miR-26a/内质网应急调控回路在非酒精性脂肪性肝病中的功能及机制研究

Nonalcoholic fatty liver disease (NAFLD) is a disease syndrome characterized by predominant macrovesicular steatosis of the liver in the absence of chronic alcohol consumption. It has become the most common cause of chronic liver disease. Recent evidence strongly supports an important role of endoplasmic reticulum stress (ER stress) in NAFLD. However, the molecular mechanism that ER stress affects NAFLD is poorly understood. Therefore, understanding the role and mechanism of ER stress in NAFLD have become topics of intense investigation. miR-26a plays a critical role in development and various human diseases, especially cancer, but its function in ER stress and NAFLD remains unknown. In this study, we propose to investigate the relationship between miR-26a and ER stress and its implications in NAFLD. Our pilot studies revealed a new feedback loop between miR-26a and ER stress in liver cells, in which ER stress induces miR-26a expression, whereas miR-26a alleviates ER stress. Our previous work also showed that miR-26a regulates insulin sensitivity and metabolism of glucose and lipids and hepatocyte-specific overexpression of miR-26a in mice alleviates HFD-induced ER stress and hepatic steatosis. These preliminary data strongly suggest a regulatory circuit between miR-26a and ER stress, as well as its role in NAFLD. To further explore the above preliminary findings, we will continue to investigate the molecular mechanism underlying miR-26a regulation by ER stress. Meanwhile, we will use a combination of bioinformatics and RNA deep sequencing to perform a high-throughput screen for miR-26a targets involved in ER stress, by which to clarify the mechanism that miR-26a modulates ER stress. Furthermore, we will utilize our previously established hepatocyte-specific miR-26a transgenic mice to examine the role of miR-26a in NAFLD. Finally, we will verify the correlation between miR-26a and ER stress, as well as their function, in NAFLD patients. The proposed work is innovative because it will establish a novel miRNA-ER stress pathway in NAFLD. In addition, we are ideally positioned to carry out these studies because of our unique miR-26a transgenic mouse lines. In summary, this project will establish a novel link between miRNAs, ER stress and NAFLD and could provide novel opportunities for treating this disease. Because ER stress is widely associated with many other diseases, including cancer and metabolic disease, our model may also provide a novel mechanistic paradigm for these diseases.

非酒精性脂肪性肝病(NhAFLD)是肝病领域的新挑战。内质网应急在NAFLD中作用重要，但分子机制不清楚。miR-26a作用广泛，但是它与内质网应急、NAFLD的关系尚未建立。我们的前期研究发现：肝细胞内质网应急诱导miR-26a，同时miR-26a缓解内质网应急，形成反馈调控回路；miR-26a调控细胞代谢，并可能抑制NAFLD发生发展。本研究旨在阐明miR-26a/内质网应急调控回路及其机制，揭示其在NAFLD中的功能和机理。拟开展以下工作：1）明确内质网应急调控miR-26a的分子机制；2）鉴定miR-26a的靶基因,确定它调节内质网应急的分子机制；3）利用前期建立的miR-26a转基因小鼠，在NAFLD小鼠模型中，验证miR-26a功能；4）临床样本分析miR-26a、内质网应急与NAFLD的关系。本研究将建立miR-26a和内质网应急的新联系，为NAFLD的诊断治疗提供潜在靶点。

NAFLD是一种无过量饮酒史，以肝细胞脂肪变性和脂肪贮积为特征的临床综合征。NAFLD的发病趋势呈低龄化且增长迅速，是发达国家和富裕地区第一大肝病，也是我国慢性肝病的首要病因。此外，NAFLD还与2型糖尿病、心血管疾病、神经性疾病等多种疾病密切相关。遗憾的是，由于NAFLD研究长期被忽视，其发病机制很不清楚，目前也缺乏有效的药物治疗。因此，鉴定NAFLD的治疗新靶点具有十分重要的理论意义和临床价值。本项目系统揭示了miR-26a/内质网应激负反馈调控回路在NAFLD发生发展中的作用及机制。主要结果如下：（1）发现了肝细胞内质网应激特异性诱导miR-26a表达：利用三种不同化合物在人肝癌细胞系及小鼠原代肝细胞中建立内质网应激模型，通过miRNA点杂交、实时荧光定量PCR（QRT-PCR）等方法，发现了内质网应激特异性上调miR-26a表达。（2） 阐明了miR-26a对肝细胞内质网应激的抑制作用及其机制：在人肝癌细胞系及小鼠原代肝细胞中，通过过表达或敲除miR-26a等手段，发现了miR-26a能显著减轻衣霉素、毒胡萝卜素以及棕榈酸引起的内质网应激及脂质积累，并进一步鉴定了内质网应激关键因子eIF2α是miR-26a的直接靶基因。（3）揭示了miR-26a/内质网应激调控回路在NAFLD的功能：利用在国际上率先构建的肝细胞特异性过表达miR-26a小鼠和miR-26a双敲小鼠（同时敲除miR-26a-1和miR-26a-2），建立高脂饮食（HFD）诱导的内质网应激及NAFLD模型，在动物、组织、细胞和分子水平系统探究了miR-26a对NAFLD发生发展的影响。研究结果表明，miR-26a能够显著降低肝脏内质网应激程度，抑制NAFLD发生发展。（4）建立了miR-26a与NAFLD的临床相关性：发现miR-26a在NAFLD患者肝脏中表达下降。在此结果基础上，延伸探究了RNA高级结构（RG4）对miR-26a表达调控的作用及机制。这些结果不仅阐明了miR-26a与内质网应激之间的负反馈调控回路，而且揭示了miR-26a在NAFLD中的重要作用和分子机理，同时也导致了miR-26a表达调控新机制的发现，为NAFLD的预防和治疗提供新靶点和新思路，具有重要的科学意义和临床应用潜力。