Mechanism Of Decitabine Regulating Sorafenib Resistance In Hepatocellular Carcinoma Cells Through Demethylation

ObjectiveTo investigate the mechanism of drug resistance in the treatment of hepatocellular carcinoma with Sorafenib from an epigenetic perspective.To examine the effect of the sensitivity effect of Sorafenib on hepatocellular carcinoma after the combination of epigenetic drug decitabine,and to provide new ideas and methods for the clinical treatment of hepatocellular carcinoma.Methods1.Using the GEPIA 2 database to retrieve clinical information of 508 patients with primary hepatocellular carcinoma,the Kaplan-Meier method was used to analyze the expression of organic anion transporting polypeptide 1B3 in different cancers and their paracancerous tissues.polypeptide 1B3 expression differences in different cancers and their paraneoplastic tissues.To analyze the correlation between OATP1B3 expression in HCC and patients’survival time.2.Detection of OATP1B3 mRNA and protein expression levels in Hep3B,HepG2,SNU182,SNU387,HUH7 and LM3 cells using RT-qPCR,Western Blot.3.Detection of OATP1B3 promoter methylation levels in Hep3B,HepG2,SNU182 and SNU387 cells using bisulfite methylation.4.Detection of the effect of DNA methyltransferase inhibitor DAC on OATP1B3expression after the action of Hep3B and HepG2 using RT-qPCR,Western Blot.5.Dynamic monitoring of the effect of Sorafenib in combination with DAC on the proliferation of Hep3B and HepG2 cells using RTCA-e Sight.6.To detect changes in Sorafenib uptake by Hep3B and HepG2 cells after Sorafenib was combined with DAC（hereinafter:combination group）using LC-MS/MS.7.Detection of changes in Hep3B,HepG2 survival and changes in IC₅₀ of Sorafenib after DAC co-administration compared to Sorafenib alone in the DAC co-administration group using MTS.8.Detection of lipid peroxide levels in each group using flow cytometry.9.A human hepatoma cell line Hep3B hepatocellular carcinoma model was prepared using SPF grade BALB/c nude mice,and when the tumors grew to about 100 mm³,the tumor-bearing mice were randomly divided into Control,DAC,Sorafenib,and DAC+Sorafenib groups.DAC（2.5mg/kg,intraperitoneal injection）,Sora（20mg/kg,gavage administration）or PBS were given respectively,and the size of the tumor and the weight of the nude mice were measured regularly after administration.At the end of the experiment,the animals were executed and tissue samples were collected for RT-qPCR,Western Blot and immunohistochemistry.Results1.The results of GEPIA2 database analysis showed that the expression of OATP1B3 in hepatocellular carcinoma and its paraneoplastic tissues differed significantly.The overall survival rate of HCC patients with high OATP1B3 expression was significantly higher than that of those with low expression.2.RT-qPCR,Western Blot results all showed that Hep3B,HepG2,SNU182 and SNU387 with relatively low expression of OATP1B3 in these four cells,which were selected for methylation rate determination.3.Bisulfite methylation sequencing results showed high promoter methylation rate of SLCO1B3 in Hep3B and HepG2,and Hep3B and HepG2 were selected for subsequent experiments.4.RT-qPCR and Western Blot results showed that the mRNA and protein expression of SLCO1B3 in hepatoma cell lines Hep3B and HepG2 were relatively low,and the expression of OATP1B3 was up-regulated after DAC incubation in both.5.The results of RTCA-e Sight assay showed that the proliferation rate of Hep3B,HepG2 was significantly lower than that of Sorafenib group after co-administration of DAC.6.LC-MS/MS results showed that the uptake of Sorafenib by Hep3B,HepG2 was increased by 1.87-fold and 2.47-fold respectively after co-administration of DAC.7.MTS results showed that Hep3B,HepG2 survival was significantly lower in the co-administered group compared to Sorafenib alone,and the IC₅₀of Sorafenib was significantly reduced after the co-administration.8.Flow cytometry showed that the levels of lipid peroxides were significantly higher in the DAC combination group than in the sorafenib alone group,and that DAC combination significantly enhanced sorafenib-induced iron death in cells.9.The results of Hep3B xenograft nude mouse model showed that after 21 days of administration,the tumor volumes in the combination group were all significantly smaller than those in the Sorafenib alone,DAC and Control groups.RT-qPCR and Western Blot results showed that the expression of OATP1B3 in the combination group and DAC group were all significantly higher than that in the Control Sorafenib group;similarly,the immunohistochemical results showed that the expression of OATP1B3 in the combination group and DAC group were significantly higher than that in the Control group and Sorafenib group.Conclusion:The epigenetic drug DAC reverses sorafenib resistance by inhibiting DNA methylation of SLCO1B3,upregulating the expression of OATP1B3,which mediates the transmembrane transport of Sorafenib,increasing the exposure level of Sorafenib in hepatocellular carcinoma cells and enhancing sensitivity to hepatocellular carcinoma cells.