Smad3C末端和连接区磷酸化位点突变对肝纤维化-肝癌发展进程的调控及丹酚酸B的干预

The pathomechanism of hepatofibrosis–hepatocarcinogenesis is related with TGF-β/Smad signal modulated by MAPK pathway. The phosphorylation of Smad3 at linker region (pSmad3L) increased in hepatocarcinogenesis period. Our previous results showed that CASE could suppress hepatocellular carcinoma progression by increasing the phosphorylation of Smad3 at C-terminal (pSmad3C) and inhibiting pSmad3L. Furthermore Salvianolic acid B has also the action of anti-hepatic fibrosis via TGF-β/Smad signal. We presume that the shift of pSmad3C/pSmad3L might be the important mechanisms of hepatocarcinogenesis, and Salvianolic acid B might effect hepatocarcinogenesis via pSmad3L/pSmad3C and their downstream target gene. Therefore, we propose to investigate the regulation of phosphorylation sites mutation of Smad3 at C-terminal and linker region in hepatofibrosis-hepatocarcinogenesis progression and the action of Salvianolic acid B by using pSmad3C and/or pSmad3L mutant gene knock-in mice in vivo and by using transfer Smad 3 mutant plasmids (3-SA or EPSM) into HepG2 cells in vitro. The expressing level of the protein and gene in the signaling pathway will be detected by using immunoprecipitate, western blot and qRT-PCR methods. This study attemps to elucidate the transducer mechanism of pSmad3C / p21 // pSmad3L / c-Myc in hepatofibrosis-hepatocarcinogenesis progression and Salvianolic acid B’s anti- hepatocarcinogenesis effect.

肝纤维化-肝癌病理机制与MAPK通路调控下的TGF-β/Smad信号相关，肝癌时Smad3连接区磷酸化（pSmad3L）增强。复方芪参提取物通过促进Smad3 C末端磷酸化（pSmad3C）和减少pSmad3L延缓肝癌发展。丹酚酸B抗肝纤维化亦与TGF-β/Smad相关。推测：pSmad3C/pSmad3L转换可能是促肝癌发展的关键机制；丹酚酸B可能影响pSmad3L/3C及下游靶基因而抑制肝癌发生。故拟采用ES打靶技术建立的Smad3C/3L磷酸化位点突变小鼠并诱导其肝纤维化→肝癌；Smad3C/3L磷酸化位点突变质粒（3-SA和EPSM）转染HepG2细胞，免疫沉淀、WB、qRT-PCR等测蛋白和靶基因，观察Smad3磷酸化位点突变对肝纤维化-肝癌发展进程的调控及丹酚酸B的干预。阐明肝纤维化-肝癌进程中的pSmad3C/p21//pSmad3L/c-Myc转换机制以及丹酚酸B作用机制。

已知肝纤维化-肝癌进程中Smad3连接区磷酸化（pSmad3L）显著增加，复方芪参提取物（CASE）促进Smad3 C-末端磷酸化（pSmad3C）、降低pSmad3L发挥抗肝纤维化-肝癌作用。本课题通过构建Smad3C/Smad3L磷酸化位点突变体内外模型，研究Smad3C/Smad3L磷酸化在肝纤维化-肝癌进程中的作用及CASE中的主要活性成分丹酚酸B（Sal B）的干预作用。结果表明：① Smad3C磷酸化位点突变促进二乙基亚硝胺（DEN）诱导的小鼠肝纤维化-肝癌进程，减弱pSmad3C/p21介导的抑癌信号、增强pSmad3L/PAI-1//c-Myc介导的促纤维化促癌信号。② 转染Smad3 EPSM质粒（Smad3 L区磷酸化位点突变）上调pSmad3C抑制肝癌HepG2细胞增殖并诱导其凋亡发挥抗癌效应，而转染Smad3 3S-A质粒（Smad3 C-末端磷酸化位点突变）上调pSmad3L促进HepG2细胞增殖且抑制其凋亡发挥促癌效应。③ Sal B延缓DEN诱导的小鼠肝纤维化-肝癌进程，抑制肝癌HepG2、SMMC-7721细胞迁移、侵袭并诱导其凋亡；Sal B增加上述肝组织/肝癌细胞中pSmad3C及其下游靶基因p21表达且显著降低pSmad3L及其靶基因PAI-1、c-Myc转录及蛋白水平。④ 敲低pSmad3C减弱Sal B对DEN诱导的小鼠肝纤维化-肝癌进程的抑制作用，上调pSmad3C增强Sal B对HepG2细胞增殖、迁移的抑制效应及促凋亡作用，上调pSmad3L则呈现相反的效应。结论：pSmad3C/pSmad3L转换是肝纤维化-肝癌进展的重要分子机制，Sal B促进pSmad3L→pSmad3C转换发挥抗肝纤维化-肝癌作用，此研究为肝癌防治提供了新的有效靶点（pSmad3C/pSmad3L）及候选药物（Sal B）。本研究达到预期目标，已发表中英文文章12篇，待发表英文论文3篇，参加国际国内学术会议十余场，提交会议摘要近二十篇，课题组多人受本课题资助获得奖励和荣誉，培养4名博士和8名硕士。