铁过负荷抑制miR-122表达的分子机制及其在肝脏炎症反应中的作用

It is indisputable that iron overload induces and aggravates non-infectious inflammation in liver. However, the underlining mechanism remains unclear. Previous reports indicated that hepatic inflammation is regulated by microRNAs through targeting multiple signaling molecules. Most recently, we observed that hepatic iron overload reduced liver-specific miR-122 expression and upregulated NF-κB pathway. Meanwhile, overexpression of miR-122 significantly treated liver inflammation upon iron overload. Therefore, we hypothesized that hepatic iron overload disrupts expression and function of microRNAs in hepatic cells and subsequently induces non-infectious inflammation. To this end, we plan to utilize methods of microarray, bioinformatics and molecular biology involving both cultured cells and experimental animals. We will further investigate the regulation of iron overload on miR-122 expression as well as the expression of its upstream transcription factors and downstream, inflammation-related target genes. Furthermore, the underlying molecular mechanisms will be fully determined. In addition, to reveal the roles of identified genes, such as miR-122 and its target genes, in iron overload-induced inflammation, adeno-associated viral vector-mediated, liver-targeted gene transfer techniques will be used to overexpress and/or knockdown specific genes in vivo. Our results will advance the knowledge of hepatic iron overload and inflammation. The new potential targets identified from these studies will provide theoretical guidance and experimental basis for the development of new anti-inflammatory treatment.

铁过负荷可引起和/或加重肝脏炎症反应已是不争的事实，但机制尚未完全阐明。近年研究证实miRNAs可通过多个靶分子影响肝脏炎症过程。最近我们发现肝铁过负荷可引起肝脏特异性miR-122表达下降和NF-КB信号通路活性增强，而提高miR-122表达则可以明显逆转铁过负荷引起的肝脏炎症反应。因此，推测铁过负荷很可能通过影响肝脏miRNAs的表达和功能，促进炎症反应。为证实此推测，本项目拟采用细胞实验和动物实验相结合的方法，利用基因芯片、生物信息学、分子生物学等技术，进一步确定铁过负荷对miR-122表达的调控作用，及其上游转录调控因子和下游与炎症有关的靶基因，阐明作用机制，并通过腺相关病毒介导定向过表达或干扰肝脏相关基因等方法，阐明miR-122及其靶基因与铁过负荷引起炎症的关系。结果将推进人们对肝铁负荷与炎症关系的认识。如发现治疗肝脏炎症潜在靶点，将为开发新的抗炎途经提供理论指导和实验依据。

为了证实铁过负荷可以引起和/或加重肝脏炎症反应，并在此基础上阐明其分子机制，在项目经费资助下的研究发现：（1）铁过负荷可不依赖巨噬细胞，通过肝实质细胞特异性的HNF4α/miR-122通路直接造成肝实质细胞炎症信号通路激活，进而造成肝脏炎症；（2）铁过负荷造成的HNF4α/miR-122/CCL2炎症通路活性变化与氧化应激反应没有明显的联系，很可能是铁过负荷的直接作用结果；（3）铁过负荷可能是通过激活泛素化连接酶TRIP12，进而影响转录因子YY1蛋白稳定性使其蛋白量减少，导致其与HNF4α的结合活性减弱，最终引起HNF4α/miR-122/CCL2炎症通路活性变化；（4）通过重组腺相关病毒AAV8靶向肝脏过表达YY1可以明显缓解铁过负荷引起的肝脏炎症反应的发生。. 本研究结果初步阐明，铁过负荷可以通过YY1/HNF4α/miR-122/CCL2通路导致肝实质细胞发生炎症反应，此信号通路独立于氧化应激。上述研究结果进一步完善了人们对肝铁负荷与炎症关系的认识。有意义的是，本研究结果还提示，通过生物技术过表达转录因子YY1很可能是缓解铁过负荷所引起的肝脏组织细胞炎症反应的重要措施。相关研究结果已发表论文4篇。