肝纤维化-肝癌发生发展进程中YAP/TAZ和pSmad3C/3L-ERK/JNK/p38交互作用及丹酚酸B干预的机制

Hippo通路中MST1/2基因缺失致YAP/TAZ异常活化可促进肝纤维化-肝癌发生发展，pSmad3C/pSmad3L转换亦是其关键病理机制。丹酚酸B经MAPK通路调控pSmad3C/pSmad3L抑制肝纤维化-肝癌进展；预实验结果：丹酚酸B可抑制肝纤维化小鼠肝脏YAP蛋白表达。推测，肝纤维化-肝癌进程中可能存在YAP/TAZ和pSmad3C/3L-ERK/JNK/p38交互作用。拟建立Smad3的C末端磷酸化位点突变小鼠、MST1/2-DKO小鼠肝纤维化-肝癌模型，Smad3的WT、3S-A、EPSM质粒、YAP1基因过表达或干扰慢病毒转染细胞，采用免疫荧光双染、WB、qRT-PCR、共聚焦显微技术等法检测通路中各成员蛋白表达及相互影响，观察YAP/TAZ和pSmad3C/3L-ERK/JNK/p38间交互作用及丹酚酸B干预，阐明丹酚酸B抗肝纤维化-肝癌机制，为其临床应用提供理论依据。

The Hippo signaling pathway and its two downstream effectors, the YAP and TAZ transcriptional coactivators, are drivers of hepatic-fibrosis and hepatocarcinogenesis in MST1/2 DKO mice, and the shift between pSmad3C and pSmad3L is also its key pathological mechanism. Our previous results have shown that Salvianolic acid B exerts anti-hepaticfibrosis-hepatocarcinogenesis effects via inhibition of MAPK mediated pSmad3C/pSmad3L. Preliminary results showed that Salvianolic acid B inhibited the expression of YAP protein in mice with DEN-induced liver fibrosis. It was speculated that the cross-talk between YAP/TAZ and pSmad3C/3L-ERK/JNK/p38 might exist in the progression of hepatic fibrosis and hepatocellular carcinoma. Therefore, it will be used in this project that transgenic animals include of Smad3 C terminal phosphorylation site mutant mice and/or MST1/2 DKO mice, as well as Smad3 WT, 3S-A, EPSM plasmid, and/or YAP 1 gene overexpression or interference with lentivirus will be transfected into HepG2 cells. The protein expression of hippo and TGF-β signaling pathway will be investigated by immunofluorescence, immunoblotting, real-time reverse transcriptase-polymerase chain reaction techniques and confocal microscopy method and so on. These studies purpose to uncover the correlative mechanisms between YAP/TAZ and pSmad3C/3L-ERK/JNK/p38 and the intervention of Salvianolic acid B in the progression of hepatic fibrosis-carcinoma, which will be helpful to clarify the possible mechanisms of hepatocarcinogenesis and provide a theoretical foundation for the clinical application of Salvianolic acid B.