Effects Of LATS1 Gene Demethylation On Hippo-YAP Signaling Pathway In Hepatocellular Carcinoma

Objective: To investigate the effects of LATS1 gene demethylation on proliferation?cell cycle regulation?nude mice xenograft and Hippo signaling pathway in human hepatocellular carcinoma cells.Methods: The protein expression level of large tumor suppressor gene 1(LATS1)and downstream cancer gene Yes-associated protein(YAP)were detected by Western Blot in human hepatocellular carcinoma cells line Hep G2 and SMMC-7721,the methylation of LATS1 in Hep G2 and SMMC-7721 were analyzed by methylation-specific PCR(MSP)and bisulfite sequencing PCR(BSP).After by using 5-Aza-2'-deoxycytidine(DAC)disposed Hep G2 and SMMC-7721,the methylation of LATS1 was detected by MSP and the protein expression level of LATS1 and YAP were detected by Western Blot in each group.The apoptosis and cycle of cells were detected by flow cytometry.The ability of tumorigenicity was evaluated in nude mice model.Result: MSP showed that LATS1 highly methylation in Hep G2 and SMMC-7721.BSP quantitative analysis showed that the degree of LATS1 methylation in Hep G2 cells(the proportion of methylated CPG island sites in all CPG island sites)was 92.4%,and the degree of LATS1 methylation in SMMC-7721 cells was 93.1%;the promoter region of LATS1 gene was highly methylated.WB showed low expression of LTAS1 and high expression of YAP(P<0.05).After Hep G2 and SMMC-7721 disposed by DAC,LATS1 was lowly methylated and the protein expression level of LATS1 was significantly increased(P<0.05),while the protein expression level of YAP was significantly decreased(P<0.05).Flow cytometry showed that the cell cycle was arrested in G1 stage(P<0.05),and the cell apoptosis rate was increased significantly(P<0.05).The size and volume of subcutaneous tumor in nude mice were significantly decreased(P<0.05).Conclusion: The LATS1 gene is highly methylated in human hepatocellular carcinoma cells.Demethylation can increase the protein expression level of LATS1 and decrease the protein expression level of YAP,reduce the proliferation ability of Hep G2 and SMMC-7721 cells and induce their apoptosis,inhibit the ability of hepatocellular carcinoma cells turning into a tumor.