肺癌中miR-449a表观遗传修饰异常抑制miR-449a-MAPK负反馈环，促进细胞转移的研究

In our previous studies, we found that miR-449a could regulate lung cancer cells invasion and metastasis through MEK1-ERK1/2-AP1-MMP2 pathway. On this basis, we will use promoter reporter gene method to study whether miR-449a could forming miR-449a-MAPK negative feedback loop through AP1. Whether miR-449a expression is affected by the promoter DNA methylation and histone methylation modification will be studied by BSP, MSP and chromatin immunoprecipitation methods. Demethylating reagent, plasmid transfection and real-time PCR methods will be used to study whether abnormal epigenetic modifications suppress the miR-449a-MAPK negative feedback loop. In addition, the interrelation among miR-449a expression, miR-449a abnormal methylation and clinicopathological parameters will be studied by real-time PCR and MSP methods. This study will analyze the molecular mechanism and signaling pathway through which miR-449a regulates lung cancer metastasis, reveal the effects of abnormal epigenetic modification on lung cancer metastasis regulated by miR-449a, and provide experimental evidence and theoretical basis for clarifying the hypothesis of "miR-449a-MAPK negative feedback loop" and "miR-449a abnormal epigenetic modifications" raised by us.

前期研究发现miR-449a可通过MEK1-ERK1/2-AP1-MMP2途径调控肺癌转移。本项目拟应用启动子报告基因等方法，研究miR-449a是否通过AP1形成miR-449a-MAPK负反馈环；应用BSP、MSP和染色质免疫共沉淀方法，研究miR-449a表达是否受启动子DNA甲基化及组蛋白甲基化修饰调控；应用去甲基化试剂处理、质粒转染及real-time PCR方法，研究表观遗传异常修饰是否抑制miR-449a-MAPK负反馈环；应用real-time PCR、MSP等方法，分析肺癌组织miR-449a表达与异常甲基化及临床病理参数关系；分析和阐明miR-449a调控肺癌转移的分子机理和信号途径，为揭示表观遗传学异常对miR-449a调控肺癌转移的影响，为阐明我们提出的"miR-449a-MAPK负反馈环"和"miR-449a表观遗传修饰异常"假说提供实验依据和理论基础。

转移是肺癌患者死亡的主要原因，探讨肺癌侵袭转移的确切机制和信号调节途径，以寻找更好的早期诊断、预测、逆转和治疗肺癌转移的有效措施具有重要的理论指导意义。我们前期通过microRNA芯片找到了与肺癌侵袭转移相关的miRNA, 其中miR-449a表达差异显著，在高转移潜能细胞中低表达；进一步研究发现miR-449a可通过MEK1-ERK1/2-AP1-MMP2途径调控肺癌细胞侵袭转移。在本项研究中，我们通过启动子报告基因和Western blot方法，证实miR-449a基因启动子处含有AP1结合位点，可通过AP1位点形成miR-449a-MAPK负反馈环；应用染色质免疫共沉淀方法发现肺癌细胞中miR-449a低表达受H3K27Me3组蛋白甲基化修饰调控，机制研究发现SUZ12介导H3K27me3调控miR-449a表达；应用裸鼠移植瘤模型发现miR-449a抑制肿瘤的形成和转移；应用qRT-PCR检测肺癌组织标本及淋巴结转移灶中miR-449a的表达，发现肺癌淋巴结转移灶中miR-449a低表达，且miR-449a低表达与肺癌患者的淋巴结转移，临床分期及患者吸烟情况密切相关；本研究发现肺癌细胞中miR-449a表达受H3K27me3修饰，miR-449a可通过 MEK1/ERK1/2/c-Jun信号途径形成自身负反馈环抑制肺癌转移。本项目研究丰富了肺癌转移的分子机理和信号途径，揭示了表观遗传学异常对miR-449a表达及miR-449a调控肺癌转移的影响，为临床寻找新的治疗靶点提供理论依据。