Department of Pharmacology, Dalian Medical University, 116044, Dalian, China.
Department of General Surgery, The Second Affiliated Hospital of Dalian Medical University, 116023, Dalian, China.
Cell Death Dis. 2020 Nov 6;11(11):953. doi: 10.1038/s41419-020-03160-y.
p66Shc, a master regulator of mitochondrial reactive oxygen species (mtROS), is a crucial mediator of hepatocyte oxidative stress. However, its functional contribution to acetaminophen (APAP)-induced liver injury and the mechanism by which it is modulated remain unknown. Here, we aimed to assess the effect of p66Shc on APAP-induced liver injury and to evaluate if circular RNA (circRNA) functions as a competitive endogenous RNA (ceRNA) to mediate p66Shc in APAP-induced liver injury. p66Shc-, miR-185-5p-, and circ-CBFB-silenced mice were injected with APAP. AML12 cells were transfected with p66Shc, miR-185-5p, and circ-CBFB silencing or overexpression plasmids or siRNAs prior to APAP stimulation. p66Shc was upregulated in liver tissues in response to APAP, and p66Shc silencing in vivo protected mice from APAP-induced mitochondrial dynamics perturbation and liver injury. p66Shc knockdown in vitro attenuated mitochondrial dynamics and APAP-induced hepatocyte injury. Mechanically, p66Shc perturbs mitochondrial dynamics partially by inhibiting OMA1 ubiquitination. miR-185-5p, which directly suppressed p66Shc translation, was identified by microarray and bioinformatics analyses, and its overexpression attenuated mitochondrial dynamics and hepatocyte injury in vitro. Furthermore, luciferase, pull-down and RNA immunoprecipitation assays demonstrated that circ-CBFB acts as a miRNA sponge of miR-185-5p to mediate p66Shc in APAP-induced liver injury. circ-CBFB knockdown also alleviated APAP-induced mitochondrial dynamics perturbation and hepatocyte injury. More importantly, we found that the protective effects of circ-CBFB knockdown on p66Shc, mitochondrial dynamics and liver injury were abolished by miR-185-5p inhibition both in vivo and in vitro. In conclusion, p66Shc is a key regulator of APAP-induced liver injury that acts by triggering mitochondrial dynamics perturbation. circ-CBFB functions as a ceRNA to regulate p66Shc during APAP-induced liver injury, which may provide a potential therapeutic target.
p66Shc 是线粒体活性氧（mtROS）的主要调节因子，是肝细胞氧化应激的关键介质。然而，其在对乙酰氨基酚（APAP）诱导的肝损伤中的功能贡献及其调节机制尚不清楚。在这里，我们旨在评估 p66Shc 对 APAP 诱导的肝损伤的影响，并评估环状 RNA（circRNA）是否作为一种竞争性内源性 RNA（ceRNA）来调节 p66Shc 在 APAP 诱导的肝损伤中的作用。p66Shc-、miR-185-5p-和 circ-CBFB 沉默的小鼠注射了 APAP。在 APAP 刺激前，AML12 细胞转染了 p66Shc、miR-185-5p 和 circ-CBFB 沉默或过表达质粒或 siRNA。APAP 反应中肝组织中 p66Shc 上调，体内 p66Shc 沉默可保护小鼠免受 APAP 诱导的线粒体动力学紊乱和肝损伤。体外 p66Shc 敲低可减弱线粒体动力学和 APAP 诱导的肝细胞损伤。机制上，p66Shc 通过抑制 OMA1 泛素化来破坏线粒体动力学。通过微阵列和生物信息学分析鉴定出 miR-185-5p，它可以直接抑制 p66Shc 的翻译，其过表达可减弱体外的线粒体动力学和肝细胞损伤。此外，荧光素酶、下拉和 RNA 免疫沉淀测定表明，circ-CBFB 作为 miR-185-5p 的 miRNA 海绵，在 APAP 诱导的肝损伤中调节 p66Shc。circ-CBFB 敲低也减轻了 APAP 诱导的线粒体动力学紊乱和肝细胞损伤。更重要的是，我们发现，circ-CBFB 敲低对 p66Shc、线粒体动力学和肝损伤的保护作用在体内和体外均被 miR-185-5p 抑制所消除。总之，p66Shc 是 APAP 诱导的肝损伤的关键调节因子，通过触发线粒体动力学紊乱起作用。circ-CBFB 作为 ceRNA 在 APAP 诱导的肝损伤中调节 p66Shc，这可能为提供潜在的治疗靶点。
