miR-130a靶向Gax调控Rap1A表达在胸主动脉瘤血管平滑肌细胞表型转化中的作用及机制研究

Vascular smooth muscle cells phenotypic modulation leading to aortic tunica media weakening is the important pathologic basis of the formation and development of thoracic aortic aneurysm. Previous studies have showed that growth arrest-specific homeobox (Gax) transcription factor plays a key role in the process of VSMCs phenotypic modulation. Our prophase researches showed that the expression of Gax in human ascending aortic aneurysm wall was significantly decreased. In previous trials, we found miR-130a was a good possibility in the prediction of miRNAs targeted to the 3’-untranslated region (3’-UTR) of Gax gene, and miR-130a expression was significantly increased in human ascending aortic aneurysm specimens and the process of VSMCs phenotypic switching induced by osteopontin (OPN). Our studies also showed the most significant change of Rap1A expression was found in VSMCs over-expressing Gax, and Rap1A had an effect on the expression of markers for VSMCs contractile phenotype, and VSMCs proliferation and migration. However, the specific roles of miR-130a targeting Gax-regulated Rap1A expression in VSMCs phenotypic modulation in thoracic aortic aneurysm are still needed to be confirmed. On this basis we will carry on macro and micro researches at the gene, cell and tissue level using the methods of ChIP, cells experiment, clinical specimens and so on to clarify that miR-130a-Gax-Rap1A axis exists in VSMCs phenotypic modulation in thoracic aortic aneurysm, which will help give further new insight into the pathogenesis of thoracic aortic aneurysm and provide new possible target molecules for prevention and treatment for thoracic aortic aneurysm.

血管平滑肌细胞（VSMCs）表型转化所导致的主动脉中层薄弱是胸主动脉瘤发生的重要病理基础。研究揭示生长终止特异性同源盒（Gax）转录因子在VSMCs表型转化中起重要作用。我们研究显示：Gax在升主动脉瘤中表达降低；在预测靶向Gax基因的miRNAs中发现miR-130a的可能性大，其在升主动脉瘤和骨桥蛋白诱导VSMCs表型转化中表达上调；在Gax过表达的VSMCs中发现Rap1A表达变化最显著，Rap1A对收缩型VSMCs标志物表达、VSMCs增殖和迁移有影响。但miR-130a靶向Gax调控Rap1A表达在胸主动脉瘤VSMCs表型转化中的具体作用仍有待于明确。本项目拟在此基础上通过采用ChIP、细胞实验及临床标本等方法从基因、细胞和组织水平上确定胸主动脉瘤VSMCs表型转化中存在miR-130a-Gax-Rap1A调控轴，将为阐明胸主动脉瘤发病机制提供新思路，为防治胸主动脉瘤提供新靶标。

胸主动脉瘤的人群发病率呈逐年上升趋势，严重威胁着人类的生命健康。血管平滑肌细胞表型转化所导致的主动脉中层薄弱是胸主动脉瘤发生的重要病理基础。本项目的主要研究内容是探讨miR-130a靶向Gax调控Rap1A表达在胸主动脉瘤血管平滑肌细胞表型转化中作用及机制。结果显示与对照相比，miR-130a和Rap1A表达能够显著抑制血管平滑细胞分化型标志蛋白Calpoin和SM-MHC的表达，促进细胞的增殖和迁移，而Gax表达能够明显促进血管平滑肌细胞分化型标志蛋白Calpoin和SM-MHC的表达，抑制细胞的增殖和迁移，差异均具有统计学意义（P<0.05），表明miR-130a，Gax和Rap1A表达在血管平滑肌细胞表型转化过程中起重要作用；在预测靶向调节Gax基因的miRNA中发现miR-130a的可能性大，细胞实验结果显示与对照组相比，miR-130a过表达能够显著下调血管平滑肌细胞中的Gax表达，miR-130a低表达能显著上调血管平滑肌细胞中的Gax表达，差异均具有统计学意义（P<0.05），表明在血管平滑肌细胞表型转化过程中，miR-130a对Gax基因表达起调控作用；本项目采用基因芯片技术检测抑制性转录因子Gax的下游靶基因，结果显示Rap1A在Gax过表达的血管平滑肌细胞中变化最为显著，细胞实验结果显示Rap1A过表达能够显著减轻Gax表达对血管平滑肌细胞增殖、迁移、以及标志性蛋白Calpoin和SM-MHC的作用，差异有统计学意义（P<0.05）；细胞实验结果还显示miR-130a过表达能够显著上调血管平滑肌细胞中Rap1A的表达，miR-130a低表达显著下调Rap1A的表达，与对照组相比，差异具有统计学意义（P<0.05）；Real-time PCR结果显示，与对照组相比，miR-130a和Rap1A在胸主动脉瘤壁中膜组织中高表达，Gax呈低表达，差异有统计学意义（P<0.05）; Pearson相关性分析方法结果显示，miR-130a，Gax和Rap1A表达之间存在相关性，有统计学意义（P<0.05）；本项目研究表明胸主动脉瘤血管平滑肌细胞表型转化过程中存在miR-130a-Gax-Rap1A调控轴，项目的完成有助于阐明胸主动脉瘤的发病机制，有助于寻找能够评估和调控胸主动脉瘤病情发展的关键分子，为防治胸主动脉瘤提供新靶标。