Snail、G9a和CDYLb在肝癌EMT和侵袭转移过程中的作用及机制研究

The high incidence of invasion and metastasis is the first leading cause of liver cancer death. Epithelial-mesenchymal transition (EMT) plays a vital role during cancer invasion and metastasis, thus, understanding the mechanisms involved in the EMT regulation is crucial for cancer metastasis research. Though Snail is the key regulator of liver cancer metastasis, the mechanism underlying Snail induced EMT in liver cancer cells has not been demonstrated yet. Clinical studies revealed that the inappropriate enrichment of H3K9 methyltransferase G9a which could bind with Snail and H3K9me2 modification contributed to hepatocarcinogenesis, however, the importance of G9a for liver cancer metastasis remains unclear. Our preliminary studies indicated that H3K9 methylation effector protein CDYLb could form a complex with G9a and moreover, they had a synergic effect on the progression of hepatocellular carcinoma. However, the cooperative function exerted by G9a and CDYLb in liver cancer metastasis is still unknown. In this study, through CHIP, siRNA interference and xenograft metastasis model experiments, we aim to confirm the critical roles of Snail, G9a and CDYLb in EMT, invasion and metastasis of liver cancer and also demonstrate that the recruitment of G9a and CDYLb by Snail is important for the transcriptional repression of E-cadherin, which would reveal the molecular mechanism of EMT, invasion and metastasis of liver cancer cells and provide novel targets for anti-metastatic liver cancer drug development.

肝癌患者早期易发生侵袭转移，是导致患者死亡的首要因素。上皮-间质转化（EMT）对于恶性肿瘤的侵袭和转移至关重要，阐明EMT过程的调控机制已成为肿瘤转移研究中的关键。作为肝癌转移的关键调控因子，Snail诱导肝癌细胞发生EMT过程的分子机制尚未阐明。能与Snail结合的组蛋白甲基转移酶G9a及其催化的H3K9甲基化修饰的异常富集与肝癌发生密切相关，但是G9a在肝癌转移中的作用仍不清楚。H3K9甲基化效应蛋白CDYLb能与G9a形成复合物且二者共同参与了肝癌的发生，然而G9a和CDYLb在肝癌转移中的协同作用尚未明确。本项目将通过CHIP、siRNA干拢和裸鼠移植瘤等技术确定Snail、G9a和CDYLb在肝癌EMT和侵袭转移过程中的关键作用以及Snail对G9a和CDYLb的募集对于E-钙粘蛋白抑制的重要性，不仅能够揭示肝癌EMT和侵袭转移的分子机制，还能为抗转移型肝癌的药物研制提供新靶点。

肝癌患者早期易发生侵袭转移，是导致患者死亡的首要因素。上皮-间质转化（EMT）对于恶性肿瘤的侵袭和转移至关重要，阐明EMT过程的调控机制已成为肿瘤转移研究中的关键。作为肝癌转移的关键调控因子，Snail诱导肝癌细胞发生EMT过程的分子机制尚未阐明。能与Snail结合的组蛋白甲基转移酶G9a与肝癌发生密切相关，但是G9a在肝癌转移中的作用仍不清楚。HDAC家族蛋白能与G9a形成复合物，然而G9a和HDAC蛋白在肝癌转移中的协同作用尚未明确。本项目通过研究Snail2在肝细胞发生EMT过程中的作用, Snail2、G9a和HDAC对肝癌细胞中E-钙粘蛋白的表达调控作用, Snail2调控肝癌细胞中E-钙粘蛋白的转录抑制的分子机制以及Snail2的缺失对于肝癌细胞侵袭转移能力的影响来确定Snail2、G9a和HDAC在肝癌EMT和侵袭转移过程中的关键作用以及Snail对G9a和HDAC的募集对于E-钙粘蛋白抑制的重要性。目前，我们成功利用TGF-β1诱导两种人肝细胞QSG-7701和HL-7702发生EMT过程。我们发现Snail1作为诱发EMT过程中导致E-钙粘蛋白表达下降的转录作用因子，表达水平应该上调，但是在诱导后其表达量并没有增加，但其家族成员Snail2表达量有显著性上调。另外，我们利用荧光酶报告基因体系证实了Snail2蛋白对E-钙粘蛋白基因启动子的直接结合以及该基因的表达抑制，同时发现G9a和HDAC对于Snail2的抑制作用是必须的。接下来Co-IP实验表明Snail2能够与G9a相互作用，Snail2也能与HDAC1，HDAC3发生相互作用，G9a和HDAC1，HDAC2，HDAC3也都有相互作用，表明Snail2可能是通过募集G9a和HDAC蛋白从而抑制E-钙粘蛋白的表达。最后我们在高转移肝癌细胞系MHCC-97H中使Snail2沉默表达，发现其体外迁移和侵袭能力均明显降低，生长速度也明显减慢。在低转移肝癌细胞系HepG2中过表达Snail2，其体外迁移和侵袭能力明显升高。我们将继续在裸鼠中研究Snail2的沉默和过表达对肝癌细胞转移的影响。因此，我们初步证实了Snail、G9a和HDAC在肝癌EMT和侵袭转移过程中的关键作用以及Snail对G9a和HDAC的募集对于E-钙粘蛋白转录抑制的重要性，对理解肝癌的形成、发展和侵袭转移的分子机制具有理论意义。