C-Myc Transactivates GP73 And Facilitates Metastasis Of Hepatocellular Carcinoma Cells Through GP73 Mediated MMP-7 Trafficking In A Mildly Hypoxic Microenvironment

Background / AimGolgi phosphoprotein 73(GOLM1/GP73)is highly expressed in hepatocellular carcinoma(HCC)cells and it serves as a clinical biomarker for diagnosis and indication of disease progression.Recently,some studies indicate that highly expressed GP73 might facilitate metastasis of HCC,but the mechanism remains poorly understood.This study aims to explain how GP73 is transactivated in HCC cells and how it facilitates metastasis of HCC in mild hypoxic microenvironment.Methods1.Confirm the correlation of c-Myc and GP73 in mild hypoxic condition using immunoblotting and wound healing assays.2.Prove the regulatory relations between c-Myc and GP73 using immunoblotting and immunohistochemical assays in vitro and in vivo.3.Prove whether c-Myc binds to the promoter of GOLM1 and transactivates GP73 using chromatin immunoprecipitation(Ch IP)assay and luciferase reporter assay.4.Investigate the regulatory relationships of the factors in c-Myc/GP73/MMP-7 axis using quantitative realtime PCR and immunoblotting assays.5.Confirm the correlation of GP73 and MMP-7 in pathological tissues derived from HCC patients using immunofluorescence staining analysis.6.Explain the mechanism how GP73 facilitates the vesicular trafficking and secretion of MMP-7 via molecular biological and protein trafficking-related assays.Results1.In mild hypoxic condition,c-Myc correlates positively with GP73 in Hep G2 cells.2.In vitro and in vivo assays demonstrate that c-Myc positively regulates GP73 expression.3.Ch IP and luciferase reporter analysis show that c-Myc transactivates GP73 through binding to the promoter of GOLM1.4.In vitro and in vivo assays demonstrate that both c-Myc and GP73 positivelyfacilitate metastasis of HCC cells and GP73 is the dominant factor.5.Trafficking-related assays demonstrate that inhibition of GP73 blocks the trafficking of intracellular MMP-7.6.Coimmunoprecipitation and live cell imaging assays indicate that GP73 interacts with intracellular MMP-7,and assists in the vesicular trafficking and secretion of MMP-7.Conclusion1.c-Myc correlates positively with GP73 in pathological tissues derived from HCC patients.2.c-Myc transactivates GP73 and promotes cell metastasis in mild hypoxic microenvironment.3.GP73 is the dominant factor facilitating metastasis of HCC cells compared with c-Myc.4.Inhibition of GP73 blocks the trafficking of intracellular MMP-7 and inhibit metastasis of HCC cells.5.GP73 interacts with MMP-7 and involves in MMP-7 trafficking.