小泛素样修饰物特异性蛋白酶3通过调节脂滴水平体外促进HepG2细胞丙型肝炎病毒复制

doi:10.3969/j.issn.1672-5069.2022.02.003

摘要/Abstract

摘要： 目的探究丙型肝炎病毒(HCV)感染细胞小泛素样修饰物特异性蛋白酶3(SENP3)对脂滴水平的调节及其对HCV复制的影响。方法以感染性HCV病毒颗粒感染人HepG2细胞,采用Western blot法检测感染前后SENP3蛋白表达水平。采用脂质体法转染SENP3-siRNA至HepG2细胞,在感染HCV后采用Western blot法检测HCV核心蛋白表达量,采用qRT-PCR法检测HCV RNA水平,采用油红O染色观察细胞脂滴水平。以软脂酸处理敲低SENP3后的HepG2细胞,采用qRT-PCR法检测感染HCV后HCV RNA水平。结果在HCV感染HepG2细胞后1 d、3 d和6 d,HCV 核心蛋白相对表达量分别为0.01±0.00、0.17±0.02和0.43±0.04,SENP3蛋白相对表达量分别为0.23±0.04、0.42±0.03和0.46±0.04,差异显著(P<0.05);转染SENP3-siRNA可有效降低SENP3表达并降低HCV感染后细胞HCV核心蛋白表达水平;SENP3敲低细胞HCV RNA相对水平为54.2±11.4%,较转染非特异性siRNA细胞显著降低(P<0.01);敲低SENP3蛋白表达后细胞脂滴数量显著减少;敲低SENP3的HepG2细胞感染HCV后HCV RNA相对水平为58.2±5.2%,较转染非特异性siRNA细胞显著降低(P<0.01),而以软脂酸处理敲低SENP3的HepG2细胞,在感染HCV后HCV RNA相对水平为74.6±6.4%,较未经软脂酸处理细胞显著升高(P<0.01)。结论在HCV感染肝细胞时SENP3可通过调节脂滴代谢促进HCV复制。

关键词: HepG2细胞, 丙型肝炎病毒, 小泛素样修饰物特异性蛋白酶3, 脂滴

Abstract: Objective The purpose of this experiment was to preliminarily explore the effects of small ubiquitin-like modifier specific protease 3 (SENP3) on lipid droplet regulation and hepatitis C virus (HCV) replication in HepG2 cells in vitro. Methods Human HepG2 cells were infected with infectious HCV particles, and SENP3 protein expression were detected by Western blot. The HepG2 cells were transfected by SENP3-siRNA and infected by HCV particles, then the HCV RNA level was detected by quantitative real-time PCR (qRT-PCR). The lipid droplets were observed by Oil red O staining. After SENP3 knocked down, the HepG2 cells were treated with free fatty acids, and the HCV RNA level was detected by qRT-PCR after HCV infection. Results The relative expression levels of HCV core protein were 0.01±0.00, 0.17±0.02 and 0.43±0.04 on the first, third and sixth days after HCV infection in HepG2 cells, and the relative expression levels of SENP3 protein were 0.23±0.04, 0.42±0.03 and 0.46±0.04, respectively, significantly different among them (P<0.05); the transfection of SENP3-siRNA in the cells effectively reduced the expression of SENP3 and HCV core protein after HCV infection; the relative level of HCV RNA in SENP3 knockdown cells was 54.2±11.4%, significantly lower than that in non-specific siRNA transfection cells (P<0.01); after SENP3 knockdown, the number of lipid droplets decreased significantly; the HCV RNA relative level in SENP3 knockdown HepG2 cells infected with HCV particles was 58.2±5.2%, significantly lower than that in non-specific siRNA transfection cells (P<0.01); the relative level of HCV RNA in HepG2 cells with SENP3 knockdown and co-cultured with palmitic acid was 74.6±6.4%, significantly higher than that in HepG2 cells with SENP3 knockdown (P<0.01). Conclusion The SENP3 could promote HCV replication by participating in regulation of lipid droplet metabolism in HCV-infected hepatocytes.

Key words: HepG2 cells, Hepatitis C virus, Small ubiquitin-like modifier specific protease 3, Lipid droplets

刘文竹, 胡欣, 江伟, 左秀静, 朱华军. 小泛素样修饰物特异性蛋白酶3通过调节脂滴水平体外促进HepG2细胞丙型肝炎病毒复制[J]. 实用肝脏病杂志, 2022, 25(2): 161-164.

Liu Wenzhu, Hu Xin, Jiang Wei, et al. Small ubiquitin-like modifier specific protease 3 promotes hepatitis C virus replication by regulating lipid droplets levels in HepG2 cells in vitro[J]. Journal of Practical Hepatology, 2022, 25(2): 161-164.

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