Expression Of YAP Confers Doxorubicin Resistance And Mechanism In Hepatocellular Carcinoma Cells

Objective:To study the effect of expression of YAP on doxorubicin resistance in hepatocellular carcinoma, explore the potecial mechanism between overexpression of YAP and activation of chemoresistance related signaling pathway.Methods:Plasmid transfection was used to over express YAP and Western blot anlaysis was used to detect the expression level of YAP in hepatocellular carcinoma cell lines. The MTT assay was used to detect the cell viability of plasmid transfected cells and the controls when exposured to doxorubicin and statistical analyzed the cell survival difference between the experimental and control group. By using RNA. interference, the expression level of endogenous YAP was inhibited, and then analyzed by Western blot both in the siYAP-transfect group and non-targeting siRNA groups. Analyze the cell viability in siYAP transfectants by MTT after exposing to doxorubicin and take data into statistical analysis. TUNEL anlaysis was used to detect cell apoptosis in siYAP-transfect cells exposing to doxorubicin, compared with the control groups. YAP-overexpress transfectants and YAP-lowexpress transfectants was analyzed by Western blot and semiquantitative WB to detect YAP and apoptosis related proteins, such as cleaved PARP, Bcl-xL and BAX. The activation of MAPK or Akt signal pathway was also detected. Treated HCC lines with different inhibitors of MAPK or Akt signal pathway and then detect the changes in expression levels of apoptosis proteins. Eventually, detect vability of these cells by MTT assay and undertook statistical analysis.Results:After exposing to doxorubicin for48and72hours, YAP-overexpress transfectants including Huh7-YAP and Hep3B-YAP showed a higher cell vability than the control groups. The half maximal inhibitory concentration (IC50) at48hours in controls was0.83μg/ml and1.14μg/ml, when in YAP-overexpress cells Huh-YAP and Hep-3B, it was1.02μg/ml and1.73μg/ml. By RNAi, the expression levels of endongenous YAP continuously decresed in siYAP transfected cells from1to3days. Treated with different concentration of doxorubicin, siYAP transfectants showed a significantly decrese in cell viability. And in the same concentration of doxorubicin after48hours, the number of apoptotic cells in siYAP transfants increased significantly by TUNEL. After pretreatment of doxorubicin, the apoptosis related proteins, cleaved PARP and Bax decresed while Bcl-Xl increased significantly in YAP-overexpress HCC lines. In contrast, apotosis proteins expression and signal pathway activationg in RNAi siYAP transfectants showed an obviously opposite trend. Pretreatment with MAPK or Akt inhibitor LY294002and U0126could inhibit corresponding activation. Moreover, only U0126and doxorubicin was used at the same time in siYAP cells could markedly decrese these cells’ vability by MTT. Downregulation of Bcl-xL expression level and upregulation of cleaved PARP and Bax level were obiously observed by WB.Conclusion:In concludsion, our data provide evidence that overexpression of YAP could inhibit the inhibition of doxorubicin to HCC. It may be one of the mechanisms on doxorubicin resistance of HCC. Inhibition of endogenous YAP can inhibit inverse doxorubicin resistance to promote apoptosis. At last, MAPK signal pathway is related to YAP confers doxorubicin resistance in HCC, and inhibiting its activation can partly inhibit the drug resistance.