miR-145、miR-21与TGF-β/Smad信号通路的相互作用在复方芪参提取物抗肝癌机制中的研究

Hepatocellular carcinoma (HCC) therapy is a difficult problem for human. Our previous results showed that Compound Astragalus and Salvia miltiorrhiza extract (CASE) could exert anti-HCC effect by interfering with TGF-β/Smad signaling pathway, especially regulating the phosphorylation of Smad3 at the C-terminal or linker region (pSmad3C、pSmad3L). In addtion, our preliminary experiment showed that CASE could increase miR-145 expression, while decrease miR-21 expression, in rats induced by diethylinitrosamine. We presume that the interplay between miR-145、miR-21 and TGF-β/Smad signaling pathway might be the important mechanisms of CASE's anti-HCC action. Therefore, we propose to investigate the interaction between miRNA and TGF-βsignaling pathway under the influence of CASE in HepG2 cells in vitro and subcutaneous hepatoma xenografts of BALB/c nude mice in vivo by using agomir、antagomir or the mutanted plasmids in which the kinase sites at the C-terminal or linker region of Smad3. The expressing level of the protein、gene in the signaling pathway、microRNA will be detected by using western blot、qRT-PCR and luciferase assay. This study attemps to elucidate the molecular mechanisms of CASE's anti-HCC effect. The results may provide a theoretical foundation for clinical application of CASE in HCC treatment.

肝细胞肝癌治疗是重大课题。前期结果：复方芪参提取物（CASE）通过干预TGF-β/Smad信号通路尤其是Samd3的C末端和连接区磷酸化（pSmad3C/3L）发挥抗肝癌作用；预实验结果：CASE促进大鼠肝癌模型中miR145表达上调和miR21表达下调。推测,TGF-β/Smad通路与miR-145/miR-21间的交互作用可能是CASE抗肝癌的重要机制。故拟在HepG2细胞、肝癌荷瘤裸鼠模型中，用Smad3C/3L磷酸化位点突变质粒及agomir/antagomir干预pSmad3C/3L及miRNA表达，采用WB、qRT-PCR、荧光素酶报告基因实验等方法检测通路中成员蛋白、miRNA的表达及其相互影响，观察TGF-β/Smad信号通路尤其是pSmad3C/3L与miR-145、miR-21的交互作用及CASE对该作用的干预，阐明CASE抗肝癌分子机制，为其临床防治肝癌提供理论依据。

已知复方芪参提取物（CASE）通过干预TGF-β/Smad通路尤其Smad3 C-末端和连接区（L）磷酸化（pSmad3C/pSmad3L）发挥抗肝癌作用，本课题在HepG2细胞、肝癌移植瘤模型中，采用Smad3C/Smad3L磷酸化位点突变质粒（Smad3 3S-A/Smad3 EPSM）、agomir/antagmir分别干预pSmad3C/pSmad3L、miR-145/miR-21表达，WB、qRT-PCR、荧光素酶报告基因等方法检测TGF-β/Smad通路成员蛋白、miR-145/miR-21表达及其相互影响，观察TGF-β/Smad通路尤其pSmad3C/pSmad3L与miR-145/miR-21的交互作用及CASE对该作用的干预。结果表明：HepG2细胞及其移植瘤中：① 上调pSmad3C或下调pSmad3L升高miR-145而降低miR-21表达水平，抑制肝癌进展；上调pSmad3L降低miR-145而升高miR-21表达水平，促进肝癌进程。② CASE升高pSmad3C/pSmad3L调控下的miR-145而降低miR-21表达水平，抑制肝癌进展。③ 上调miR-145升高pSmad3C而降低pSmad3L及其上游MAPK磷酸化蛋白水平，下调miR-21显著抑制MAPK通路活化；上调miR-145或下调miR-21抑制肝癌进展。提示，促进pSmad3L→pSmad3C信号转换可能是miR-145调控TGF-β/Smad信号转导的重要分子机制，而miR-21通过干预MAPK通路活化而间接调控TGF-β/Smad信号转导。④ CASE抑制miR-145/miR-21干预下的MAPK通路活化，促进pSmad3L→pSmad3C信号转换而抑制肝癌进展。结论：pSmad3C/pSmad3L与miR-145/miR-21间存在交互作用，CASE通过干预该交互作用而发挥抗肝癌效应，此为将CASE开发用于临床肝癌防治提供了理论依据。该研究达到预期目标。有10篇摘要参加国内外学术会议交流，课题主持人受邀在BIT's 9th International Symposium of Cancer Immunotherapy-2016会议上做了大会专题报告。已发表论文10篇，其中SCI论文7篇，待发表论文2篇。课题组多人受本课题资助获得奖励和荣誉，培养5名博士和4名硕士。