LncRNA-GMAN调控胃癌细胞侵袭和转移的分子作用机制研究

Gastric cancer is one of the most malignant cancers. Invasion and metastasis is the major cause of death of gastric cancer patient. However, the molecular mechanisms of gastric cancer cell invasion and metastasis still remain unclear. Accumulated evidence indicates that long non-coding RNA (lncRNA) plays an important role in the regulation of tumor progression and metastasis. In order to identify the key lncRNA involved in regulating gastric cancer invasion and metastasis, we detected the expression profile of lncRNA in gastric cancer samples compared with their adjacent normal gastric mucosa. Our microarray data identified a series of differentially expressed lncRNAs in gastric cancer. Finally, we identified an uncharacterized lncRNA, renamed as GMAN (Gastric cancer metastasis associated long non-coding RNA), which is upregulated in the gastric tumor tissues and associated with gastric cancer invasion and metastasis. Using CRISPR/Cas9 genome editing technique, our preliminary study showed that GMAN promotes cell invasion and metastasis, but has no significant effect on the cell proliferation, adhesion and cell cycle. We elucidated the molecular mechanism for GMAN in regulating gastric cancer cell invasion and metastasis. Ephrin was identified as a target gene of GMAN. Interestingly, GMAN enhanced the protein expression of Ephrin but not its mRNA expression. Our in vitro and in vivo data demonstrated that Ephrin mediated the function of GMAN in promoting gastric cancer cell invasion and metastasis. Based on these new findings, this project aims to study the role of GMAN in regulating gastric cancer cell invasion and metastasis, the molecular mechanism for regulating the protein expression of Ephrin, and explore the therapeutic potential for the gastric cancer metastasis by targeting GMAN/Ephrin axis. The launching of this study will help to further understand the molecular regulation mechanism of tumor cell migration, and provides a new potential therapeutic target for the diagnosis and treatment of gastric cancer.

胃癌的高发病率和死亡率严重危害我国人民的健康。胃癌细胞侵袭和转移，是胃癌患者死亡的主要原因之一，然而其分子调控机制不明确并缺少有效的预后治疗靶点。我们在前期工作中，结合胃癌临床样本和LncRNA表达谱芯片技术，筛选胃癌侵袭和转移相关LncRNA。其中，胃癌转移相关LncRNA-GMAN在胃癌组织中高表达，且与胃癌侵袭转移和病人预后显著相关。后续研究结合CRISPR/Cas9技术表明GMAN显著促进胃癌细胞侵袭和体内转移，对胃癌细胞增殖等生物学行为无明显影响。进一步研究显示，GMAN促进靶蛋白Ephrin的蛋白表达，而不影响其mRNA水平。本课题我们将以胃癌细胞为模型，通过分子、细胞、动物和临床四个层面，研究GMAN/Ephrin轴在胃癌细胞侵袭转移中的功能，阐释GMAN的分子作用机制。本课题的完成将有助于深入了解胃癌细胞侵袭转移的分子机理，为胃癌的诊断治疗提供新的理论依据与潜在的分子靶点。

胃癌是严重影响我国人民生命健康的恶性肿瘤。胃癌患者就诊时大多处于中晚期，出现侵袭和转移，预后极差，我们迫切需要深入了解胃癌细胞转移的关键基因和调控机制。本项目利用长链非编码RNA表达谱芯片技术，结合多个胃癌样本队列验证，鉴定了首个胃癌转移相关lncRNA GMAN，发现GMAN可能作为胃癌不良预后的独立风险因素；GMAN能促进胃癌细迁移和转移，对胃癌细胞增殖、细胞周期、平板克隆、基质粘附等生物学行为无明显影响；GMAN对靶基因EphrinA1 mRNA的转录表达、mRNA稳定性没有显著作用，核糖体谱实验显示GMAN能促进EphrinA1 mRNA的翻译，挽救实验表明EphrinA1介导了GMAN在促胃癌细胞侵袭和转移中的作用；我们揭示了GMAN存在反义转录本GMAN-AS，GMAN-AS抑制EphrinA1 mRNA的翻译，进而抑制胃癌细胞迁移侵袭能力；通过RNA pull-down技术，揭示了GMAN、GMAN-AS、EphrinA1 mRNA三者之间的互作关系，GMAN-AS能直接结合并抑制EphrinA1 mRNA，而GMAN通过竞争性结合抑制GMAN-AS，促进EphrinA1的表达，同时结合domain mapping，解析了这些分子之间互作的关键片段序列；利用CRISPR-Cas9技术，敲除GMAN关键序列MFR，显著抑制EphrinA1 mRNA的翻译和蛋白表达，抑制胃癌细胞的迁移侵袭和体内转移能力，外源过表达EphrinA1能挽救GMAN敲除细胞的表型；利用CRISPR-Cas9技术靶向GMAN的体内给药技术，实现在动物水平治疗胃癌转移，显著改善荷瘤小鼠的生存。本项目的研究发现了“三个碱基互补RNA分子间的相互作用调节靶基因的翻译过程”，阐释了GMAN/GMAN-AS/EphrinA1轴调控胃癌细胞转移的新机制，为胃癌转移提供了新的治疗靶点和治疗策略。