Department of Public Health, School of Medicine, Shihezi University, Shihezi, 832000, Xinjiang, China.
School of Public Health, Capital Medical University, No.10 Xitoutiao, You AnMen Wai, Beijing 100069, China.
Life Sci. 2020 Jun 15;251:117607. doi: 10.1016/j.lfs.2020.117607. Epub 2020 Mar 30.
Arsenic trioxide (ATO) can bind directly to the human promyelocytic leukemia (PML) protein, leading to modification of PML by SUMOs. UBC9 is the only known E2-conjugating enzyme involved in SUMOylation. PML degradation via RNF4, an E3 ubiquitin ligases family member. PML is key organizer of nuclear bodies (NBs) that regulate many biological processes such as senescence, and DNA damage. ATO can activate the TGFβ/Smad signaling pathway, causing liver fibrosis. However, the roles of PML Sumoylation in ATO-induced liver fibrosis remain unclear.
This study aimed to investigate the role of PML Sumoylation in the ATO-induced HSCs activation and to improve the mechanism of ATO-induced liver fibrosis.
Hepatic stellate cells (HSCs) were treated with 2 μmol/L ATO. Cell viability was detected by CCK-8 analysis. Immunoblot analysis and real-time quantitative PCR were used to detect the expression of IL-1β, TNF-α, TGF-β1, p-Smad2/3, α-SMA, Collagen I and PML SUMOylation after silencing PML, UBC9, and RNF4, respectively. The formation of PML-NBs was observed by immunofluorescence staining.
2 and 5 μmol/L ATO intervention increased HSCs cell viability. ATO was able to significantly trigger PML SUMOylation and the formation of PML-NBs. Inhibition of SUMOylated PML by silencing UBC9, subsequently preventing the downregulation of HSCs activation indicators induced by ATO (P < 0.05). Conversely, enhancing SUMOylated PML accumulation by silencing RNF4, activating TGFβ/Smad signaling pathway, eventually promoting the induction of liver fibrosis.
These results indicated that PML SUMOylation plays a critical role in the development of liver fibrosis induced by ATO.
三氧化二砷（ATO）可以直接与人类早幼粒细胞白血病（PML）蛋白结合，导致 PML 通过 SUMO 修饰。UBC9 是唯一已知参与 SUMO 化的 E2 连接酶。PML 通过 RNF4 降解，RNF4 是一种 E3 泛素连接酶家族成员。PML 是调节衰老和 DNA 损伤等许多生物学过程的核体（NBs）的关键组织者。ATO 可以激活 TGFβ/Smad 信号通路，导致肝纤维化。然而，PML 泛素化在 ATO 诱导的肝纤维化中的作用尚不清楚。
本研究旨在探讨 PML 泛素化在 ATO 诱导的 HSCs 激活中的作用，并阐明 ATO 诱导肝纤维化的机制。
用 2μmol/L ATO 处理肝星状细胞（HSCs）。用 CCK-8 分析检测细胞活力。用免疫印迹分析和实时定量 PCR 检测沉默 PML、UBC9 和 RNF4 后，IL-1β、TNF-α、TGF-β1、p-Smad2/3、α-SMA、Collagen I 和 PML SUMOylation 的表达。通过免疫荧光染色观察 PML-NBs 的形成。
2 和 5μmol/L ATO 干预增加了 HSCs 的细胞活力。ATO 能够显著触发 PML SUMOylation 和 PML-NBs 的形成。沉默 UBC9 抑制 SUMO 化的 PML，随后阻止 ATO 诱导的 HSCs 激活标志物的下调（P<0.05）。相反，沉默 RNF4 增强 SUMO 化的 PML 积累，激活 TGFβ/Smad 信号通路，最终促进肝纤维化的诱导。
这些结果表明，PML SUMOylation 在 ATO 诱导的肝纤维化发展中起关键作用。
