Relationship Between The Expression Of Aquaporin 9 And The Clinical-pathological Features In Hepatocellular Carcinoma Patients And The Underlying Mechanisms

Objective: The expression level of aquaporin-9(AQP9)was evaluated in patients with hepatocellular carcinoma,and the association between AQP9 and the clinical-pathological features/prognosis was investigated.Furthermore,the regulatory roles of AQP9 in the pathogenesis of hepatocellular carcinoma were assessed in this study.Methods: A total of 50 hepatocellular carcinoma and matched para-carcinoma samples were obtained from patients underwent surgery at our hospital.The levels of AQP9 in paired samples were examined.Additionally,the relationship between the expression of AQP9 and the clinical-pathological features/five-year survival rate was studied using immunohistochemistry analysis,western blotting and Real-time quantitative polymerase chain reaction,RT-qPCR(RT-qPCR).Huh-7 and SMMC-7721 cells were transduced with lentiviral vector overexpressing AQP9(LVAQP9)and the control(LV-NC).The cell proliferation was evaluated by cell counting kit-8(CCK-8)assay kit.In addition,the expression of proliferating cell nuclear antigen(PCNA)was determined using immunofluorescence staining.The migratory and invasive abilities of cells were investigated by Transwell assay.Cell apoptosis was evaluated using flow cytometry.Furthermore,RT-qPCR and western blot analysis were employed to access the expression levels of Wnt/?-catenin signaling-and Epithelial-mesenchymal transition(EMT)-related molecules.In vivo xenograft model was generated,and infected SMMC-7721 cells were injected into nude mice.Tumor volume and weight were monitored,and the levels of Wnt/?-catenin-and EMT-associated markers were examined.Results: The protein levels of AQP9 was significantly reduced in tumor samples compared with the non-tumor control(P<0.05).The downregulation of AQP9 mRNA was remarkably correlated with tumor size,the number of tumor,tumor lymph nodes metastasis(TNM)stage,lymphatic and distal metastasis(P<0.05),but not associated with patient age,gender,tumor grade,portal venous emboli and liver fibrosis(P>0.05).The five-year survival rate of the patients with low AQP9 expression(35.7%,10/28)was significantly decreased compared with the high AQP9 expression group(63.6%,14/22;P<0.05).The results also revealed that AQP9 was notably downregulated in hepatocellular carcinoma cells compared to normal hepatocytes L02(P<0.05).In addition,overexpression of AQP9 inhibited the proliferation,migration and invasion of Huh-7 and SMMC-7721 cells(P<0.05).The levels of PCNA,E-cad,N-cad,?-SMA,DVL2,GSK-3?,cyclinD1 and ?-catenin were downregulated by overexpressed AQP9,and cell apoptosis was significantly promoted in cells overexpressing AQP9(P<0.05).Furthermore,following the treatment with inhibitor of Wnt/?-catenin signaling(XAV939),the proliferative activity of Huh-7 cells were significantly suppressed;PCNA and EMT-related molecules were downregulated;migration and invasion of cells were remarkably inhibited;and cell apoptotic rate was reduced(P<0.05).Vice versa,after treating the cells with the inducer of Wnt/?-catenin signaling(SKL2001),the effects of overexpressed AQP9 were reversed(P>0.05).In vivo studies indicated that tumor volume and weight were remarkably decreased in AQP9 overexpression group,where the levels of Wnt/?-catenin signaling-and EMT-related molecules were also reduced(P<0.05).Conclusion: AQP9 was significantly downregulated in hepatocellular carcinoma tissues and cells,which was correlated with tumor size,number of tumor,TNM stage,five-year survival rata,lymphatic and distal metastasis of patients.Overexpressed AQP9 was able to inhibit the proliferation,migration and invasion of hepatocellular carcinoma cells via Wnt/?-catenin pathway.In summary,AQP9 could be a promising therapeutic target for the treatment of patients with hepatocellular carcinoma.