GOLPH3 Gene Regulates Hepatocellular Carcinoma Through The MTOR Signal Pathway And Its Relationship With TCM Syndrome

ObjectiveTo investigate the function and mechanism of Golgi phosphorprotein3(GOLPH3)in hepatocellular carcinoma(HCC),and to explore the relationship between GOLPH3 and HCC pathological features and TCM syndromes,in order to provide new therapeutic strategies for HCC patients.Methods1.Immunohistochemistry,western blotting and real-time quantitative PCR were used to detect the expression of GOLPH3 mRNA and protein in HCC clinical samples and HCC cell lines MHCC97 L and MHCC97 H.And Chi-square test was used to analyze the correlation between the protein expression level of GOLPH3 and clinicopathological features of HCC.2.Using SiRNA silencing HCC cell lines MHCC97 L,MHCC97H GOLPH3 gene to study the function of GOLPH3.Cells proliferation were analyzed by MTT;cells apoptosis were analyzed by Annexin-V-FITC /PI staining and hoechst 33342 staining,and cells caspase 3/7 activity were detected in MHCC97 L and MHCC97 H cells transfected with GOLPH3 siRNA.3.The lentiviral transfection method was used to construct and screen GOLPH3 stable low-expressing MHCC97 L and MHCC97 H cells,and to establish GOLPH3 stable and low expression MHCC97 L and MHCC97 H subcutaneous tumor models to study the role of GOLPH3 in tumor growth in vivo.4.Western blotting and immunohistochemistry were used to investigate the function of GOLHP3 in the mTOR signaling pathway.5.MHY1485,an activator of the mTOR signaling pathway,was used to verify the role of GOLHP3 in the mTOR signaling pathway.6.Fisher's exact test was used to analyze the correlation between HCC TCM syndromes and GOLPH3 expression and clinicopathological features.Results1.The expression of GOLPH3 mRNA and protein in HCC clinical samples and HCC cell lines MHCC97 L and MHCC97 H were significantly upregulated compared with the normal control group(P <0.05).Chi-square test results showed that GOLPH3 protein expression was significantly associated with serum AFP levels in HCC patients(P = 0.006);GOLPH3 expression levels were not significantly associated with gender,age,HBsAg,tumor size,vascular invasion,and cirrhosis(P >0.05).2.SiRNA silencing of GOLPH3 expression in MHCC97 L and MHCC97 H inhibited cell proliferation and promoted apoptosis.3.The knockdown of GOLPH3 by lentivirus resulted in the limited growth of MHCC97 L and MHCC97 H subcutaneous xenografts.4.The phosphorylation levels of mTOR,AKT,and S6K1(p70 ribosomal protein S6 kinase),the key proteins involved in the mTOR signaling pathway in HCC clinical samples,were significantly higher than those in normal liver tissues(P <0.05).In vitro and in vivo experiments demonstrated that the phosphorylation levels of mTOR,AKT,and S6K1 were significantly reduced after silencing GOLPH3 in MHCC97 L and MHCC97 H cells compared with the normal control group(P <0.05).5.The mTOR activator MHY1485 partially abolished the apoptosis of MHCC97 L and MHCC97 H cells caused by GOLPH3 silencing.6.Fisher's exact test analysis showed that there was no significant correlation between HCC TCM syndrome type and GOLPH3 protein expression level(P >0.05),and there was no obvious correlation with HCC clinical pathological features(P <0.05).ConclusionThe current study suggests that GOLPH3 contributes to the development of HCC by activating mTOR signaling pathway and is a promising diagnose biomarker and therapeutic target for HCC.This might provide a scientific basis for developing effective approaches to treat the HCC patients with GOLPH3 overexpression.