ZNF143 inhibits hepatocyte mitophagy and promotes non-alcoholic fatty liver disease by targeting increased lncRNA NEAT1 expression to activate ROCK2 pathway

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorders worldwide. The mitophagy is suggested to be repressed in NAFLD, but the mechanism remains to be elucidated. Methods: NAFLD cell and mouse models were established by treating with free fatty acid (FFA) and feeding a high fat diet (HFD), respectively. QRT-PCR, Western blotting, or IHC measured the expression of ZNF143, lncRNA NEAT1, ROCK2, and lipid formation/mitophagy-related proteins. Cell viability and mitophagy were evaluated by MTT and immunofluorescence. The chloroform-methanol extraction method measured triglyceride and total cholesterol levels. ELISA detected ALT and AST levels. The interactions among ZNF143, lncRNA NEAT1 and SND1 were analysed by ChIP, dual-luciferase reporter, pull-down, and RIP. The lipid droplets were determined by Oil-red O and HE staining. Results: ZNF143 and lncRNA NEAT1 were upregulated in hepatic cells treated with FFA (<i>p</i> &lt; 0.01 and <i>p</i> &lt; 0.001). Knockdown of ZNF143 or lncRNA NEAT1 inhibited lipid droplets formation, while promoting mitophagy (<i>p</i> &lt; 0.01 and <i>p</i> &lt; 0.001). ZNF143 promoted lncRNA NEAT1 transcriptional expression through binding to its promoter. LncRNA NEAT1 increased ROCK2 mRNA stability by targeting SND1. LncRNA NEAT1 or ROCK2 overexpression reversed the effect of ZNF143 or lncRNA NEAT1 knockdown on hepatic steatosis and mitophagy (<i>p</i> &lt; 0.01 and <i>p</i> &lt; 0.001). ZNF143 or lncRNA NEAT1 knockdown inhibited HFD-induced steatosis and promoted mitophagy <i>in vivo</i> (<i>p</i> &lt; 0.01 and <i>p</i> &lt; 0.001). Conclusion: The upregulation of lncRNA NEAT1 caused by ZNF143 promoted NAFLD through inhibiting mitophagy via activating ROCK2 pathway by targeting SND1, providing potential targets for NAFLD therapy.

背景：非酒精性脂肪性肝病（Nonalcoholic fatty liver disease, NAFLD）是全球范围内最常见的肝脏疾病。现有研究提示线粒体自噬（mitophagy）在NAFLD中受到抑制，但其具体调控机制仍有待阐明。
方法：本研究分别采用游离脂肪酸（free fatty acid, FFA）处理细胞、高脂饮食（high fat diet, HFD）喂养小鼠，成功构建NAFLD细胞模型与小鼠模型。通过实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, QRT-PCR）、蛋白质印迹（Western blotting）、免疫组化（immunohistochemistry, IHC）检测ZNF143、长链非编码RNA（long non-coding RNA, lncRNA）NEAT1、ROCK2以及脂质生成与线粒体自噬相关蛋白的表达水平。采用MTT法与免疫荧光法评估细胞活力与线粒体自噬水平。通过氯仿-甲醇萃取法测定甘油三酯与总胆固醇含量。利用酶联免疫吸附试验（enzyme linked immunosorbent assay, ELISA）检测谷丙转氨酶（alanine transaminase, ALT）与谷草转氨酶（aspartate transaminase, AST）的血清水平。通过染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）、双荧光素酶报告基因实验、pull-down实验及RNA免疫沉淀（RNA immunoprecipitation, RIP）分析ZNF143、lncRNA NEAT1与SND1三者间的相互作用。采用油红O染色与苏木精-伊红（hematoxylin-eosin, HE）染色观察细胞内脂滴沉积情况。
结果：经FFA处理的肝细胞中，ZNF143与lncRNA NEAT1的表达水平显著上调（*p* < 0.01 与 *p* < 0.001）。敲低ZNF143或lncRNA NEAT1可显著抑制脂滴形成，并促进线粒体自噬（*p* < 0.01 与 *p* < 0.001）。ZNF143可通过结合lncRNA NEAT1的启动子区域，正向调控其转录表达。lncRNA NEAT1可通过靶向结合SND1，增强ROCK2的mRNA稳定性。过表达lncRNA NEAT1或ROCK2，可逆转敲低ZNF143或lncRNA NEAT1对肝脏脂肪变与线粒体自噬的调控效应（*p* < 0.01 与 *p* < 0.001）。体内实验结果显示，敲低ZNF143或lncRNA NEAT1可抑制HFD诱导的肝脏脂肪变，并促进线粒体自噬（*p* < 0.01 与 *p* < 0.001）。
结论：ZNF143介导的lncRNA NEAT1上调，通过靶向SND1激活ROCK2通路以抑制线粒体自噬，进而促进NAFLD的发生发展，该研究为非酒精性脂肪性肝病的临床治疗提供了潜在的分子靶点。