Explore The Mechanism Of ZYX Protein Promoting Hepatocellular Carcinoma Progression Based On AKT/mTOR Signaling Pathway

Objective: To investigate the expression of ZYX in HCC and its regulatory effect on the course of HCC.The effects of abnormal expression of actin interaction protein(ZYX)on proliferation,migration,invasion and apoptosis of Hepatocellular carcinoma(HCC)cells were investigated by cytological experiments.To investigate the activation of AKT/ m TOR pathway by ZYX and its mechanism of mediating HCC progression through molecular biology.At the zoological level,the effect of ZYX abnormal expression on solid HCC tumors was verified through subcutaneous tumor formation model of nude mice,which expanded the new mechanism of HCC progression,made ZYX potentially become a new target for HCC treatment.Methods: 1.The expression of ZYX in HCC patients,human HCC tissues and human HCC cell lines were detected by bioinformatics method and Western blot(WB).2.Plasmid construction and lentiviral transfection techniques were used to construct stable over-expression of ZYX human HCC cell lines(PRF/PLC/5 and HCCLM3)and stable low-expression of ZYX human HCC cell lines(SK HEP-1 and Huh-7)as tools to detect the effects of abnormal ZYX expression on various aspects of human HCC.3.The effects of ZYX abnormal expression on the proliferation and colony formation of human HCC cells were detected by CCK-8 assay,Ed U staining and clonal formation assay.4.The effect of ZYX abnormal expression on the migration and invasion ability of human HCC cells was observed by cell scratch assay and transwell assay.5.The effect of ZYX abnormal expression on apoptosis of human HCC cells was observed by flow cytometry.6.The effect of ZYX abnormal expression on subcutaneous tumor formation in nude mice was analyzed in tumor xenotransplantation model.7.Western blot(WB)was used to detect the abnormal expression of ZYX on proteins related to proliferation,Epithelial mesenchymal transition(EMT)and AKT/m TOR signaling pathway in cells and tumor tissues.8.H&E and immunohistochemical staining(IHC)were used to observe the effect of ZYX on the morphological changes of tumor tissues and the expression of related proteins in tissues.9.Human HCC cells with abnormal ZYX expression were treated with an AKT specific activator(SC79)and an AKT specific inhibitor(MK2206)to detect the expression of AKT,p-AKT,m TOR and p-m TOR,and to re-examine the phenotypes of cell proliferation,migration and invasion and the changes of related proteins.Results: 1.It was found that ZYX was highly expressed in HCC patients through the bioinformatics database,and the expression of ZYX in human HCC tissues was higher than that in control normal tissues by Western blot(WB).2.CCK-8 assay,Ed U staining and clonal formation assay showed that high ZYX expression could promote the proliferation and cloning of human HCC cells,and low ZYX expression inhibited the proliferation and cloning of human HCC cells.WB results showed that the expression of cell proliferation-related proteins(PCNA,Cyclin D1,CDK2,CDK4)increased with the increase of ZYX expression,and decreased with the decrease of ZYX expression.3.Through the cell scratch and transwell assay,it was observed that high ZYX expression promoted human HCC cell migration and invasion,while low ZYX expression inhibited human HCC cell migration and invasion.The expression of EMT-related proteins(E-cadherin,Vimentin,Snail,MMP-2,MMP-9)also changed with the change of ZYX expression.4.Flow cytometry further confirmed the inhibitory effect of high expression of ZYX on the apoptosis of human HCC cells,and low expression of ZYX promoted the apoptosis of human HCC cells,and the expressions of BAX and Bcl-2 showed the same trend.5.WB results showed that ZYX over-expression promoted the phosphorylation of AKT/m TOR signaling pathway protein,and ZYX interference decreased the phosphorylation of AKT/m TOR signaling pathway protein,while the background expressions of AKT and m TOR had no significant changes.6.ZYX over-expression promoted the growth of HCC tumor,and the growth of HCC tumor in the low ZYX expression group was significantly slower than that in the control group.Immunohistochemistry confirmed that the expression of Ki-67 and MMP-2 in the tumor increased with the increase of ZYX expression,and decreased with the decrease of ZYX expression.The expression of tumor protein also showed the same trend.7.The AKT specific agonist SC79 restored the proliferation,migration and invasion ability of human HCC cells which had been weakened due to low ZYX expression.The phosphorylation levels of AKT and m TOR proteins were also restored,and the expressions of cell proliferation,migration and invasion related proteins were increased compared with those in the low ZYX expression group.The AKT specific inhibitor MK2206 inhibited cell proliferation,migration,and invasion enhanced by ZYX over-expression,as well as the phosphorylation level of AKT/m TOR signaling pathway proteins.The expression of proteins associated with cell proliferation,migration,and invasion,which were increased by ZYX overexpression,was restored under MK2206.Conclusion: 1.ZYX is highly expressed in human HCC,which can promote the proliferation,migration and invasion of HCC cells and inhibit cell apoptosis,thus promoting HCC tumor growth and disease progression.2.ZYX plays a role in disease progression of HCC by promoting phosphorylation and activation of AKT/m TOR signaling pathway and regulating expression of downstream related proteins.3.ZYX may be an effective therapeutic target for HCC and provide a new idea for clinical treatment of HCC.