同源异型盒基因HOXA5抑制肝癌的侵袭和迁移通过调控UBC9的表达

目的:检测人肝癌及癌旁组织中HOXA5基因的表达情况,观察稳定高表达HOXA5基因的肝癌细胞MHCC97H迁移和侵袭能力的变化,探讨HOXA5调控肝癌侵袭迁移的机制,为肝癌的分子靶向治疗提供新的靶点。方法:通过实时定量PCR(qRT-PCR)、Western blot以及免疫组织化学等技术检测肝癌及癌旁组织中HOXA5基因的表达。构建稳定高表达HOXA5的肝癌细胞MHCC97H,利用划痕和Transwell侵袭实验检测细胞迁移、侵袭能力的变化。应用CHIP-Seq筛选出HOXA5潜在的靶基因,在稳定高表达HOXA5基因细胞中应用qRT-PCR、Western blot验证HOXA5与靶基因的关系。结果:新鲜的肝癌组织以及相对应的癌旁组织中,免疫组织化学显示相比于癌旁组织的表达情况,肝癌组织中HOXA5的表达低于癌旁;qRT-PCR、Western blot检测发现相对于癌旁组织,肝癌组织中HOXA5的表达水平显著下调。过表达了HOXA5基因的MHCC97H细胞的侵袭能力下降。CHIP-Seq的结果发现,HOXA5结合在UBC9的上游启动子区域。在稳定高表达HOXA5的MHCC97H细胞中,UBC9的mRNA和蛋白表达均下调。当在稳定高表达HOXA5的同时过表达UBC9时,肝癌细胞的侵袭和迁移能力部分回复。结论:在肝癌组织中HOXA5的表达下调;UBC9介导了HOXA5抑制肝癌细胞侵袭迁移的作用,这为进一步阐明肝癌发生侵袭转移过程中的分子机制提供一个新的思路,为精准治疗肝癌提供一个可能的靶点。

Homeobox Gene HOXA5 Inhibits Invasion and Metastasis of Hepatocellular Carcinoma by Regulating Expression of UBC9

Objeetive:Through Detecting the expression of HOXA5 in human liver carcinoma;observing the changes of hepatocellular carcinoma cell line's migration and invasion ability in stable high expression HOXA5's MHCC97H.And exploring the mechanism of how HOXA5 regulate the invasion and migration of hepatocellular carcinoma,eventually to provide a new target for the molecular targeted therapy of liver cancer.Methods:Real-time quantitative PCR (qRT-PCR),western blot and immunohistochemistry were performed to detect the expression of HOXA5 gene in liver cancer tissues.The stable high expression of HOXA5 gene in hepatocellular carcinoma cell line MHCC97H was constructed,and then the changes of migration and invasion ability of MHCC97H cells were detected by wound healing assays and Transwell invasion assays.The potential target genes of HOXA5 were screened out by using CHIP-Seq,what's more,Western and blot qRT-PCR were performed to verify the relationship between HOXA5 and its target genes in the cells which stable high expression of HOXA5 gene.Results:In the fresh liver cancer tissue and the corresponding adjacent non-tumourous tissue,immunohistochemistry showed that HOXA5 was lower in liver cancer tissues than in adjacent non-tumourous tissue.Detection of qRT-PCR and Western blot showed that the expression of HOXA5 was significantly lower in liver cancer tissues than in adjacent non-tumourous tissue.In MHCC97H cells,the overexpression of HOXA5 caused cells' invasion ability decreasing.CHIP-Seq shows that HOXA5 binding to the upstream promoter region of UBC9 gene.UBC9's mRNA and protein expression were down regulated in stably high express HOXA5's MHCC97H cells.When UBC9 was overexpressed in stably expressing HOXA5's MHCC97H cells,the invasion and migration ability of hepatocellular carcinoma cells were partially recovered.Conclusions:The expression of HOXA5 in liver carcinoma was downregulation.UBC9 mediated HOXA5's inhibition affect of cancer cell's invasion and migration,which provide a new way of thinking to further elucidate the molecular mechanism of HCC invasion and metastasis,what's more our finding may demonstrate that HOXA5 as a possible target for precision treatment of hepatocellular carcinoma.