MicroRNA-29b/142-5p调控干扰素-γ基因高表达促进胆道闭锁发病的机制研究

Biliary atresia (BA) is a life-threatening cholestatic disease and its cause is still unkown. The overexpression of INF-γ and its epigenetic regulation have been found to play an important role in the pathogenesis. Our previous study found in BA the phenomenon of DNA hypomethylation in the genome and the promoter region of the INF-γ gene, and DNA methylases (DNMTs) were obviously down-regulated. Epigenetics demonstrated that DNA methylation and DNMTs could be regulated by micro ribonucleic acid (microRNA, miR). Our microRNA microarray study found many up-regulated microRNAs in BA,including miR-29b and miR-142-5p, which could regulate DNMTs by targeting DNMTs mRNAs. Therefore,we assume that there exits an molecular mechanism in the pathogenesis of BA, that is " microRNA overexpression - DNMTs downregulation - DNA hypomethylation in the promoter region of INF-γ gene - INF-γ over-expression". The following study is to verify the mechanism in three levels of human tissue,cell and animal model. This study will be important for unclosing the mechanism of pathogenesis in BA.

胆道闭锁（biliary atresia,BA）是严重威胁儿童健康的胆汁淤积性疾病，研究其发病机制具有重大意义。现已知INF-γ高表达及其表观遗传学调控在BA发病中具有重要作用。本项目组前期研究发现，BA患儿存在基因组整体水平和甲基化敏感基因INF-γ启动子区低甲基化，并且DNA甲基转移酶(DNMTs)表达下降。表观遗传学研究发现DNA低甲基化和DNMTs受到微核糖核酸（miR）调控。本项目组通过miR芯片发现BA患儿存在多种miR高表达，包括miR-29b和142-5p，而miR-29b和142-5p可靶向抑制DNMTs mRNA翻译。因此我们推测，在胆道闭锁发病中存在"miR高表达-DNMTs抑制-INF-γ启动子区低甲基化-INF-γ高表达"这一机制。本课题将从人体组织、细胞和动物模型三个层次验证miR抑制DNMTs促进INF-γ高表达的机制。本课题对揭示BA的发病机制具有重要意义。

胆道闭锁（Biliary atresia, BA）是发生在婴幼儿时期最常见的胆汁淤积性疾病，然而其发病机制未知。课题前期通过 miRNA 芯片筛查，发现 miRNA-29b/142-5p在胆道闭锁患儿肝脏及外周血中均升高，且与表观遗传密切相关。通过大样本验证后，明确胆道闭锁肝脏中 miRNA-29b/142-5p升高，DNMTs 明显降低，基因组 DNA 整体甲基化水平降低， IFN-γ 启动子区的甲基化水平减低，IFN-γ 蛋白表达明显升高，且miRNA-29b/142-5p 升高的水平与 IFN-γ 的表达呈现出正相关关系。通过荧光报道素酶实验进一步确定 miRNA-29b/142-5p 对DNMTs 的靶向作用，通过 5-氮杂-2’-脱氧胞苷对 Jurkat 细胞和 LO2 细胞的作用，确定去甲基化对 IFN-γ分泌的促进作用；通过分别转染 siRNA、miRNA mimic 和 miRNA inhibitor 明确了 miRNA-29b/142-5p 通过靶向抑制 DNA 甲基转移酶对 IFN-γ 基因启动子区甲基化的调控，使得 IFN-γ 表达增加，促使胆道闭锁的发生和发展。这将为表观遗传学在胆道闭锁发病中提供一定的启示，为胆道闭锁病因的研究提供新的思路。