CircRNA_007585-miRNA-326-UCP2调控通路在非酒精性脂肪性肝炎中的作用及机制研究

Uncoupling protein (UCP) guided mitochondrial dysfunction plays a pivotal role in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the mechanism is still unclear. Currently, based on the capacity of post transcriptional regulation and effect of“microRNA sponge”, miRNA and circRNA become hot research spot but their role in NASH has not been reproted. Based on previously established UCP, miRNA/circRNA research platform and preliminary experiment result, the applicants propose that circRNA_007585 efficiently binds miRNA-326 to increase UCP2 expression and finally promotes NASH progression. To verify this hypothesis, the applicants plan to use UCP2, the important representative of UCP, as the target gene and test the expression and regulation of circRNA_007585, miR-326 and UCP2 using RIP, luciferase report gene and other related methods, on the basis of sample enlargement and addtion of in vitro research. Furthermore, respective antagonization of circRNA, miRNA and UCP2 expression would be carried out to explore their influence on NASH phenotype and potential down stream mechanisms, including expression of lipid deposition and inflammation related genes. The anticipated results may provide new clues for the pathogenesis and therapy of NASH that shows increased global prevalence.

解偶联蛋白(UCP)介导的线粒体功能失调在非酒精性脂肪性肝炎(NASH)中起重要作用，但机制未明。miRNA因其对下游基因的转录后调控作用、circRNA因其对miRNA的高效“海绵样吸附”作用成为研究热点, 但在NASH中的作用未见报道。申请人在既往建立的UCP和miRNA/circRNA研究平台基础上，结合预实验结果，提出“circRNA_ 007585通过高效竞争性吸附miR-326来增加UCP2表达以促进NASH发生发展”这一假说。申请人计划以解偶联蛋白中的重要代表UCP2为靶基因，在扩大样本及增加细胞层面研究的基础上，运用RIP、荧光素酶报告基因等技术验证circRNA_007585、miR-326和UCP2在NASH中的表达及调控情况，再分别对其进行过表达或拮抗，以探索对NASH程度的影响和可能下游机制(包括脂质沉积和炎症反应相关基因)，为NASH发病机制研究和诊治提供新思路。

非酒精性脂肪性肝病（nonalcoholic fatty liver disease, NAFLD）是指除外酒精和其他明确肝损因素所致的以弥漫性肝大泡性脂肪变、伴或不伴炎症为主要特征的临床病理综合征。其中NASH可逐渐进展为终末期肝病，成为NAFLD发展进程中的重要阶段和分水岭，是隐匿性肝硬化的主要诱因。在国家自然科学基金委面上课题的资助下，申请人建立包括RIP， FISH、荧光素酶报告基因检测等技术平台，聚焦circRNA及细胞自噬在NASH中的作用， 在体内外层面通过系列研究，发现前期所预测的circRNA_007585-miR-326-UCP2这条通路调控证据不充分，遂改选当时预测的另一条circRNA调控通路，并完成下述实验结果：阐明circRNA_002581及CPEB1的差异性表达，并证实circRNA_002581-miR-122-CPEB1通路存在；发现干扰circRNA_002581后可以改善动物和细胞模型中NASH程度，包括脂肪沉积（HE和油红染色）、炎症指标（ALT，AST，TNF-a，白介素系列等）、细胞凋亡、能量代谢（ATP）、脂质过氧化（H2O2）等；通过检测自噬相关蛋白（P62、LC3-I，II）、电镜评估自噬体数量、免疫荧光检测LC3表达及定位，自噬流测定等，证明自噬在NASH中受到抑制，而干扰circRNA_002581后可以部分缓解该抑制，这可能是以circRNA_002581为靶点进行NASH治疗的潜在机制；进一步发现自噬中的经典AMPK-mTOR通路为circRNA_002581-miR-122- CPEB1调控的下游通路，后者通过调控PTEN进一步发挥作用。上述研究为NASH的发病机制及诊治提供了新思路和新方法。在本课题资助下，负责人发表课题资助标注SCI论文3篇，成功晋升为主任医师、博士生导师，培养一名硕士研究生毕业，目前四名硕士研究生在读，一名联合培养博士生在读。同时部分研究的延续内容申请到2020年浙江省自然科学基金重点项目(LZ21H030002)资助。