低氧微环境下长链非编码 RNA RP11-70F11.8 促进肝癌侵袭转移的分子机制

There is a close relationship between hypoxic microenvironment and tumor metastasis. Previous studies paid more attention to the expression and function of mRNAs associated with hypoxic microenvironment, but not the noncoding RNAs especially for long noncoding RNAs (lncRNAs). lncRNAs play important roles in multiple biological processes, including carcinogenesis, migration and invasion of hepatocellular carcinoma (HCC). There is a lot of lncRNAs in hepatocellular carcinoma to be revealed for the complexity of numbers, functions and mechanisms of lncRNA. In our preliminary study, we identified differentially expressed lncRNAs and mRNAs in SMMC-7721 cells cultured in hypoxia and normoxia by Human LncRNA Microarray analysis. We found that one of the differentially expressed lncRNAs, RP11-70F11.8, was positively related with HIF-1 alpha, and knowckdown of RP11-70F11.8 significantly inhibited invasion and migration induced by hypoxia in HCC cells. Therefore, we conclude that RP11-70F11.8 may promote the invasion and metastasis of HCC by regulating mRNAs expression in hypoxia. We attempt to perform our study for RP11-70F11.8 by molecular and cellular experiments and animal model in vivo and in vitro. We will investigate the effect and regulatory mechanism of RP11-70F11.8 on the invasion and migration of HCC cells in hypoxic microenvironment, analyze the relationship between levels of the lncRNA expression and clinical pathology of HCC patients, and explore its potential capacity in HCC prognosis.

低氧微环境与肿瘤转移高度相关，以往研究多关注与低氧相关的蛋白编码基因，对非编码RNA、特别是长链非编码RNAs（lncRNAs）认识不足。lncRNAs在肝细胞癌的发生发展过程中具有重要作用，其数量、功能及作用机理繁复，仍有大量的与肝细胞癌相关的lncRNAs有待发现和揭示。我们前期采用lncRNA表达谱芯片对低氧处理的人肝癌细胞分析，发现低氧可诱导lncRNA RP11-70F11.8表达，且与HIF-1α的表达呈正相关；干扰lncRNA RP11-70F11.8显著抑制低氧诱导的肝癌细胞侵袭和迁移，提示该lncRNA可能在低氧微环境下，通过调控肝癌转移相关mRNAs的表达继而促进肝癌侵袭转移。我们拟通过分子、细胞及动物模型等体内外实验，研究低氧微环境下该lncRNA对肝癌细胞侵袭迁移的作用机制，分析该lncRNA表达水平及肝癌患者临床病理之间的关系，探讨其在肝癌预后判断中的价值。

低氧微环境与肿瘤转移高度相关，以往研究多关注与低氧微环境相关的蛋白编码基因，对非编码 RNA、特别是长链非编码 RNAs（lncRNAs）认识不足。近年来研究发现lncRNAs 在肝细胞癌的发生发展过程中发挥着重要的调控作用，但是由于lncRNAs数量、功能及作用机理繁复，仍有大量的与肝细胞癌相关的 lncRNAs 有待被发现，尤其是低氧微环境下lncRNAs调控肝癌转移的作用及机制有待进一步的揭示。基于以上背景，我们首先采用 lncRNA 表达谱芯片对低氧处理的人肝癌细胞的30,586个lncRNAs和26,109个编码蛋白质转录本进性表达分析，通过筛选获得表达上调的lncRNAs 154个（antisense LncRNAs=22， enhancer LncRNAs=36， lincRNAs=71，其他25个）、下调的lncRNAs 55个（antisense LncRNAs=8， enhancer LncRNAs=21，lincRNAs=18，其他8个）；上调mRNA 169个、下调mRNA 39个。其中，lncRNA RP11-70F11.8在低氧条件下表达差异最为显著。我们通过分子、细胞及动物模型等体内外实验研究发现，体外低氧微环境下lncRNA RP11-70F11.8显著促进肝癌细胞增殖、侵袭和转移，干扰 lncRNA RP11-70F11.8 显著抑制低氧诱导的肝癌细胞增殖、侵袭和迁移。以上结果显示，低氧微环境促进lncRNA RP11-70F11.8表达，该上调的lncRNA通过调控肝癌细胞增殖、侵袭和转移促进肝癌进展。应用real-time qPCR在临床样本中对比检测lncRNA RP11-70F11.8表达与肝癌临床生存的关系发现，lncRNA RP11-70F11.8高表达患者总体生存期和无病生存期均显著降低。通过本研究，我们明确了lncRNA RP11-70F11.8受低氧微环境调控，通过促进肝癌细胞增殖，增加裸鼠皮下成瘤率及促进肿瘤组织的生长并增强肝癌细胞的侵袭、转移，高表达lncRNA RP11-70F11.8与肝癌患者预后不良相关。本研究明确lncRNAs作为肝癌预后标志物的潜能，为HCC的治疗和预后判断提供新靶点和新思路。