MicroRNA-A6增效的HEV体外瞬时转染HepG2复制体系的建立及鉴定

摘要/Abstract

摘要： 目的验证HEV编码的MicroRNA-A6对全长HEV瞬转HepG2细胞系中病毒复制的增效作用。方法按照已报道序列合成miR-A6,利用重组PCR、基因克隆以及体外转录技术获得单一高浓度的HEV RNA。HEV RNA与miR-A6按比例采用脉冲电流转染HepG2细胞,转染后24、48和96 h后观察细胞上清IgG分泌和HEV RNA 表达载量,与不加miR-A6的HepG2细胞进行比较,分析miR-A6对HEV复制的作用,然后加入anti-A6抑制miR-A6表达,分析其对HEV表达和复制的影响。结果 MiR-A6∶HEV RNA质量比为0.5∶1和1∶1,共转染HepG2细胞后48 h,与单转染HEV RNA相比,lg值升高了1.57和2.44。HEV-IgG先于HEV RNA表达。体外实验显示,Ⅰ型和Ⅳ型HEV复制力没有明显差别,都在转染后48 h达到峰值,加入anti-A6抑制miR-A6后HEV-IgG抑制47.12%,HEV RNA抑制43.3%。结论 MiR-A6可以在体外显著增加HEV RNA瞬转HepG2细胞系中HEV病毒的复制力,为后续HEV分子病毒学和表型耐药研究奠定基础。

关键词: MicroRNA, 肝炎病毒, 戊型, 全长扩增, 瞬时转染, 复制力

Abstract: Objective To investigate the synergistic effect of hepatitis E virus (HEV)-encoded microRNA-A6 (miR-A6) on viral replication in full-length HEV transiently transfected-human hepatocellular carcinoma cell line (HepG2).Methods miR-A6 was synthesized according to the reported sequence. Technologies of recombinant polymerase chain reaction (PCR), gene cloning and in vitro transcription were used to obtain a high concentration of HEV RNA. Proportional mixed HEV RNA and miR-A6 were transiently transfected into HepG2 using pulse current. Immunoglobulin G (IgG) secretion and HEV RNA load in supernatants of cultured HepG2 were measured at hour 24, 48 and 96 after transfection, respectively. Replication of HEV was compared between HepG2 cells with and without miR-A6 transfection. Moreover, anti-A6 was further used to confirm the effect of miR-A6 on the replication of HEV.Results Compared with that in HepG2 transfected with HEV RNA alone, lg HEV RNA increased by 1.57 and 2.44 at hour 48 after co-transfection of miR-A6 and HEV RNA with mass ratio of 0.5∶1 and 1∶1, respectively. Detection of IgG in supernatants was prior to HEV RNA. There was no obvious difference of replication in vitro between HEV type Ⅰ and type Ⅳ, in which HEV RNA replication both peaked up at hour 48 after transfection. The expression of HEV-IgG and HEV RNA were inhibited by 47.12% and 43.3% after addition of anti-A6.Conclusion miR-A6 could significantly increase the replication ability of HEV RNA in full-length HEV transiently transfected HepG2 in vitro, which may lay a foundation for further research on HEV molecular virology and phenotype resistance.

Key words: microRNA, Hepatitis E virus, Full-length amplification, Transient transfection, Replication ability

张伟,游绍丽,刘鸿凌,朱冰,臧红,辛绍杰,荣义辉. MicroRNA-A6增效的HEV体外瞬时转染HepG2复制体系的建立及鉴定[J]. 肝脏, 2017, 22(10): 899-903.

ZHANG Wei, YOU Shao-li, LIU Hong-ling, ZHU Bing, ZANG Hong, XIN Shao-jie, RONG Yi-hui. Identification of synergistic effect of microRNA-A6 on transiently transfected-HepG2 replication system of HEV in vitro[J]. Chinese Hepatolgy, 2017, 22(10): 899-903.

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