Lysine tRNA fragments and miR-194-5p co-regulate hepatic steatosis via β-Klotho and Perilipin 2 (small RNA I)

Non-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5’tRF transfer RNA fragments and microRNA miR-194-5p. Unlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5’tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5’tRF levels while increasing lipid accumulation. Importantly, transfecting fattened cells with a synthetic LysTTT-5’tRF mimic elevated mRNA levels of the metabolic regulator β-Klotho while decreasing triglyceride amounts by 30% within 24 hours. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5’tRF levels. The different yet complementary roles of miR-194-5p and LysTTT-5’tRF offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis. We assessed the expression of short noncoding RNAs (microRNAs ans transfer RNA fragments) in hypothalamus samples from diet-induced obese mice after anti-miR-132-treatment, which showed reduced liver steatosis and control animals (treated with inert miR molecule and lean animals on regular chow diet). C57Bl/6J mice were fed a regular chow diet (RCD) or a high fat diet (Harlan Teklad, Madison, Wisconsin, USA) for 11 weeks to reach diet-induced obesity (DIO). Injected oligonucleotides were modified by locked nucleic acid protection and complementary to mature miR-132 (AM132, 16-mer) or to mature primate-specific miR-608 (AM608, 15-mer, as a control with no predicted complementary sequences in mice) (LNA, Exiqon, Qiagen). DIO mice were injected intravenously with 3.3 mg/kg oligonucleotide for three consecutive days and were sacrificed 7 days post-treatment. Liver and hypothalamus samples were collected, snap frozen in liquid nitrogen, and stored at -80°C. Age matched RCD mice were sacrificed and samples collected as above

非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）是指肝细胞内出现细胞内脂滴蓄积，但其具体分子机制尚未完全阐明。本研究报道了LysTTT-5’ tRNA衍生片段（transfer RNA fragments, tRF）与微小RNA miR-194-5p（microRNA miR-194-5p）在NAFLD进程中既独立又协同的调控作用。与瘦型动物不同，饮食诱导的NAFLD小鼠肝细胞内LysTTT-5’tRF与miR-194-5p的表达水平同时下调，而通过可抑制肝脂肪变性的miR-132反义寡核苷酸治疗后，二者的表达水平可得到恢复。进一步研究发现，将人源Hep G2细胞置于油酸环境中培养7天，可同时抑制miR-194-5p与LysTTT-5’tRF的表达，并促进细胞内脂滴蓄积。值得注意的是，向脂变细胞中转染合成的LysTTT-5’tRF模拟物，可在24小时内上调代谢调控因子β-Klotho的mRNA水平，并使细胞内甘油三酯含量降低30%。与之相反，通过反义技术抑制miR-194-5p的表达，可诱导其新靶标——与NAFLD相关的脂滴包被蛋白PLIN2的蓄积。此外，在15种经筛选可减轻脂肪变性的药物重定位化合物中，达那唑（Danazol）与拉坦前列素（Latanoprost）可分别上调miR-194-5p或LysTTT-5’tRF的表达水平。miR-194-5p与LysTTT-5’tRF的差异化但互补的调控作用，为理解小非编码RNA在NAFLD发病机制中的复杂调控功能及参与的多条信号通路提供了新视角。本研究检测了接受抗miR-132治疗（可减轻肝脂肪变性）的饮食诱导肥胖小鼠、接受无活性miR分子处理的对照小鼠及常规饲料饲养的瘦型小鼠下丘脑样本中的小非编码RNA（微小RNA与tRNA衍生片段）表达水平。将C57BL/6J小鼠分别饲喂常规饲料饮食（regular chow diet, RCD）或高脂饲料（Harlan Teklad, 美国威斯康星州麦迪逊市）11周，以构建饮食诱导肥胖（diet-induced obesity, DIO）模型。注射用寡核苷酸均经锁核酸修饰，分别靶向成熟miR-132（AM132，16碱基）或成熟灵长类特异性miR-608（AM608，15碱基，作为对照，在小鼠中无预测的互补结合序列）（锁核酸试剂购自Exiqon、Qiagen）。对DIO小鼠连续3天静脉注射3.3 mg/kg的寡核苷酸，于治疗后7天处死小鼠。采集肝脏与下丘脑样本，经液氮快速冷冻后保存于-80℃。将同周龄的RCD小鼠处死并按上述方法采集样本。