调控miR-181a的ceRNA鉴定及其在胃癌发生发展中的作用

ceRNAs can sequester miRNAs, thereby protecting their target RNAs from repression. In the previous study, we found that miR-181a is over expressed in human gastric cancer tissues, and miR-181a functions as an oncomir by repressing the expression of tumor suppressor KLF6. However, the regulation of miR-181a still need further investigated. Based on the bioinformatics survey, we found a KLF6 neighboring gene, long non-coding RNA CR749391, contains four binding sites with the seed of miR-181a. Based on our preliminary study, we further found an inverse correlation between the expressions of miR-181a and CR749391 in gastric cancer tissues. These resultings indicatd that CR749391 can function as ceRNA to sequester miR-181a and attenuates miR-181a mediated KLF6 inhibition and relieve the progress of gastric cancer. In this study, we used in situ hybridization, LncRNA overexpression and LncRNA siRNA assay, luciferase assay, RNA immunoprecipitation， RNA pulldown assay and xenograft assay to reveal that LncRNA CR749193 can regulate biological activity of miR-181a and reactivate the miR-181a target gene KLF6.

内源性竞争性RNA(ceRNA)学说认为，ceRNA能竞争性结合miRNAs从而调节miRNAs靶基因的表达。我们前期研究证实miR-181a在胃癌组织中高表达，并发现它可抑制KLF6的蛋白表达来促进胃癌的发生发展。然而miR-181a又是如何被调控的尚不清楚。生物信息学分析发现KLF6基因下游的长链非编码RNA CR749391与miR-181a种子区有4个串联的结合位点，预实验发现miR-181a和CR749391在胃癌组织中的表达呈负相关，提示CR749391可作为ceRNA竞争性结合miR-181a，减少miR-181a对KLF6的抑制作用，从而抑制胃癌的进展。本课题拟采用原位杂交、LncRNA过表达及沉默技术、荧光素酶实验、RNA免疫共沉淀技术、RNA pulldown技术和裸鼠成瘤实验等，阐明lncRNA调控miR-181a的分子机制及在KLF6活化中的作用，指导临床实践。

长链非编码RNA是生命活动的调节者，它的表达紊乱在包括肿瘤等多种疾病中发挥重要作用。然而，胃癌中lncRNA的功能尚不明确。在本实验中，我们发现一个新的重要功能lncRNA，CR749391，位于KLF6基因下游，与miR-181a存在四个串联的结合位点。我们发现CR749391能与miR-181a能互相结合调控KLF6基因的表达，功能实验证实CR749391缺失能促进胃癌细胞增殖、迁移和浸润，并能抑制凋亡，过表达CR749391作用则相反，过表达CR749391能抑制体内肿瘤生长。CR749391在胃癌组织中表达是下调的，与KLF6表达是相关，但与miR-181a表达则是负相关。该研究提示CR749391依赖ceRNA（miRNA分子海绵）作用机制，吸附miR-181a，正向调控KLF6表达。该发现对探索胃癌的分子机制提供的新的视角，并对lncRNA的靶向治疗提供理论基础。另一个新的lncRNA，linc00483，它在胃癌中表达上调，并与成瘤性、肿瘤大小、转移和预后相关。linc00483能在体内外促进胃癌细胞增殖、迁移和转移。上调linc00483能吸附内源性肿瘤抑制因子miR-30a-3p，进而上调SPAG9的表达，进一步激活MAPK信号通路。因此，linc00483作为致瘤性lncRNA，linc00483或者其下游通路可能为胃癌的治疗靶点。本研究揭示CR749391和linc00483在胃癌致癌中是两个重要的调控因子，并可能为预测胃癌患者预后的生物标记物。