lncRNA-uc001ylu在缺氧诱导EMT促进胃癌转移中的机制研究

We prevously found that hypoxia could prompt gastric cancer cell epithelial mesenchymal transtition, which is an indispensable participant in the cancer metastasis. However, the mechanism still remains unclear.Long non-coding RNAs (lncRNAs) are a novel class of non-coding transcripts, which are more than 200 nucleotides in length and involved in the regulation of gene expression in various ways.It has been a popular topic in tumor study. Funded by The National Natural Science Fund, we previously performed that differentially expressed lncRNAs between gastric cancer cells exposed to hypoxia and normoxia; normal human gastric epithelia,primary gastric carcinomas, and distant metastatic tissues and cell lines were identified by microarray . LncRNA-uc001ylu expression was markedly upregulated in gastric cancer cell lines exposed to hypoxia compared with normoxia and also overexpressed in distant metastatic tissues and cell lines compared with normal's and non- metastatic's by RT-PCR.Here, we demonstrated the role of lncRNA-uc001ylu in hypoxic metastasis and invasion in gastric cancer.Further bioinformatics analysis and systematic literature review, we identify its location in the chromosomes coincides with the EMT-transcriptional factor ZEB-1.This project will further explore the mechanism of how ZEB-1 reguated lncRNA-uc001ylu and it mediate target gene in hypoxia condition by RT-PCR, CHIP, EMSA, DNA methylation techniques.To reveal the mechanisms of lncRNA-uc001ylu-EMT contributes to hypoxia-induced invasion in gastric cancer, and provide a new thinking of blocking tumor metastasis in clinical practice.

我们前期的研究发现缺氧诱导上皮间质转化是促进胃癌转移的关键途径，但其机制尚不清楚。LncRNA是一类长度大于200nt的非编码RNA，通过多层面表观遗传调控编码基因成为肿瘤转移研究的热点。我们在已结题国科金资助下通过高通量筛选和功能研究首次发现：缺氧通过上调lncRNA-uc001ylu诱导EMT的发生。进一步的生物信息学分析和初步的研究发现其与EMT-转录因子ZEB-1密切相关。本项目拟借助RNA-CHIP，EMSA、甲基化、乙酰化阻断等技术进一步探讨缺氧条件下ZEB-1调控lncRNA-uc001ylu的机制，以及lncRNA-uc001ylu对靶基因的多层面调控机制及其对胃癌转移的作用，完善lncRNA-EMT-靶基因在缺氧诱导胃癌转移中的作用机制，为提出lncRNA通过多层面表观遗传调控机制诱导EMT发生的新机制奠定理论依据，更为发掘阻断胃癌转移的关键"枢纽"分子奠定理论基础。

肿瘤特殊微环境在肿瘤转移中发挥重要作用，我们前期证实缺氧是促进胃癌转移的关键因素。我们首次用高通量lncRNA芯片进行了缺氧诱导的胃癌细胞以及高转移胃癌组织、细胞中的lncRNA的差异筛选，发现lncRNA-uc001ylu在缺氧条件下和高转移胃癌组织中显著上调，进一步通过胃癌转移组织进行验证发现其与癌旁相比，在胃癌组织中高表达，并与预后显著相关，通过克隆形成，transwwll功能学研究证实lncRNA-uc001ylu在缺氧诱导胃癌细胞转移中发挥重要作用。为了探讨其在缺氧条件下上调的机制，利用生物信息学分析发现其位于EMT-转录因子ZEB-1附近，利用siRNA干扰ZEB-1的表达观察lncRNA-uc001ylu的影响，发现抑制ZEB-1的表达能够下调lncRNA-uc001ylu的表达，初步证实缺氧是通过上调ZEB-1的表达从而上调lncRNA-uc001ylu的表达，为了进一步探讨ZEB-1对lncRNA-uc001ylu的调控机制，通过启动子分析实验发现在lncRNA-uc001ylu启动子上游存在ZEB-1的结合位点，进一步利用双标免疫荧光实验证实ZEB-1是通过与lncRNA-uc001ylu启动子上的结合位点相互作用转录活化上调其表达的。进一步的功能研究发现lncRNA-uc001ylu通过调控下游基因TNPAIP2参与缺氧诱导胃癌细胞侵袭转移的发生。本项目深入探讨缺氧条件下ZEB-1调控lncRNA-uc001ylu及其对下游靶基因的的调控机制，揭示了lncRNA-uc001ylu—EMT在缺氧诱导胃癌转移中的作用机制， 该研究填补了lncRNA在胃癌侵袭和转移作用中的空白，并首次发现了lncRNA-uc001ylu能够参与肿瘤微环境下的EMT的发生。为临床阻断胃癌转移提供新思路。