miRNA线粒体-胞浆分布异常在亚临床甲减肝脏脂代谢紊乱中的作用及机制研究

TSH升高是亚临床甲减的主要特征，常引起患者肝脏脂质聚积等脂代谢紊乱。线粒体是脂代谢的枢纽，也是巨大的miR存储池；其中大量细胞核编码的miR是协调线粒体和细胞功能的重要分子，但迄今对其转运和调控机制及病理意义仍知之甚少。我们前期研究表明TSH能使肝脏/肝细胞脂质聚积，并使大量miR在线粒体和胞浆间的分布异常，且两者显著相关，但其具体机制不清，国内外也未见报道。因此，本研究拟用亲和纯化与质谱和二代核酸测序、报告基因、普通或靶向线粒体/肝脏的微环载体等技术，1）阐明TSH引起miR线粒体-胞浆分布异常的机制；2）揭示分布异常的miR在TSH导致的肝脏脂代谢紊乱中的作用和机制；3）在患者样本、野生型和肝脏特异TSHR敲除的亚甲减小鼠中验证并揭示这些机制的临床意义。本项目有助于填补对miR线粒体转运机制认识的空白，拓展对线粒体和miR功能调控方式的认识，加深对TSH导致肝脏脂代谢紊乱机制的理解。

Elevated TSH is the major feature of subclinical hypothyroidism, resulting in disorders of hepatocyte lipid metabolism. Mitochondria are the hub of lipid metabolism and a huge storage pool for miRNAs. A large number of nuclear-encoded miRNAs have regulatory functions inside/outside the mitochondria, which are important mechanisms for the coordinated function between mitochondria and cells. However, little is known about the mechanisms for their transport, regulation and pathological abnormalities until now. Our preliminary studies have shown that TSH can induce lipid accumulation in hepatocytes and abnormal mitochondrial-cytoplasmic distribution of a large number of miR, but their roles in TSH-induced hepatocyte lipid metabolism disorder, especially the general mechanism of miR trans-mitochondrial transport have not been reported so far. Therefore, this project will use biotinylated microRNA pull-down, biotin affinity purification plus mass spectrometry and second-generation DNA sequencing, dual luciferase reporter gene and expression/siRNA/sponge mini-circle vector and other functional research techniques to reveal the mechanism of hepatocyte miR trans-mitochondrial transport. Moreover, this project will elucidate the roles and signaling networks of abnormal distribution of miR mitochondria-cytoplasm in TSH-induced hepatocyte lipid metabolism disorders. Furthermore, its clinical significance will be evaluated and revealed in mouse model and patient samples of subclinical hypothyroidism. This project will help us to understand the mechanism of TSH-induced lipid accumulation in hepatocytes from the perspective of miR trans-mitochondrial transport, and expand the understanding of regulation methods for mitochondria and miRNA function. This project will also fill the gap in understanding the mechanism of miR mitochondrial transport.