miR-152通过调节Wnt信号通路致非酒精性脂肪肝病的机制研究

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of triglycerides, which are formed from the esterification of FFA and glycerol within hepatocytes. Although nowdays the "three hit hypothesis" was considered as the dominant theory, the pathogenesis of NAFLD is still vague and needs further exploration. In our previous study, we found that miR-152 was highly expressed in NAFLD rat liver tissue by deep sequencing technology. We also found that miR-152 was highly conserved between human and rats, and Wnt10b was the target gene through bioinformatics software and database analysis. Further preliminary experiments disclosed that over-expression of miR-152 in human hepatocyte not only significantly promoted lipid accumulation but also dramatically down-regulated Wnt10b, which indicated that miR-152 may induce lipid accumulation in hepatocyte through the mechanism of regulation of Wnt signal pathway. This study is to elucidate the biological characteristics of the miR-152 and its effect in the hepatocyte lipid synthesis and in the Wnt signal pathway by overexpression and knockdown miR-152 in L-02 hepatocytes. We also intend to investigate the function and mechanism of miR-152 in NAFLD by looking at the starting point of Wnt signal pathway. So far, the role and mechanism of the miR-152 in NAFLD lipid synthesis has not been reported, thus this study is initially innovation. It may provide new targets for the prevention and treatment of children of NAFLD.

肝细胞内脂质异常积聚是非酒精性脂肪肝病（NAFLD）的重要病理学基础，但其机制不明。前期研究中，我们通过二代测序技术发现miR-152差异高表达于NAFLD大鼠肝组织中；生物信息学发现，miR-152在人与大鼠间高度保守，Wnt10b是其靶基因；预实验发现在肝细胞中过表达人miR-152不但显著促进脂质积聚，而且显著下调Wnt10b，提示miR-152可能通过调节Wnt信号通路的机制致肝细胞内脂质积聚。本研究拟阐明miR-152的生物学特征；采用miRNA mimics及inhibitors评价miR-152对NAFLD细胞脂质合成、Wnt信号通路的影响；以Wnt信号通路为切入点，分析miR-152影响脂质合成的机制，论证miR-152在NAFLD中的作用与机制。miR-152影响NAFLD脂质合成的作用与机制均未见报道，本研究具有源头创新性，可为儿童NAFLD的防治提供新靶标。

MiRNAs参与肝脏脂质积聚、胰岛素抵抗等过程调节，与非酒精性脂肪肝病（NAFLD）的发生发展密切相关。本课题通过高脂饮食诱导SD大鼠建立NAFLD模型，运用二代测序技术得到肝组织中异常表达miRNAs，结合运用生物信息学、细胞生物学、分子化学等方法与技术，对miR-152通过WNT信号通路参与NAFLD疾病发生发展的分子机制进行了初步研究。首先构建miR-152过表达慢病毒和miR-152沉默慢病毒，分别转染人正常肝细胞和人肝癌细胞中，得到miR-152过表达细胞和miR-152沉默细胞；以空载为对照，棕榈酸和油酸干预三组肝细胞，测定肝细胞脂质积聚情况；同时，通过构建荧光素酶报告基因质粒，结合生物信息学分析结果验证miR-152靶基因为Wnt10b；进而利用RT-PCR及Western blot技术检测WNT信号通路及下游脂质合成关键转录因子的表达水平，结果显示miR-152增加GSK-3β磷酸化水平，促进β-Catenin降解，抑制β-Catenin进入肝细胞核内，上调脂质合成关键转录因子SREBP-1c和PPARγ的表达。最后通过检测肝细胞内ATP含量和线粒体拷贝数，发现miR-152对线粒体功能的负调控作用。综合上述结果，本研究为探讨miR-152调控WNT信号通路参与NAFLD发生的分子机制奠定了坚实的工作基础，同时对NAFLD防治新靶标提供了重要的启示性线索。项目资助发表SCI论文3篇，核心论文1篇，国际会议论文2篇，待发表SCI论文2篇。培养硕士生4名。项目实际投入经费52万元，支出34.03万元，各项支出基本与预算相符。剩余经费17.9万元，剩余经费计划用于本项目研究后续支出。