Huaier Enhanced The Chemotherapeutic Sensitivity Of Oxaliplatin Via SOX9/YAP Signaling Axis In Hepatocellular Carcinoma

Objectives: Hepatocellular carcinoma(HCC)is one of the common maligancies.The oxaliplatin-based FOLFOX4 chemotheraphy is one of the indispensable treatment strategies in hepatocellular carcinoma.However,chemotheraphy may contribute to liver injury.Hence,it is essential to reduce the dosage of oxaliplatin and enhance the curative effect of oxaliplatin.This research mainly explore the effect of Huaier on the chemotherapy curative effect of oxaliplatin and its mechanism.Methods: CCK-8 assays were used to detect the half maximal inhibitory concentration(IC50)of oxaliplatin and Huaier in hepatocellular carcinoma.Flow Cytometry was used to detect cell apoptosis.We screened the significantly differential genes associated with the chemotherapy sensitivity of oxaliplatin by analysing the data of GEO database.We used Calcusyn 2.0 software to calculate combination index between Huaier and oxaliplatin.WB were used to detect the expression of cyclin D1、Bcl-2、Cleaved Caspase Substrate、YAP and Cyr61.Quantitative real-time PCR was used to detected the correlation between SOX9 and YAP m RNA expression levels.Nude mouse tumorigenicity assays were used to analyse whether the expression levels of SOX9 affected the growth of hepatocellular carcinoma in vivo.Immunohistochemical assays were used to analyse the correlation between SOX9 and YAP protein expression levels in tissues of hepatocellular carcinoma.We explored the correlation between SOX9 and the degradation or generation of YAP after the treatment of cyclohexane or proteasome inhibitor MG132.Immunofluorescence assays were used to observe the localization of SOX9 and YAP in hepatoma cells.Co-immunoprecipitation were use to explore whether there is interaction between SOX9 and YAP.Results: CCK-8 assays revealed that the half maximal inhibitory concentration of oxaliplatin is 10μg/ml and the half maximal inhibitory concentration of Huaier is 12mg/ml.By analyzing the data of GEO Database,we found that oxaliplatin promotes the expression of Cyr61;The combination index between oxaliplatin and Huaier was less than one,which indicated that there are synergistic effect between oxaliplatin and Huaier.Flow Cytometry found that Huaier enhanced the apoptotic effect of oxaliplatin.WB assays revealed that oxaliplatin combined with Huaier decreased the expression levels of Bcl2 and YAP.Blockade of YAP enhanced the chemotherapy sensitivity of oxaliplatin.In vivo and in vitro studies proved the correlation between SOX9 and YAP protein expression levels.We treated hepatoma cells with cyclohexane or proteasome inhibitor MG132 and found that the expression of SOX9 promoted degradation of YAP and showed no significant influence on the generation of YAP.Immunofluorescence assays observed that there is colocalization between SOX9 and YAP in hepatoma cells.Coimmunoprecipitation revealed that there is interaction between SOX9 and YAP.Conclusions: The expression of YAP correlated with the chemotherapy sensitivity of oxaliplatin.Huaier combined with oxaliplatin promoted the apoptosis by reducing the expression of YAP in hepatoma cells.SOX9 promoted the expression of YAP by inhibiting the degradation of YAP.