MUC5B基因启动子区多态性与IPF的相关性及分子机制研究

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive and ultimately fatal disease with unknown aetiology and unclear pathogenesis. At present, there is no effective treatment except lung transplantation. Recently, several studies have reported that rs35705950 located in the promoter region of the MUC5B gene is associated with IPF in the European and American population, but there is no data of Chinese population. Previously we have genotyped 49 IPF patients and 50 controls in Han Chinese, and found that rs35705950 confers risk of IPF, but its mechanism is still unclear. By bioinformatic analysis, we discovered that rs35705950 located in the CpG islands of MUC5B promoter region, suggesting that epigenetic mechanisms may be involved in the development of IPF. In the present study, we intends to validate the association between the MUC5B gene and IPF by fine mapping the gene promoter region in a larger Chinese population, and to analyze the correlation between gene polymorphisms and clinical features; to detect MUC5B expression level as well as the promoter methylation status in the lung tissues of the IPF patients and controls; in the human lung epithelial cells, to check how the promoter methylation status affect the transcriptional factor binding condition via ChIP assay; to observe the effect of MUC5B overexpression on the lung epithelial cells secretion; and to explore whether the MUC5B gene is a new therapeutic target for IPF treatment in a mouse model. The present research from the perspective of epigenetics, will provide new theoretical basis for the clinical prevention and treatment..

特发性肺纤维化（IPF）病因不明，目前除肺移植外无其他有效方法。最近研究发现MUC5B基因启动子rs35705950与欧美人群的IPF相关，但无中国人群的数据。课题组前期对中国汉族人群49例IPF患者和50例对照分析发现rs35705950等位基因与IPF相关，但其作用机制不明。我们经生物信息学发现rs35705950位于MUC5B启动子区CpG岛，提示表观遗传机制可能参与IPF发生发展。本项目拟进一步扩大样本量对MUC5B启动子区进行精细定位并分析多态性与临床特征的相关性；在IPF及对照肺组织检测MUC5B表达水平，BSP法检测MUC5B启动子甲基化状态；以人肺上皮细胞为模型，ChIP法检测不同甲基化状态对转录因子结合的影响；观察MUC5B过表达对肺上皮细胞分泌细胞因子的影响；在IPF小鼠模型中观察MUC5B是否能成为新的治疗靶点。本项目从表观遗传学角度，为IPF防治开辟新思路。

特发性肺纤维化（IPF）是一种病因不明、进行性发展的间质性肺疾病，目前除肺移植外无其他有效治疗方法。遗传因素在IPF发病过程起重要作用。近期研究发现MUC5B基因启动子区多态性位点rs35705950与欧美人群IPF的发生和预后相关，但在中国人群中尚未充分验证。本项目从IPF的遗传易感性分析入手，采用“病例-对照”的关联分析，在中国北方汉族人群的162例IPF患者和200例对照受试者中，检测了MUC5B基因启动子区rs35705950多态性及其附近的三个常见变异位点（rs2672794,rs868903,rs12804004）与IPF的易感性，发现仅rs35705950位点与疾病相关（p = 0.015）。同时收集了IPF家系寻找疾病相关的罕见变异，在2个家系中分别发现了MUC5B基因的一个错义突变c.9977C>T (p.Thr3326Met)，和TERT基因的一个错义突变c.2146G>A (p.Ala716Thr)。MUC5B基因表达调控方面，发现在IPF患者肺组织上皮细胞中MUC5B基因表达量增高；博来霉素诱导的小鼠肺纤维化模型中，发现经5-氮杂胞苷处理的肺纤维化组MUC5B基因表达量有下降趋势，推测MUC5B基因的甲基化状态可能影响其表达调控。综上，本研究在遗传学水平验证了MUC5B基因的变异与散发型和家族性IPF相关性，并在基因表达水平探索了该易感基因的功能。