Huang Xiaogang, He Xiongzhi, Qiu Rongxian, Xie Xuemei, Zheng Fengfeng, Chen Feihua, Hu Zhenting
Department of Infectious Diseases, The Affiliated Hospital of Putian University, Putian City, Fujian Province, 351100, China.
Department of Infectious Diseases, The Affiliated Hospital of Putian University, No. 999 Dongzhen East Road, Licheng District, Putian City, Fujian Province, 351100, China.
Open Med (Wars). 2023 Jun 16;18(1):20230718. doi: 10.1515/med-2023-0718. eCollection 2023.
Unfolded protein response (UPR) plays an important role in the pathogenesis of many liver diseases. BMI1 has a liver protection effect, but whether it participates in the regulation of hepatocyte death through UPR is not well defined. Herein, the endoplasmic reticulum stress model was established by inducing hepatocyte line (MIHA) with tunicamycin (TM, 5 µg/ml). Cell counting kit-8 assay and flow cytometry were used to evaluate the viability and apoptosis of hepatocytes. The expression levels of BMI1, KAT2B, and proteins related to UPR (p-eIF2α, eIF2α, ATF4, and ATF6), NF-κB (p65 and p-p65), apoptosis (cleaved caspase-3, bcl-2, and bax) and necroptosis (p-MLKL and MLKL) were determined by Western blot. The relationship between KAT2B and BMI1 was determined by co-immunoprecipitation and ubiquitination assay. The results showed that TM not only promoted UPR, apoptosis, and necroptosis in hepatocytes but also upregulated the expression levels of BMI1 and KAT2B and activated NF-κB pathway. BAY-117082 reversed the effects of TM on viability, apoptosis, NF-κB pathway, and BMI1 but strengthened the effects of TM on KAT2B/MLKL-mediated necroptosis. BMI1 promoted the ubiquitination of KAT2B, and BMI1 overexpression reversed the effects of TM on viability, apoptosis, and KAT2B/MLKL-mediated necroptosis. In summary, overexpression of BMI1 promotes the ubiquitination of KAT2B to block the MLKL-mediated necroptosis of hepatocytes.
未折叠蛋白反应（UPR）在多种肝脏疾病的发病机制中起重要作用。BMI1具有肝脏保护作用，但其是否通过UPR参与肝细胞死亡的调控尚不清楚。在此，通过用衣霉素（TM，5μg/ml）诱导肝细胞系（MIHA）建立内质网应激模型。使用细胞计数试剂盒-8检测法和流式细胞术评估肝细胞的活力和凋亡。通过蛋白质免疫印迹法测定BMI1、KAT2B以及与UPR相关的蛋白（p-eIF2α、eIF2α、ATF4和ATF6）、NF-κB（p65和p-p65）、凋亡（裂解的半胱天冬酶-3、bcl-2和bax）和坏死性凋亡（p-MLKL和MLKL）的表达水平。通过免疫共沉淀和泛素化检测确定KAT2B与BMI1之间的关系。结果表明，TM不仅促进肝细胞中的UPR、凋亡和坏死性凋亡，还上调BMI1和KAT2B的表达水平并激活NF-κB通路。BAY-117082逆转了TM对活力、凋亡、NF-κB通路和BMI1的影响，但增强了TM对KAT2B/MLKL介导的坏死性凋亡的影响。BMI1促进KAT2B的泛素化，BMI1过表达逆转了TM对活力、凋亡和KAT2B/MLKL介导的坏死性凋亡的影响。总之，BMI1的过表达促进KAT2B的泛素化以阻断MLKL介导的肝细胞坏死性凋亡。
