ARHGAP9基因在肝癌侵袭转移中的作用及机制研究

Liver cancer as one of common malignancies is characterized by high invasion ang migration, which results in high mortality. Although various therapies are employed for liver cancer, the final effect is not very ideal. Our previous studies displayed that the expression of ARHGAP9 was lower in the liver cancer tissue, and the prognosis of low ARHGAP9 expression patients was poor. High expression of ARHGAP9 also inhibited the activation of signalings associated with invasion and migration in liver carcinoma. In this study, we aims to clarify the relevance between ARHGAP9 and the clinical prognosis and to elucidate the effect of this gene on the invasion and metastasis of liver carcinoma by means of high-throughput sequencing, data excavation, Q-RT-PCR, western blot, gene interference, gene overexpression, co-immunoprecipitation, laser confocal microscopy, etc. on the basis of bioinformatics, cell biology and mathematical statistics. This research will provide new sights for understanding the effect and mechanism of ARHGAP9 in invasion, migration and clinical prognosis of liver carcinoma and clarify the possibility of ARHGAP9 to be the clinical prognosis monitoring index and molecular therapeutic target.

肝癌是最常见的恶性肿瘤之一，高侵袭转移是其重要的生物学特性，是导致病人死亡的重要原因。尽管有较多的治疗方法，但效果并不理想。我们前期初步研究发现，ARHGAP9基因在肝癌组织中明显低表达，且低表达患者较高表达患者预后差。ARHGAP9高表达还抑制肝癌侵袭转移相关信号通路的激活。本项目拟综合应用生物信息学、细胞分子生物学和数理统计学等多学科知识，采用高通量测序、数据挖掘、Q-PCR、Western blot、基因干扰、基因过表达、免疫共沉淀和激光共聚焦等技术，通过临床大样本检测分析和体内外试验，揭示ARHGAP9基因与临床预后的相关性，阐明该基因对肝癌侵袭转移的影响及机制，探讨其作为肝癌临床预后监测指标和分子治疗靶点的可能性。

Rho GTPase酶活化蛋白(RhoGAPs) 是一类功能域专一结合Rho家族的蛋白，通过促进绑定到Rho上的GTP酶水解，使Rho蛋白恢复到GDP结合的失活状态，能使Rho GTPase酶水解活性提高超过100倍，发挥着关闭Rho信号通路的重要作用，目前正成为一类新的肿瘤生物标志物。本课题通过收集临床肝癌患者手术切除肿瘤组织及正常肝组织，运用RNA-seq测序、TCGA数据库挖掘、基因富集分析（GSEA）和Q-RT-PCR技术，发现ARHGAP9在肝癌组织中低表达，且低表达患者预后差。ARHGAP9过表达后，肝癌增殖、侵袭迁移及肝癌肿瘤转移的能力都受到了显著抑制。通过RNA二代测序发现一个转录子FOXJ2在ARHGAP9过表达的肝癌细胞中显著增加，且FOXJ2的表达显著抑制肝癌细胞侵袭、迁移，且当FOXJ2被干扰后，ARHGAP9抑制肝癌细胞侵袭迁移的作用被阻断。荧光素酶报告基因检测发现ARHGAP9通过FOXJ2促进了E-cadherin的转录。ChiP实验发现FOXJ2直接与E-cadherin的启动子CDH1相结合而促进E-cadherin转录的。以上结果表明，ARHGAP9通过上调FOXJ2/E-cadherin抑制肝癌细胞侵袭迁移的，有望成为肝癌治疗的新靶点，预后新指标。