VASH2基因在肝细胞癌中转录激活并通过Sonic Hedgehog信号通路促进肝癌细胞上皮-间质转化的机制研究

The early invasion and metastasis of hepatocellular carcinoma(HCC) accounted for the poor prognosis of HCC. It was reported epithelial-mesenchymal transition came to the key regulator to promote invasion and metastasis of HCC. But the underlying mechanisms were unclear. Our team found that Vasohibin 2(VASH2) gene was transcriptionally activated in HCC for the first time. Further results showed VASH2 could indcue the changes of the phenotype of HCC cells. VASH2 overexpression up-regulated mesenchymal marker Vimentin and down-regulated epithelial marker E-cadherin. Deep invastigation detected that VASH2 could bind to Gli-1, one of the key transcriptional factors in Shh signal pathway. So we raised our prediction that VASH2, transcriptionally activated in HCC, promoted EMT of HCC via Shh signal pathway. To confirm our hypothesis, bioinformatics and luciferase reporter gene assay are used to make clear of the regulation of miR200 on VASH2 transcription. In the aspect of functions, we will explore the roles of VASH2 on EMT of hepatocellular carcinoma from the molecular, cellular, tissue and animal levels. Finally, to investigate the molecular mechanisms in the process of VASH2-induced EMT. Our study around the novel VASH2 gene will provide new thought and target for treatment of progressive HCC.

肝细胞癌预后差的根源是其早期即发生侵袭转移，研究表明上皮-间质转化（EMT）是促进肝癌侵袭转移的关键因素，但具体机制尚未明确。申请者前期发现VASH2基因在肝癌中转录激活，并能促进肿瘤生长及血管生成；进一步实验证实VASH2过表达能够上调EMT间质表型标志物Vimentin而下调上皮表型标志物E-cadherin，并增加肿瘤的侵袭能力；机制探讨发现VASH2能够结合并上调Shh信号通路核心转录因子Gli-1的表达。据此提出假说：VASH2基因在肝癌中转录激活并通过Shh信号通路促进肝癌细胞EMT。为验证此假说，我们将利用生物信息学结合报告基因实验等手段从转录后水平明确miR200对VASH2的调控；从分子、细胞、组织及动物水平明确VASH2对肝癌EMT的作用；并探讨VASH2诱导EMT的分子机制。围绕VASH2这一全新基因为揭示肝癌EMT的机制奠定基础，为进展期肝癌的治疗提供理论依据。

目的：肝细胞癌是最难治的恶性肿瘤之一，手术是可能获得治愈的手段，且预后仍不容乐观，需要术后辅助治疗的支持。Vasohibin 2（VASH2）最初被认为是一种促血管生成因子，本研究前期预实验证实VASH2在肝癌组织中高表达，但是它在肿瘤发生发展过程中的作用尚不明确。方法：通过定量PCR和western blot等方法检测肝癌组织及细胞株中VASH2基因的表达情况，并统计分析miR-200a/b/c与VASH2在肝癌细胞及组织中的相关性；体外转染miR-200a/b/c模拟物及抑制物，观察其对VASH2转录和表达的影响；3’UTR荧光报告基因实验检验miR-200a/b/c与VASH2的相关性。筛选出VASH2过表达及干扰的稳定转染细胞株，通过定量PCR、western blot、免疫荧光等方法检测细胞中EMT相关表型标志物的改变。通过Transwell、凋亡、干细胞比例等实验验证VASH2对肝癌细胞株侵袭、抗凋亡及干细胞比例的影响。结果：VASH2在肝癌组织及癌旁组织中存在差异性表达，呈现癌组织表达明显升高的现象。通过转染miR-200模拟物、抑制物及3’UTR萤光报告基因实验验证VASH2为miR-200a/b/c的特异性靶基因之一。过表达VASH2的细胞株倾向于间质细胞发展：波形蛋白表达明显上调，而E-钙粘蛋白表达下降；干扰VASH2的细胞株倾向于上皮细胞发展：波形蛋白表达下降，而E-钙粘蛋白表达升高。功能实验证实VASH2具有促进肝癌细胞侵袭、抗凋亡及提升干细胞的比例等作用。进一步探讨发现，VASH2促进MMP2及CXCR4的表达，从而促进肿瘤细胞的侵袭、转移的能力。在凋亡实验中，过表达VASH2上调Bcl-2、ABCC1等抗凋亡基因的表达，从而促进肝癌细胞的耐药。结论：VASH2在肝癌组织中的表达呈明显升高趋势；miR-200a/b/c作为VASH2的特异性microRNA，在转录后水平特异性调控VASH2的表达； VASH2能够促进肝癌细胞上皮间质转化，从而促进肝癌细胞的恶性转化，具体表现为促进肝癌细胞侵袭、抗凋亡及提升干细胞的比例等。本研究的意义在于明确VASH2在肝癌发展过程中的作用及机制，为肝癌的靶向治疗提供理论基础。