SUMO特异性蛋白酶3在HBV复制中的作用机制

The role of SUMO specific protease 3 in HBV replication

目的初步探讨SUMO特异性蛋白酶3(SENP3)在HBV感染中的作用机制。方法体外培养HepG2.2.15细胞,或利用HBV转基因小鼠肝脏组织,通过表达SiRNA干扰等,经蛋白质印迹、实时PCR、免疫组织化学染色等实验技术,检测SENP3在HBV感染细胞模型中的表达。结果 HBV DNA与其PBMC中SENP3的含量呈反比。健康对照组PBMC中SENP3的平均蛋白水平是CHB患者的7.4倍(P<0.05)。HBV转基因小鼠肝脏中SENP3表达显著降低,小鼠肝脏组织中AKT磷酸化水平增高,是健康对照组的4.1倍(P<0.01)。SENP3在HBV DNA全基因重组质粒转染的HepG2受体细胞(HepG2.2.15)中表达量显著低于未转染HBV DNA的HepG2细胞。与HepG2细胞相比,HepG2.2.15细胞中AKT磷酸化水平显著增高,其含量平均是HepG2细胞的5.2倍(P<0.01);在HepG2.2.15细胞中转染SENP3质粒使其过表达,则AKT磷酸化水平降低,HBV DNA含量降低,并与SENP3质粒浓度呈负相关性;分别向HepG2.2.15细胞转染100ng和500ng的SENP3质粒,则细胞中HBV DNA含量由对照组中的7.983.03×106 IU/mL降低至3.45×106 IU/mL和1.92×106 IU/mL;应用siRNA干扰SENP3在HepG2.2.15细胞中的表达,则Akt磷酸化水平升高,其浓度是对照组的5.6倍(P<0.01)。结论机体HBV DNA载量与SENP3的表达量呈负相关,SENP3可通过抑制肝细胞AKT磷酸化水平,降低HBV DNA载量。

Objective To investigate the role of small ubiquitin-related modifier (SUMO) specific protease 3 (SENP3) in hepatitis B virus (HBV) infection. Methods To explore the mechanism of SENP3, HepG2.2.15 cell and HBV transgenic mouse were applied for experiments in vivo and vitro using western blot, real-time polymerase chain reaction and immunohistochemistry. Results The HBV DNA of patients was negatively correlated with the content of SENP3 in peripheral blood mononuclear cells. The average protein concentration of SENP3 in healthy control group was 7.4 times higher than that in patient group ( P <0.05). In the liver of HBV transgenic mice, the expression of SENP3 was significantly decreased and phosphorylation of protein kinase B (Akt) was 4.1 times higher than that of control group ( P <0.01). In HepG2.2.15 cells transfected with HBV DNA whole-genome plasmid, the expression of SENP3 was significantly lower, while the phosphorylation of Akt was 5.2 times higher than that in HepG2 cells ( P <0.01). Overexpression of SENP3 in HepG2.215 cells resulted in decreases of Akt phosphorylation and HBV DNA content. Moreover, SENP3 plasmid concentration was negatively correlated with HBV DNA content. When transfecting HepG2.2.15 cells with 100 ng and 500 ng SENP3 plasmids, the concentrations of HBV DNA were reduced from 7.983.03×10^6 IU/ml to 3.45×10^6 IU/ml and 1.92×10^6 IU/ml, respectively. While silencing SENP3 in HepG2.2.15 cells, phosphorylation of Akt was increased 5.6 times ( P <0.01). Conclusion The expression of HBV DNA is negatively correlated with the expression of SENP3, which can reduce the level of HBV DNA by inhibiting the phosphorylation of Akt in hepatocytes.