mRNA选择性剪切调控分子Fox1基因突变参与原发性胆汁性肝硬化发病机制的研究

mRNA alternative splicing is one of key regulating molecular events to make various proteins, whose abnormal function might contribute to the aetiology and development of primary biliary cirrhosis(PBC). Our recent studies showed that: 1) expression of Fox1, splicing regulatory molecule, significantly increased in the liver tissues of PBC patients compared with normal liver; 2) mutations of Fox1 rs78684827 C/A was correlated with PBC by mass array analysis; 3）its mutations was confirmed to result in sequence variant of amino acid position 322 on the nuclear localization signal of Fox1 protein.Therefore, mutations of Fox1 rs78684827 C/A might contribute to abnormal mRNA alternative splicing of its regulatory target genes and hepatic dysfunction in PBC patients. In this study, we plan to study Fox1 participating in mRNA alternative splicing regulation of its target genes and the effect of its mutations of rs78684827 C/A on its gene and hepatic function in PBC patients using reporter gene assay，electrophoretic mobility shift assay（EMSA），exon array, single base substitution and so on. Our study would contribute to further disclosing the molecular mechanism of PBC aetiology and its biological therapeutic targets.

mRNA选择性剪切是产生蛋白质多样性的关键调控环节，其功能异常与自身免疫性疾病的发生、发展密切相关。本所前期研究发现，在原发性胆汁性肝硬化（PBC）患者肝组织中mRNA选择性剪切调控分子Fox1较正常肝组织表达明显增高；Mass array结果显示Fox1基因rs78684827 C/A突变与PBC发病高度相关；同时该位点突变可使Fox1蛋白C端核定位信号区的第322氨基酸发生错译。以上结果提示：PBC患者Fox1基因rs78684827 C/A突变可能会使其对靶基因的选择性剪切出现异常，进而导致肝细胞功能障碍。本课题拟通过外显子表达谱芯片、报告基因、RT-PCR、EMSA、点突变等方法研究PBC患者肝细胞中Fox1对靶基因的选择性剪切调控，探讨rs78684827 C/A突变对Fox1基因选择性剪切调控能力及肝细胞功能的影响，为阐明PBC发病机制和发现治疗新靶点提供试验依据。

mRNA选择性剪切是产生蛋白质多样性的关键调控环节，其功能异常与自身免疫性疾病的发生、发展密切相关。本项目发现原发性胆汁性肝硬化（PBC）患者肝组织中mRNA选择性剪切调控分子Fox1较正常肝组织表达明显增高，对4个PBC家系的Fox1基因外显子测序发现MAF>0.05的FOX1基因SNPs共有33个，两两间连锁不平衡分析共发现7组完全连锁位点；Fox1基因rs7 8684827 C/A突变与PBC发病高度相关，该位点突变可使Fox1蛋白C端核定位信号区第322氨基酸发生错译，但不影响其选择性剪切功能的功能。Fox1基因敲除大鼠呈现胚胎致死性，说明其具有重要功能，发现Fox1基因参与了自噬相关Lamp2基因亚型Lamp2A选择性剪切的调控，Lamp2A介导的CMA自噬通过脂代谢和胆汁酸代谢参与了PBC疾病的发生发展，一方面直接降解脂滴包被蛋白plin5参与脂滴的分解代谢，另一方面直接或间接参与了肝细胞胆管侧细胞膜胆红素转运体MRP2的细胞内运输，本项目为阐明PBC发病机制和发现治疗新靶点提供试验依据。研究结果已发表SCI论文3篇，培养博士研究生1人，硕士研究生4人。

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