Aquaporin 9 Inhibit Hepatocellular Carcinoma By Down-regulating β-catenin In The Nucleus

Objective: To investigate the effects of aquaporin 9 on the proliferation and its probable mechanism in hepatocellular carcinoma.Methods: SMMC-7721 cells were transfected by lentiviral vector with luciferase and lentiviral vector targeting the coding region of human AQP9 respectively,and then we selected them by puromycin to obtain both stable cells with luciferase and cells with lentiviral vector targeting the coding region of human AQP9.Further,the expression of AQP9 in SMMC-7721 cell line was tested by Western blot at protein level.There were two groups.The one is the negative control group(NC group),which was the SMMC-7721 cell transfected by empty carrier.In contrast,the other one is overexpressed AQP9 group that contained the cells transfected by lentiviral vector targeting the coding region of human AQP9(AQP9 group).Flow cytometry was used to evaluated the effects on the cell cycle;expression of cell cycle regulators at mRNA levels were measured by RT-PCR in two both group;Western blotting techniques were adopted to test the expression of protein levels of cell cycle regulators,PCNA,pser675-β-catenin andβ-catenin.Simultaneously,expression of PCNA and β-catenin were detected by immune fluorescence staining.Finally,we use t-test to compare these two sets of data.Results: By the detection through Western blotting,it showed that,compared to the negative control group,the protein level in the overexpressed AQP9 group was much higher,where there were statistically significant differences(p<0.01).By means of the flow cytometry detection,we could learn about the cell cycle,which indicated that the overexpressed gene of AQP9 affected the distribution of cell cycle of SMMC-7721.To be more concrete,the proportion of SMMC-7721 cells in G0-G1 and G2-M periods increased from 52.24% ± 0.83% to 65.68% ± 0.63% and from 13.46% ± 0.2% to 17.4% ± 1.12%,respectively,and the proportion in S period dropped from 34.3% ± 0.65% to 16.56% ± 0.85%,where there were statistically significant differences(p<0.05).Using Western blotting to detect cell cycle regulators,PCNA and β-catenin respectively,contrasted with the negative control group,it manifested that the cell cycle regulators,which contained Cyclin D1,CDK2,CDK4,had a low expression level after the overexpression of AQP9,while P27 had a high expression level.Moreover,the expression of PCNA,intra-nuclear β-catenin and pser675-β-catenin all reduced remarkably.There were statistically significant differences(p<0.05).Similarly,the experimental results which obtained from immunofluorescence staining and density analysis showed that the overexpressed AQP9 group had a lower PCNA level than negative control group,it has statistically significant differences(p<0.05).Also,overexpression of AQP9 affected the distribution of β-catenin in SMMC-7721 cells.Conclusion: Over-expression AQP9 gene can significantly inhibit SMMC-7721 cell proliferation.According to the experimental results,the mechanism is quite possibly that the gene of AQP9 may suppress the expression of β-catenin in the nucleus,which inhibit the expression of Cyclin D1.All these led to G1/S phase cell cycle arrest.