Department of Gastroenterology.
Department of Ultrasonic Medicine, The First Affiliated Hospital of Bengbu Medical College, No.287 Changhuai Road, Longzihu District, Bengbu, Anhui 233004, China.
J Biochem. 2020 Nov 1;168(5):445-453. doi: 10.1093/jb/mvaa068.
RING finger protein 8 (RNF8) is an E3 ligase that is pivotal for DNA repair. However, the role of RNF8 in ulcerative colitis (UC) remains unclear. The aim of this study is to investigate the effect and the mechanism of RNF8 on UC model induced by trinitrobenzene sulfonic acid (TNBS) in mice. Lentiviruses overexpressing RNF8 were injected into mice after the induction of UC. The histopathological changes in colon tissues were assessed by haematoxylin and eosin staining. The mRNA level of RNF8 was detected by real-time quantitative polymerase chain reaction. The protein levels of RNF8, autophagy-related proteins (LC3 and P62) and AKT/mammalian target of rapamycin (mTOR) signalling-related proteins were measured by Western blot. The pro-inflammatory cytokines (tumour necrosis factor-α and interleukin-1β) were examined by immunohistochemical analysis. Immunoprecipitation was performed to analyse the interaction between RNF8 and AKT1. The TNBS-induced UC mice exhibited colonic damage and inflammation, accompanied by decreased RNF8 expression, impaired autophagy and increased phosphorylation levels of AKT and mTOR in the colon. However, these alterations were reversed by RNF8 overexpression. Furthermore, RNF8 bound to AKT1 and mediated its ubiquitination. Collectively, RNF8 overexpression protects against TNBS-induced UC, which might be due to its enhancement of autophagy by suppressing the AKT/mTOR signalling via AKT1 ubiquitination.
环指蛋白 8（RNF8）是一种 E3 连接酶，对 DNA 修复至关重要。然而，RNF8 在溃疡性结肠炎（UC）中的作用尚不清楚。本研究旨在探讨 RNF8 对三硝基苯磺酸（TNBS）诱导的 UC 模型小鼠的作用及其机制。在 UC 诱导后，向小鼠注射过表达 RNF8 的慢病毒。通过苏木精和伊红染色评估结肠组织的组织病理学变化。实时定量聚合酶链反应检测 RNF8 的 mRNA 水平。通过 Western blot 检测 RNF8、自噬相关蛋白（LC3 和 P62）和 AKT/哺乳动物雷帕霉素靶蛋白（mTOR）信号相关蛋白的蛋白水平。通过免疫组化分析检测促炎细胞因子（肿瘤坏死因子-α和白细胞介素-1β）。免疫沉淀用于分析 RNF8 与 AKT1 之间的相互作用。TNBS 诱导的 UC 小鼠表现出结肠损伤和炎症，同时伴有 RNF8 表达降低、自噬受损以及 AKT 和 mTOR 在结肠中的磷酸化水平增加。然而，过表达 RNF8 可逆转这些改变。此外，RNF8 与 AKT1 结合并介导其泛素化。总之，过表达 RNF8 可预防 TNBS 诱导的 UC，这可能是通过 AKT1 泛素化抑制 AKT/mTOR 信号通路增强自噬所致。
