长链非编码RNA AP000695.4在肝细胞癌进展中的作用及机制研究

Our previous work found that long non-coding RNA AP000695.4 was high regulated in the hepatocellular carcinoma (HCC) tissues, and the proliferative ability of HCC cell lines decreased after silencing AP000695.4. Our preliminary mechanism exploration indicated that AP000695.4 bound with miR-7 to promote HCC proliferation. Based on these results, we hypothesized that AP000695.4 may play an proliferative role in HCC via regulating some RNAs (including mRNA, miRNA, lncRNA) at post-transcriptional level or at protein level. Next, we plan to perform MS2-RNA IP, RNA-Seq and the other assays to investigate the RNAs or proteins that can bind with AP000695.4, and then explore whether AP000695.4 and these associated molecules can form a mutually regulated network to promote HCC progression. Finally, we will analyze the prognosis predictive value of this regulatory network in HCC patients. This project aims to the mechanism of AP000695.4 in HCC progression, thus providing clinical indication for HCC prognosis and novel therapeutic targets for HCC treatment.

我们前期工作发现长链非编码RNA AP000695.4在肝癌组织中高表达,沉默其表达肝癌细胞的增殖能力明显下降；初步的机制探讨发现其可以与miR-7直接结合发挥促癌作用。据此我们提出以下假设：AP000695.4可能在转录后水平调控RNA（包括mRNA、miRNA、lncRNA等）或蛋白表达以发挥促癌作用。我们拟通过MS2-RNA IP、RNA-Seq等实验寻找与AP000695.4结合的RNA、蛋白等,并探讨其是否调控所结合RNA、蛋白等的表达,它们之间是否存在相互调控的网络关系以促进肝癌的进展；最后通过临床大样本检测AP000695 .4及其相关蛋白质、miRNA在判断肝癌预后方面的作用。本项目的完成可望揭示AP000695.4 在肝癌发生发展中的作用机制，对肝癌增殖、预后判断具有重要的理论及临床价值，为肝癌治疗的新靶点提供理论依据。

研究表明lncRNA与肿瘤的发生发展密切相关，lncRNA AP000695.4是新发现的lncRNA，我们探讨其在肝癌中的生物学功能并探讨相应分子机制。我们首先利用芯片筛选出7对癌及癌旁组织中差异表达的lncRNA，随后利用qRT-PCR、原位杂交等方法检测肝癌组织中AP000695.4的表达，分析AP000695.4表达与肝癌患者预后的关系。接着，分别构建AP000695.4稳定过表达及敲除的肝癌细胞株，利用CCK8、EdU增殖实验、裸鼠皮下成瘤等实验明确AP000695.4在肝癌中的生物学功能，最后利用双荧光素酶报告实验、RIP、RNA pull down、western blot等实验探讨AP000695.4促进肝癌增殖的分子机制。结果表明lincRNA-AP000695.4在肝癌中高表达，其高表达与肝癌患者不良预后相关，体内外实验证明lncRNA AP000695.4促进肝癌细胞增殖。AP000695.4与miR-7直接结合，促进靶基因Hif1α的表达和稳定，肝癌组织中AP000695.4与Hif1α及其下游糖酵解相关靶基因表达呈正相关关系。与此同时，AP000695.4可以增强Hif1α的蛋白稳定性。.上述结果表明AP000695.4与Hif1α形成正反馈环路调控肝癌糖酵解及增殖，这为肝癌治疗提供新的靶点。