长链非编码RNA-MEG3调控EMT影响胃癌侵袭迁移分子机制研究

Gastric cancer is the second most common malignant tumor in China. The major obstacle towards improved survival from gastric cancer is in the development of metastatic disease.Techniques in cellular and molecular biology have now advanced to the point to allow an examination of specific biomolecules in processes related to gastric cancer cell invasion. Latest research suggests the epithelial mesenchymal transitions（EMT）play an important role in gastric cancer metastatic development. miRNA as well as lncRNA were reported to regulate EMT processes, but there is not report about lncRNA and its role in gastric EMT processes.Our previous studies have found that long noncoding RNA MEG3 expressed in gastric cancer cut or missing, overexpression of MEG3 could inhibit the gastric cell growth and invasion, and display typical EMT phenotype characteristics.Further research shows that MEG3 expression was induced by TGF-β.Therefore, we presumed that MEG3 induced by TGF-βplay an important role in the metastases development of gastric carcinoma,and mechanisms is still unkwow. On the basis of our prior experiments, this study is intended to illustrate the role and molecular mechanism of MEG3 in process of tumorigenesis and development by as gene ChIP and ChIP, using cell line and tissue. Moreover, we intend to primarily indicate the MEG3 regulation mechanisms involved in the process of tumorigenesis and metastases development. The scientific meaning of this study lays emphases on enriching the understanding of tumorigenesis and development of gastric carcinoma, providing new target with high-effecacy for clinical treatment.

胃癌是中国第二大常见恶性肿瘤，上皮间质转化(EMT)是胃癌侵袭转移研究的热点。研究表明，miRNA和长链非编码RNA(lncRNA)参与调控肿瘤EMT过程，但有关lncRNA调控胃癌EMT的研究甚少。在前一自然基金资助下，我们发现lncRNA-MEG3在胃癌组织中表达下调，进一步研究表明MEG3在TGF-β诱导后表达升高，并且过表达MEG3的胃癌细胞获得了EMT表型特征。这提示MEG3可能在胃癌EMT过程中发挥重要作用，但MEG3调控胃癌EMT的机制尚不清楚。本项目拟在前期工作基础上，以胃癌细胞株、胃癌细胞及病人癌组织来源裸鼠移植瘤模型、胃癌组织为研究对象，通过基因芯片、ChIP等技术，观察MEG3调控EMT对胃癌侵袭转移的影响，阐明MEG3调控胃癌EMT的分子网络机制，探讨MEG3与miRNAs的协调作用，为揭示胃癌侵袭转移机理、开发基于RNA的新型分子靶向药物提供理论依据。

胃癌是中国第二大常见恶性肿瘤，上皮间质转化(EMT)是胃癌侵袭转移研究的热点。在前期工作中，我们发现lncRNA-MEG3在胃癌组织中表达下调，MEG3在TGF-β诱导后表达升高，并且过表达MEG3的胃癌细胞获得了EMT表型特征。本项目拟在前期工作基础上阐明MEG3调控胃癌EMT的分子网络机制，探讨MEG3与miRNAs的协调作用。本项目中，我们获取胃癌及癌旁组织标本，利用基因芯片及生物信息学技术建立lncRNA以及circRNA差异表达谱，筛选出胃癌中表达差异显著的lncRNA以及circRNA，其中显著差异表达的lncRNA共1217条（上调751，下调466）；circRNA共49条（上调23，下调26）。MEG3在胃癌中显著下调（FC=3.06, P=0.0275）。KEGG通路分析发现TGF-β通路是胃癌相关的重要信号通路，我们利用QSAR方法构建了TGF的DNA 和lncRNA MEG3 相互作用的分子模型，通过分子模拟评价两者相互作用的亲和力，发现了使MEG3 RNA与染色体的DNA结合形成RNA-DNA联体结构发挥其功能。TGF-β信号通路中的一些基因确实可作为MEG3的目的基因，如：TGF-β2、TGF-βR-1和SMAD2等，并且在这些结合位点上也同样含有丰富的GA序列。这说明GA序列引导了MEG3与目的基因的结合，从而形成RNA-DNA三联体结构，导致TGF-β2、TGF-βR-1、SMAD2等基因表达下调，发挥其抑癌作用。还发现用于体外模拟靶向药物瑞戈非尼如何作用于miR-21前体，从而阻断成熟miR-21形成，目的是为开发胃癌靶向药物提供一种新的研究方法和方向。在研究具体的生物lncRNA生物功能方面，我们发现LncRNA NNT-AS1在胃癌肿瘤组织中表达要高于正常组织，进一步进行靶基因研究，发现LncRNA NNT-AS1可以调控miR-424，进而影响转录因子E2F1的功能，从而促进胃癌的发生和细胞周期失调。本项目将为深入理解胃癌上皮间质转化的机理、开发基于 RNA 的新型分子靶向药物提供坚实基础。