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