sCLU and downstream proteins.It is speculated that sCLU can regulate apoptosis resistance through endoplasmic reticulum(ER)stress key protein GRP78 under the condition of tunicamycin(TN)induction.GRP78 and AKT may be connected through TRIB3.By constructing sCLU overexpression and sCLU RNAi model and further using Western Blot and other methods,the expression changes of each pathway protein and the relationship between sCLU and downstream proteins were explored.5.Explore the relationship between GRP78 and downstream proteins.In order to further explore whether GRP78 is directly related to TRIB3,GRP78-RNAi was constructed,and the effect of changing GRP78 on upstream and downstream proteins was explored through Western blot and other experiments.6.Explore the effect of sCLU on apoptosis resistance of HCC.In order to verify that sCLU plays an anti-apoptotic role in the progression of HCC,flow cytometry was used to detect whether the difference in the expression of sCLU had an effect on the apoptosis of HCC.Secondly,by down-regulating GRP78 and TRIB3 respectively,and detecting apoptosis by flow cytometry,we explored whether the regulation of sCLU on downstream proteins was consistent with the effect of downstream proteins on apoptosis resistance,and then speculated the establishment of sCLU-GRP78-TRIB3 pathway.Results:1.The expression of CLU in HCC was higher than that in normal tissues,and the expression of CLU in cancer tissues was higher than that in adjacent tissues,and CLU was highly expressed in all pathological stages of HCC.Survival analysis showed that patients with low expression of CLU had a poor prognosis compared with patients with high expression of CLU(p=0.035).2.Among the selected HCC cell lines,Huh-7,SK-Hep-1 and Hep3B all had high expression of sCLU protein.TN can cause the up-regulation of key ER proteins GRP78 and CHOP.Selecting cells treated with 50 μM tunicamycin MTT assay,We found that the expression levels of sCLU,GRP78 and TRIB3 were significantly increased under the induction condition of TN.3.sCLU-RNAi and GRP78-RNAi were transfected into Huh7 cell line,and treated with 50 μM tunicamycin for 48 hours respectively.The expressions of sCLU,GRP78 and TRIB3 were detected by Western blot.In the sCLU-RNAi group,the expressions of sCLU,GRP78 and TRIB3 were significantly increased after inducted by TN.The expressions of sCLU and GRP78 in the sCLU-RNAi+TN group after induction were lower than those in the NC+TN group,while the expression of TRIB3 was up-regulated compared with the NC+TN group,but the increase was not obvious.In the GRP78-RNAi group,the expression of GRP78 in the GRP78-RNAi+TN group was decreased compared with that in the NC+TN group,but the expression of TRIB3 was not significantly up-regulated compared with the NC+TN group,and there was no significant change in sCLU.4.The apoptosis test by flow cytometry showed that the down-regulation of the expression of sCLU and GRP78 could increase the apoptosis rate of cells.Under the condition of TN induction,the apoptosis rates of sCLU-RNAi+TN and GRP78-RNAi+TN groups were significantly higher than that of NC+TN group.While the down-regulation of TRIB3 expression could reduce cell apoptosis under the induction of TN.On the basis of down-regulation of sCLU expression,down-regulation of TRIB3 expression could reduce the apoptotic response caused by the decrease of sCLU.Conclusion:1.In this study,the high expression of sCLU protein in hepatocellular carcinoma was predicted by biogenesis analysis.And its expression was verified by molecular biology techniques in a variety of HCC cell lines.2.In HCC,down-regulation of sCLU protein expression can affect the protein expression in the downstream apoptosis pathway,including down-regulation of GRP78 and up-regulation of TRIB3 expression.3.Under the induction of TN,sCLU may mediates the apoptosis resistance of HCC through GRP78-TRIB3 and plays a cytoprotective role.