Lysine tRNA fragments and miR-194-5p co-regulate hepatic steatosis via ß-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 ß-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 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）以肝细胞内脂质滴蓄积为主要特征，但其具体发病机制尚未完全阐明。本研究揭示了LysTTT-5'转运RNA片段（LysTTT-5'tRF）与微小RNA miR-194-5p在NAFLD中各自独立且协同发挥的作用。与正常瘦型小鼠相比，饮食诱导的NAFLD小鼠肝脏中LysTTT-5'tRF与miR-194-5p的表达水平同时下调，而通过抑制肝脂肪变的miR-132反义寡核苷酸治疗后，二者的表达水平可恢复至正常水平。此外，将人源Hep G2细胞暴露于油酸环境中培养7天，可同时抑制miR-194-5p与LysTTT-5'tRF的表达，并促进细胞脂质蓄积。值得注意的是，向脂肪变细胞中转染合成的LysTTT-5'tRF模拟物，可在24小时内上调代谢调节因子β-Klotho的mRNA表达水平，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，则会诱导其新靶标——与NAFLD相关的脂质滴包被蛋白PLIN2的蓄积。进一步而言，在15种经筛选可缓解肝脂肪变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各自独立却又相互补充的作用机制，为阐明小型非编码RNA的复杂功能以及NAFLD发病过程中涉及的多条信号通路提供了全新的研究视角。

实验整体设计：本研究评估了经抗miR-132治疗后肝脂肪变得到缓解的饮食诱导肥胖小鼠，以及对照组小鼠（包括给予无义miRNA分子处理的小鼠和正常饲料喂养的瘦型小鼠）的下丘脑组织中短链非编码RNA（微小RNA与转运RNA片段）的表达水平。将C57Bl/6J小鼠分为两组，分别给予正常饲料饮食（RCD）与高脂饲料饮食（购自美国威斯康星州麦迪逊市Harlan Teklad公司），喂养11周以构建饮食诱导肥胖（DIO）模型。注射用寡核苷酸均经过锁核酸（LNA）修饰，分别靶向成熟miR-132（AM132，16聚体）或灵长类特异性成熟miR-608（AM608，15聚体，作为对照，其序列在小鼠中无预测互补靶点；购自Exiqon、Qiagen公司）。对饮食诱导肥胖小鼠连续3天经静脉注射3.3 mg/kg的寡核苷酸，并于治疗后7天处死小鼠。收集肝脏与下丘脑组织样本，经液氮快速冷冻后保存于-80℃冰箱。将年龄匹配的正常饲料喂养小鼠按上述流程处死并收集样本。