BACKGROUND 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. OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSION These results indicated that PML SUMOylation plays a critical role in the development of liver fibrosis induced by ATO.

中文翻译：

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PML SUMOylation在三氧化二砷诱导的HSC纤维化中的作用。

背景技术三氧化二砷（ATO）可以直接结合人早幼粒细胞白血病（PML）蛋白，导致SUMO修饰PML。UBC9是唯一参与SUMOylation的E2偶联酶。通过RNF4（一种E3泛素连接酶家族成员）降解PML。PML是调节许多生物过程（例如衰老和DNA损伤）的核机构（NB）的主要组织者。ATO可以激活TGFβ/ Smad信号通路，引起肝纤维化。但是，PML Sumoylation在ATO诱导的肝纤维化中的作用仍不清楚。目的本研究旨在探讨PML Sumoylation在ATO诱导的HSC激活中的作用，并改善ATO诱导的肝纤维化的机制。方法用2μmol/ L ATO处理肝星状细胞（HSC）。通过CCK-8分析检测细胞活力。PML，UBC9和RNF4沉默后，使用免疫印迹分析和实时定量PCR检测IL-1β，TNF-α，TGF-β1，p-Smad2 / 3，α-SMA，I型胶原和PML SUMO酰化的表达， 分别。通过免疫荧光染色观察到PML-NBs的形成。结果2和5μmol/ L ATO干预可增加HSCs细胞活力。ATO能够显着触发PML SUMOylation和PML-NB的形成。通过沉默UBC9抑制SUMO化的PML，随后防止ATO诱导的HSC激活指标下调（P <0.05）。相反，通过沉默RNF4，激活TGFβ/ Smad信号通路来增强SUMO化的PML积累，最终促进肝纤维化的诱导。