Department of Gastroenterology, the First Affiliated Hospital, School of Medicine, Zhejiang University, 310003, Hangzhou, China.
School of Clinical Medicine, Hangzhou Medical College, 310053, Hangzhou, China.
Cell Death Dis. 2020 Feb 13;11(2):123. doi: 10.1038/s41419-020-2293-7.
Circular RNAs (circRNAs) have been shown to play critical roles in cancer biology, but their functions in nonalcoholic steatohepatitis (NASH) remain unexplored. Full length of circRNA_002581 was amplified and sequenced, followed by RNA immunoprecipitation, RNA-Fluorescence in Situ Hybridization and dual luciferase reporter gene analysis to confirm the existence of the circRNA_002581-miR-122-CPEB1 regulatory axis in vitro. CircRNA_002581 knockdown was used to study its roles in high concentration of free fatty acids-induced NASH-like cell model and a methionine and choline deficiency (MCD) diet-induced NASH mice model. Autophagy flux and related potential PTEN-AMPK-mTOR pathway were tested by western blot. CircRNA_002581 overexpression significantly relieved the inhibitory role of miR-122 on its target CPEB1 by sponging miR-122. CircRNA_002581 knockdown markedly attenuated lipid droplet accumulation, reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), pro-inflammatory cytokines, apoptosis, HO, and increased ATP level in both mice and cellular models of NASH. Mechanistically, circRNA_002581 interference significantly rescue the defective autophagy evidenced by increased autophagosome number, upregulated LC3-II/I level, and decreased p62 level. Further chloroquine-mediated total autophagy inhibition antagonizes the protective effect of circRNA_002581 knockdown. Finally, CPEB1-PTEN-AMPK-mTOR pathway is shown to link the autophagy and circRNA_002581 knockdown-mediated NASH alleviation. CircRNA_002581-miR-122-CPEB1 axis actively participates in the pathogenesis of NASH through PTEN-AMPK-mTOR pathway-related autophagy suppression. Targeting circRNA_002581 is a potential therapeutic strategy for NASH through partial autophagy restoration.
环状 RNA（circRNA）已被证明在癌症生物学中发挥关键作用，但它们在非酒精性脂肪性肝炎（NASH）中的功能仍未被探索。扩增并测序全长 circRNA_002581，随后进行 RNA 免疫沉淀、RNA 荧光原位杂交和双荧光素酶报告基因分析，以体外证实 circRNA_002581-miR-122-CPEB1 调控轴的存在。使用 circRNA_002581 敲低研究其在高浓度游离脂肪酸诱导的 NASH 样细胞模型和蛋氨酸和胆碱缺乏（MCD）饮食诱导的 NASH 小鼠模型中的作用。通过 Western blot 检测自噬流和相关潜在的 PTEN-AMPK-mTOR 通路。circRNA_002581 的过表达通过海绵 miR-122 显著缓解了 miR-122 对其靶标 CPEB1 的抑制作用。circRNA_002581 敲低明显减轻了脂滴堆积，降低了丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、促炎细胞因子、细胞凋亡、HO 和 ATP 水平在 NASH 的小鼠和细胞模型中。机制上，circRNA_002581 干扰显著挽救了自噬缺陷，表现为自噬体数量增加、LC3-II/I 水平上调和 p62 水平降低。进一步用氯喹介导的总自噬抑制拮抗 circRNA_002581 敲低的保护作用。最后，CPEB1-PTEN-AMPK-mTOR 通路表明自噬和 circRNA_002581 敲低介导的 NASH 缓解与相关。circRNA_002581-miR-122-CPEB1 轴通过 PTEN-AMPK-mTOR 通路相关自噬抑制积极参与 NASH 的发病机制。靶向 circRNA_002581 是通过部分自噬恢复治疗 NASH 的潜在治疗策略。
