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

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. Overall design: We assessed the expression of short noncoding RNAs (microRNAs ans transfer RNA fragments) in liver 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 livers collected as above

非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）是指通过尚未完全阐明的分子机制，发生肝细胞内脂滴蓄积的疾病。本研究报道了LysTTT-5’tRF（转运RNA片段，transfer RNA fragments）与微小RNA miR-194-5p在NAFLD中各自独立且协同发挥的作用。与瘦型动物不同，饮食诱导的NAFLD小鼠同时出现肝组织中LysTTT-5’tRF与miR-194-5p水平的下调，而经可抑制肝脏脂肪变性的miR-132反义寡核苷酸治疗后，二者水平均可恢复。此外，将人源Hep G2细胞暴露于油酸环境中培养7天，可同时下调miR-194-5p与LysTTT-5’tRF的水平，并增加脂滴蓄积。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小时内升高代谢调节因子β-Klotho的mRNA水平，并使甘油三酯含量降低30%。与之相反，抑制miR-194-5p的反义寡核苷酸处理可诱导其新靶标——与NAFLD相关的脂滴包被蛋白PLIN2的蓄积。进一步筛选发现，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）可分别升高miR-194-5p或LysTTT-5’tRF的水平。miR-194-5p与LysTTT-5’tRF的差异化且互补的作用，为理解小非编码RNA的复杂功能及参与NAFLD发病的多条通路提供了新见解。

整体实验设计：我们评估了抗miR-132治疗后饮食诱导肥胖小鼠肝脏样本中短链非编码RNA（微小RNA及转运RNA片段）的表达水平，该治疗可减轻肝脏脂肪变性，同时设置两类对照组：给予惰性miRNA分子处理的小鼠，以及普通饲料喂养的瘦型小鼠。将C57Bl/6J小鼠分为两组，分别饲喂普通饲料日粮（regular chow diet, RCD）或高脂日粮（Harlan Teklad, Madison, Wisconsin, USA），持续11周以构建饮食诱导肥胖（diet-induced obesity, DIO）模型。注射用寡核苷酸经锁核酸（locked nucleic acid, LNA）修饰，分别靶向成熟miR-132（AM132，16聚体）或靶向灵长类特异性成熟miR-608（AM608，15聚体，作为无小鼠预测互补序列的阴性对照）（LNA，Exiqon，Qiagen）。饮食诱导肥胖小鼠经静脉注射3.3 mg/kg的寡核苷酸，连续给药3天，于治疗后7天处死动物。采集肝脏及下丘脑样本，经液氮快速冷冻后保存于-80℃。选取年龄匹配的普通饲料日粮小鼠处死并采集肝脏样本，操作步骤同上。