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

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: RNA was extracted from steatotic cells treated with LysTTT-5'tRF mimic or NC (two replicates per treatment). 150,000 cells/ml were plated and 24 hours later medium was replaced with oleic acid (OA)-rich medium prepared as described in the manuscript. 24 hours after this cells were transfected with tRF mimic oligonucleotide or negative control (IDT, Syntezza Bioscience) using HiPerFect transfection reagent (Qiagen, 301705) and harvested after an additional 24 hours.

非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）以肝细胞内细胞内脂质滴沉积为核心病理特征，其具体分子机制尚未完全阐明。本研究揭示了LysTTT-5' tRF（转运RNA片段，transfer RNA fragments, tRF）与微小RNA miR-194-5p（microRNA, miR）在NAFLD进程中各自独立且协同的调控作用。与瘦型对照动物不同，饮食诱导的NAFLD小鼠模型中，肝细胞内LysTTT-5' tRF与miR-194-5p的水平同时降低，而通过可抑制肝细胞脂肪变性的miR-132反义寡核苷酸处理后，二者的表达水平可得到恢复。

此外，将人源Hep G2细胞系暴露于富含油酸（oleic acid, OA）的培养基中培养7天后，细胞内miR-194-5p与LysTTT-5' tRF的表达被同时抑制，同时伴随脂质沉积增加。值得注意的是，使用合成的LysTTT-5' tRF模拟物转染脂变细胞后，代谢调节因子β-Klotho的mRNA水平显著升高，且在24小时内将细胞内甘油三酯含量降低了30%。与之相反，对miR-194-5p进行反义沉默会诱导其新靶标——与NAFLD相关的脂质滴包被蛋白PLIN2（perilipin 2, PLIN2）的积累。进一步研究发现，在经筛选可缓解脂肪变性的15种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的表达水平。

miR-194-5p与LysTTT-5' tRF既存在功能差异又互为补充，这为理解小型非编码RNA（small non-coding RNAs, sRNA）在NAFLD发病机制中的复杂调控作用及相关多通路提供了新的视角。

整体实验设计如下：从经LysTTT-5' tRF模拟物或阴性对照（NC）处理的脂变细胞中提取RNA，每组设置2个生物学重复。以1.5×10^5 个细胞/毫升的密度铺板培养，24小时后更换为依照本研究手稿所述方法制备的富含油酸（OA）的培养基。于此操作24小时后，使用HiPerFect转染试剂（Qiagen，货号301705）将tRF模拟物寡核苷酸或阴性对照（购自IDT、Syntezza Bioscience）转染细胞，继续培养24小时后收集细胞。