肝癌细胞中CDK2调控宿主限制性因子SAMHD1的磷酸化机制研究

CDK2 regulates HBV replication by controlling SAMHD1 phosphorylation in hepatoma cells

目的:主要研究乙肝病毒(hepatitis B virus,HBV)感染肝细胞后,细胞周期激酶2(cyclin-dependent kinase 2,CDK2)调控宿主限制性因子SAMHD1(sterile alpha motif and histidine/aspartic acid domain-containing protein 1)磷酸化的分子机制。方法:利用si RNA干扰技术,特异性处理肝癌细胞Huh7.0的对照组、干扰CDK1组和干扰CDK2组,分别用Southern blot检测这3组中乙肝病毒复制的变化,Western blot检测这3组中SAMHD1磷酸化水平的变化,流式细胞仪检测这3组中细胞周期的变化;进一步通过免疫共沉淀技术鉴定肝癌细胞中CDK2激酶和SAMHD1的相互作用。结果:在肝癌细胞Huh7.0中,与对照组相比,干扰CDK1组和干扰CDK2组的细胞周期明显有差异,分别停滞在G_2期(P=0.001)或G1期(P=0.001)。Western blot结果表明,干扰CDK2后,宿主限制性因子SAMHD1磷酸化水平下降48%,Southern blot结果表明病毒复制水平降低57%(P=0.003),而干扰CDK1后病毒复制水平没有明显变化(P=0.325)进一步通过免疫共沉淀发现在肝癌细胞Huh7.0中,CDK2激酶可与SAMHD1发生相互作用,并且相互作用发生在细胞核内。结论:HBV感染肝细胞后,募集CDK2调控宿主限制性因子SAMHD1的磷酸化,拮抗其抗病毒作用。

Objective：To investigate the molecular mechanism of phosphorylation of sterile alpha motif and histidine/aspartic acid domain-containing protein 1（SAMHD1）in hepatoma carcinoma cell. Methods：By knocking down CDK1 or CDK2 expression in hepatoma carcinoma cell,how CDK1 or CDK2 regulated cell cycle,HBV replication and phosphorylation of SAMHD1 was investigated.Furthermore,the interaction of SAMHD1 and CDK2 was analyzed by coimmunoprecipitation assays（Co-IP）and immunofluorescence.Results：Flow cytometry assay confirmed most Huh7.0 cells were arrested at S/G2 phase（P=0.001）or G1 phase（P=0.001）,respectively when knockdown CDK1 or CDK2. Meanwhile,HBV replication and SAMHD1 phosphorylation levels were significantly influenced by knocking down CDK2 expression（P=0.003）,not by CDK1 expression（P=0.325）. In hepatoma cells,SAMHD1 can bind to CDK2,and this interaction located in nucleus. Conclusion ：Our results provide evidences that HBV employs CDK2,not CDK1 to regulate SAMHD1 phosphorylation during cell cycle,thus contribute to viral replication.