LncRNA-uc003uxs通过表观遗传机制调控EMT过程在胃癌侵袭转移中的机制研究

It is becoming clear that epigenetic changes are involved in the metastasis and invasion of gastric cancer. However, the mechanism still remains unclear. lncRNAs, which are more than 200 nucleotides in length and involved in the regulation of gene expression in various ways. It has proved that lncRNA play a key role in epigenetic regulation.Differentially expressed lncRNAs between metastasis and no-metastasis gastric cancer tissues and cells were identified by microarray. LncRNA-uc003uxs expression was markedly upregulated in gastric cancer tissues and cells compared with with controls by RT-PCR and it invovled in the metastasis of GC. We then conducted a methylation microarray using uc003 interfered and control GC cell line.After the verification and analysis,we preliminary confirmed that uc003 might regulate the process of EMT via methylation changes,and then influence the metastasis and invasion of lncRNAs. We will further conduct an in-depth study of the regulation mechanisms of EMT mediated by uc003 using RIP、CHIP、RNA pull-down assays、deletion mapping experiments in this project in order to reveal the concrete mechanism that regulation of GC metastasis mediated by uc003/EMT regulators/ E-cadherin molecules signaling pathway in the metastasis of GC.

表观遗传学改变贯穿了胃癌转移的整个过程，但其机制尚不清楚。LncRNA通过表观遗传机制调控蛋白编码基因活性是当前肿瘤研究热点。我们首次用高通量lncRNA芯片对高、低转移的胃癌组织及细胞系进行lncRNA差异筛选。进一步实验证实lncRNA-uc003uxs在胃癌高转移组织及细胞中均上调并参与了胃癌侵袭转移。为深入探索其发挥作用的机制，我们利用前期的稳转细胞系及对照组进行了甲基化芯片筛选，结果显示uc003的下调可引起EMT相关分子的甲基化改变。形态学实验证实uc003促进了EMT，而转染甲基化抑制剂后EMT过程被逆转。由此推测其可能通过甲基化作用调控EMT促进胃癌侵袭转移。本项目拟借助CHIP、EMSA、BSP、RIP等技术进一步探讨uc003对E-cadherin的调控机制，以揭示uc003—EMT转录因子-E-cadherin-EMT在胃癌转移中的作用机制，为临床阻断转移提供新思路。

我们前期已经证实缺氧可以促进胃癌的侵袭转移，并证实lncRNA可以参与缺氧诱导的胃癌的侵袭转移，此次我们利用高通量lncRNA芯片进行了高、 低转移胃癌组织、细胞中的lncRNA的差异筛选，通过生物信息学分析及验证，证实lncRNA –uc003uxs（即lncRNA-BC005927）在高转移的胃癌细胞中显著上调，并在胃癌组织及癌旁组织中验证后证实其在胃癌组织中显著上调，在胃癌细胞SGC7901及MKN45中构建了lncRNA–BC005927的siRNA 载体，慢病毒包装感染SGC7901及MKN45 后，进行细胞转移的相关表型研究，进一步证实了lncRNA –BC005927参与了胃癌的侵袭转移，并探讨HIF-1a-lncRNA- BC005927—EPHB4的调控机制，利用生物信息学分析和文献回顾发现BC005927上有HIF-1的结合位点，验证并探讨HIF-1调控lncRNA- BC005927的机制， 揭示HIF-1a—uc001ylu—EPHB4在胃癌侵袭中的作用，共转染lncRNA –BC005927和EPHB4的siRNA, 进行侵袭和转移相关实验观察二者在缺胃癌侵袭转移中的作用。