LncRNA AC002454.1近程调控CDK6基因的表达影响胃癌增殖与侵袭的机制研究

Gastric cancer is a complex disease with aberrant molecular network. Abnormal regulation of non coding RNAs is a critical point. In the previous study, we found that hypomethylation of the promoter in lncRNA AC002454.1 results in high expression of AC002454.1, but the biological significance is still obscure. Based on the bioinformatics analysis, we found an upstream gene of AC002454.1, CDK6, can cross-talk with AC002454.1 via competing shared for miR-33a/b. Based on our preliminary study, we further found an positive correlation between the expression of CDK6 and AC002454.1 in gastric cancer tissues. These results indicatd that AC002454.1 can function as ceRNA (competing endogenous RNA) to enhance the expression of miR-33a/b target gene CDK6 through competitively sequester miR-33a/b and promotes gastric cancer cell proliferation and invasion. In this study will illustrate a regulatory network involving aberrant methylation of AC002454.1→ceRNA→miRNAs→CDK6. The results may contribute to a better understanding of the molecular basis of gastric cancer development. The study will also investigate the relationship between the methylation status of AC002454.1 promoter and clinical indicators of gastric cancer to find a new evaluation of gastric cancer biomarkers, thus provide a new basis for early diagnosis and prognosis of gastric cancer.

胃癌是一种分子网络疾病，非编码RNA调控异常是其重要节点。我们前期研究发现胃癌lncRNA AC002454.1启动子处于异常低甲基化状态，并导致高表达，但其在胃癌中的生物学功能尚不明确。生物信息学分析发现AC002454.1与其上游邻近的癌基因CDK6可竞争结合mi-33a/b，预实验发现AC002454.1与CDK6在胃癌组织中表达呈正相关，提示AC002454.1可能通过ceRNA活性结合有限的miR-33a/b，减少miR-33a/b对CDK6的抑制作用，近程调控CDK6基因的表达，促进胃癌的增殖侵袭。本项目将阐明AC002454.1甲基化异常→ceRNA→miRNAs→CDK6通路为核心的分子机制，以拓展对胃癌增殖侵袭的分子基础认识。并通过对AC002454.1启动子甲基化与胃癌临床指标的相关性研究，找到一种新的评估胃癌恶性程度的生物标记，为胃癌的早期诊断及预后判断提供新的依据。

通过扩大病例数检测发现，AC002454.1在胃癌及癌旁组织中表达差异无统计学意义。结合项目组前期研究基础，选取另一LncRNA HOXA11-as进行深入研究。HOXA11-as与多种肿瘤的发生发展密切相关，本研究探讨HOXA11-as在胃癌侵袭进展中的机制。在31对胃癌及癌旁正常组织中，HOXA11-as在胃癌中的表达明显高于癌旁组织，HOXA11-AS 启动子区低甲基化是其表达量增高的可能机制之一。体内外功能获得性及缺失性研究表明HOXA11-as能促进胃癌细胞的增殖及侵袭能力。RNA pull-down及RNA免疫共沉淀技术表明HOXA11-as能与IMP3结合，并增强IMP3及CD44 mRNA的结合，进而增强CD44 mRNA的稳定性并促进其蛋白表达。此外，放线菌素D实验表明过表达HOXA11-as能促进IMP3的靶基因CD44 mRNA的稳定性，但不影响对照GAPDH mRNA的稳定性。敲低IMP3能部分的逆转HOXA11-as介导的促胃癌侵袭转移的能力。综上，这些发现表明HOXA11-as在胃癌的侵袭转移中占据非常重要的作用，胃癌中分子靶点HOXA11-as具有重要的诊断及治疗价值。