Study On The Mechanism Of AZT Coordination With As₂O₃ Against Hepatocellular Carcinoma By Targeting Wnt/β-catenin Pathway And DNMTs

Objective:To clarify whether the regulation process of 3’-azide-3’-deoxythymine（AZT）and Arsenic trioxide（As₂O₃）on proliferation and apoptosis of hepatocellular carcinoma affects Wnt/β-catenin signaling pathway by targeting DNA methyltransferase（DNMT）,which provides the theoretical basis for clinical treatment of hepatocellular carcinoma to find new demethylation drugs.Methods:（1）Using The Cancer Genome Atlas（TCGA）,Gene Expression Profiling Interactive Analysis（GEPIA）and PCViz datebase to analyze the expression and survival of DNMT3b in hepatocellular carcinoma.The methylation level of SFRP2 promoter region was analyzed using UALCAN database.（2）Using CCK8 to detected the cell proliferation rates of Hep G2 and MHCC97-H cells after 24h,48h and 72h respectively by using As₂O₃（2μmol/L）,AZT（20μmol/L）or two-drug Intervention,separately.（3）Conventional methods for culturing human hepatoma Hep G2 and MHCC97-H cells is used to treat the cells with As₂O₃（2μmol/L）,AZT（20μmol/L）alone or in combination in 72h,quantitative reverse transcription PCR（RT-q PCR）was used to detect DNMT3b and secreted frizzled-related proteins,SFRP2)m RNA expression.The protein expressions ofβ-catenin,DNMT3b and SFRP2 were detected by Western blotting.（4）Using Methylation specificity PCR technique to detect changes of the methylation levels of Cp G island in the SFRP2 promoter region in As2O3（2μmol/L）,AZT（20μmol/L）or AZT coordination with As₂O₃,to investigate the DNA methylation changes of AZT coordination with As₂O₃ in treatment of HCC.（5）After transfecting DNMT3b with overexpression vector,hepatocellular carcinoma cells are jointly treated with As₂O₃ and AZT.The expression ofβ-catenin and SFRP2 was assayed by RT-q PCR and Western blotting assay to analyze whether AZT synergistically with As₂O₃affects Wnt/β-catenin pathway against hepatocellular carcinoma through targeting DNMT3b.Result:（1）Biogenic analysis showed that the expression of DNMT3b was up-regulated in hepatocellular carcinoma.In addition,the methylation degree of SFRP2 promoter region was significantly increased in HCC tissues according to UALCAN data（P<0.05）.（2）CCK8 assay showed that at the same time point,the four groups of drug interventions could inhibit the proliferation of hepatocellular carcinoma cells to different degrees in the combination group,but only at 72h（P<0.0001）.In combination with As₂O₃（2μmol/L）and AZT（20μmol/L）,the proliferation rate of hepatocellular carcinoma cells decreased most significantly at 72h with the extension of the intervention time,and the discrepancy was found to be statistically significant（P<0.0001）.（3）The results of MSP assay showed that SFRP2 demethylation occurred significantly in Hep G2 and MHCC97-H cells after the combined drug intervention,suggesting that AZT combined with As₂O₃ can reduce the methylation level of SFRP2 promoter in liver cancer cells.（4）The expression levels of DNMT3b m RNA and protein after As₂O₃（2μmol/L）and AZT（20μmol/L）drug treatment were significantly lower（P<0.01）,SFRP2 m RNA and protein were significantly higher（P<0.01）andβ-catenin protein level was significantly lower（P<0.001）in the blank group by RT-q PCR and Wb.was significantly lower than that of the blank group（P<0.001）.（5）After transfection with DNMT3b,the SFRP2 gene and protein expression levels were obviously decreased（P<0.001）andβ-catenin protein levels were dramatically increased（P<0.001）in the mimic-DNMT3b group compared with the NC group after the drug intervention.Conclusion:（1）AZT coordination with As₂O₃ can affect the changes of DNA methylation level of SFRP2.（2）AZT coordination with As₂O₃ can inhibits hepatocellular carcinoma cell proliferation possibly by targeting DNMT3b to regulate the Wnt/β-catenin signaling pathway.