FOXA1调控肝星状细胞SF2-FN1可变剪切介导肝纤维化的分子机制

Liver cirrhosis or even hepatic carcinoma developed from liver fibrosis is the leading cause of death from liver disease. Hepatic stellate cells (HSC) activation plays a key role in liver fibrosis, and the splicing factor SF2 in HSC mediates the formation of EDA+FN1 spliceosome that promotes the formation of liver fibrosis. In our previous studies, we found the expression of linc00663, FN1 and SF2 were up-regulated in the human liver fibrosis tissue; furthermore, the expression level of linc00663 was positively correlated to the expression levels of FN1 and SF2. Thus, we suspected the miR-3916 could bind to the 5’-UTR region of SF2 and the FOXA1 could bind to the promoter of linc00663. It remains unclear whether or not FOXA1 could activate the linc00663 to compete with the 5’-UTR-IREs of SF2 to bind miR-3916, then further regulate the FN1 alternative splicing and produce effect on live fibrosis. The present project aims to study that FOXA1 actives linc00663 and regulates the formation of SF2-FN1, therefore affects the activation of HSC by EDA+FN1 spliceosome, which is evidenced by PBC rodent model to further clarify the underlying mechanisms. The findings will explain the novel molecular mechanism that FOXA1 regulates the SF2-FN1 alternative splicing to mediate the activation of HSCs, and will provide the theoretical and experimental basis for the discovery of new molecular markers of liver fibrosis.

肝纤维化发展为肝硬化甚至肝癌是导致肝病死亡的最主要原因。HSC活化是肝纤维化的关键环节，其含有的剪切因子SF2介导形成EDA+FN1剪切体促进肝纤维化形成。课题组检测到人纤维化肝组织高表达linc00663、FN1和SF2，且linc00663与FN1、SF2表达正相关；预测到miR-3916与SF2的5’-UTR及FOXA1与linc00663启动子结合。FOXA1能否激活linc00663、竞争miR-3916与SF2的5’-UTR-IREs结合、调控SF2-FN1可变剪切，影响肝纤维化？有待阐明。本项目拟通过FOXA1激活linc00663，调控SF2-FN1可变剪切体，调节EDA+FN1的生成影响HSC活化，经PBC肝纤维化模型小鼠试验验证，从而阐明FOXA1调控SF2-FN1可变剪切介导HSC活化的全新分子机制，为发现新型肝纤维化分子标志物奠定理论基础和试验依据。

胆汁淤积性肝病是由各种原因引起的胆汁不能正常流入十二指肠而进入血液的病理状态，如果病程超过6个月，则成为慢性胆汁淤积，后期会引起胆汁性肝纤维化、肝硬化甚至肝癌的发生。肝纤维化（Hepatic Fibrosis，HF）是机体针对各种原因导致的的慢性肝损伤后的一种修复反应，表现为细胞外基质（Extracellular Matrix，ECM）成分尤其是胶原等的过度形成和沉积。有研究报道，ECM的主要来源均为活化的肝星状细胞（HSCs，Hepatic Stellate Cells）。长链非编码RNA（Long non-coding RNA，LncRNA）已被证明参与多种重要生物过程的调节，然而，lncRNA参与胆汁淤积性肝纤维化的机制仍不清楚。我们通过加权基因共表达网络分析（Weighted Gene Correlation Network Analysis，WGCNA）分析来自胆管结扎（BDL，Bile Duct Ligation）小鼠或对照组的HSCs的RNA测序数据，基于WGCNA分析，构建了LINC00663相关的竞争性内源性 RNA（competing endogenous RNA，ceRNA）网络，我们通过划痕试验、双荧光素酶报告基因试验、RNA结合蛋白免疫沉淀和染色质免疫沉淀等试验评估LINC00663的功能。我们发现，LINC00663可以促进LX-2细胞的活化水平、迁移能力和上皮-间质转化（EMT，Epithelial-Mesenchymal Transition）功能，并促进了BDL小鼠肝纤维化进展。在机制方面，我们证明了LINC00663通过吸附hsa-miR-3916调节剪接因子2（Splicing Factor 2，SF2）-纤连蛋白（Fibronectin，FN）交替剪接。此外，叉头盒A1（Forkhead box A1，FOXA1）可以与LINC00663的启动子特异性相互作用。总之，我们阐述了LINC00663对人肝星状细胞LX-2和胆管结扎胆汁淤积症小鼠的纤维化作用。我们建立了FOXA1/LINC00663/hsa-miR-3916/SF2-FN轴，为胆汁淤积性肝纤维化的诊断和靶向治疗提供了潜在靶点。