Gli1/HOXA5/Twist1新通路在高转移肝癌细胞中的作用及分子机制研究

Hedgehog signaling promotes hepatic cellular cancer cells invasion and metastasis, but mechanism is unclear. We found the Gli1 could affect the transcriptional activity of Twist in the subpopulation of highly invasive hepatoma cells. These results demonstrated that the Gli1 may enhance these subpopulation cells invasion and metastasis by inducing EMT process through regulation the expression of Twist. Moreover,(Co-Immunoprecipitation) Co-IP showed that Gli1 could interact with HOXA5, bioinformatics suggests that there is a HOXA5 binding site during Twist1 promoter region. Taken together, we hypothesize that Gli1 induce Twist1 expression and eventually promote highly invasive hepatoma cells invasion via interaction with HOXA5. We would like to further confirm whether Twist1 is a novel target gene of HOXA5 using duel luciferase reporter assay, electrophoresis mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP). It was reported there was the binding site of Twist in Gli1 promoter region. On this occasion, activated Twist could further enhance the expression of Gli1, and form a positive feedback control mechanism to maintain and amplify the effect of invasion and metastasis by activated Hh signaling. Based on above hypothesis, this study will illustrate the molecular mechanism of activation of the Gli1/HOXA5/Twist1 signaling axis promoting highly invasive hepatoma cells progression using luciferase experiment, EMSA and ChIP, which will provide a new theoretical basis for intervening hepatic cellular cancer through Hh signaling.

原发性肝癌的侵袭转移具有重要研究意义。前期研究发现Hedgehog信号通路关键蛋白Gli1异常高表达在高转移肝癌亚群细胞的侵袭转移中发挥了重要作用（J Hepatol 2011）。有研究表明Gli1通过Snail调控细胞EMT发生，我们进一步研究发现在高转移肝癌亚群细胞内Gli1可以通过调控Twist1的表达引起EMT发生（Oncogene2015）。前期预实验发现Gli1可以和HOXA5形成复合物，生物信息学预测Twist1启动子上存在HOXA5结合位点。综上我们推测Gli1调控高转移肝癌亚群细胞的分子机制可能为Gli1和HOXA5形成复合物，转录调控Twist1表达，而Twist1又是Gli1的转录因子，正反馈放大促进肝癌细胞侵袭转移。根据以上假说拟采用双荧光素酶实验、EMSA和ChIP等实验，阐明Gli1/HOXA5/Twist1信号通路对高转移肝癌亚群细胞侵袭转移调控的分子机制。

Gli1(glioma-associated oncogene homolog 1)在肿瘤中存在异常表达，并在肿瘤细胞发生EMT的过程中发挥重要作用，然而其在肝癌中的作用及其分子机制仍不是很清楚。本课题发现在肝癌细胞中Gli1与ATF4（activating transcription factor 4;ATF4）的表达存在显著正相关，其表达的高低与肝癌细胞的侵袭转移存在密切的关系。我们首先在4株肝癌原代细胞中发现转移能力强的细胞Gli1的表达也相对较高。进一步通过Transwell的方法挑选出稳定的高转移肝癌亚群细胞株7721，发现在高转移的肝癌细胞亚群中Gli1的表达明显高于低转移的肝癌亚群细胞。然后在Gli1表达相对较高且转移能力较强的肝癌细胞PLC中通过慢病毒干扰Gli1，结果显示PLC细胞的侵袭转移能力明显减弱，同时通过Q-PCR和蛋白免疫印记实验发现ATF4的表达也随之降低。在Gli1表达相对较低的HepG2细胞中过表达Gli1，结果显示HepG2细胞的侵袭转移能力明显增强，同时通过Q-PCR和蛋白免疫印记实验发现ATF4的表达也随之升高。在裸鼠中发现注射过表达Gli1的肝癌细胞组中形成的肺转移结节数明显高于对照组。此外通过基因芯片分析，我们发现在干扰Gli1的PLC肝癌细胞中，有1702个基因存在差异表达，其中与转移相关的基因162个，其中下调的有109个，上调的有53个。由此我们得出结论Gli1在肝癌的侵袭转移中发挥了极其重要的作用并且有可能是通过调控ATF4的表达从而促进肝癌的转移。