过表达/干扰表达miR-122对HBV稳转优势单克隆株HepGA14表达功能的影响

Effects of overexpression/interference expression of miR-122 on stable and dominant mono⁃clonal strains HepGA14 of HBV

目的:采用miR-122过表达/干扰表达慢病毒质粒干预HBV稳转优势单克隆株HepGA14,观察其乙肝表面抗原(HBsAg)、乙肝e抗原(HBeAg)、乙肝病毒DNA(HBV DNA)表达功能的变化。方法:构建miR-122过表达和干扰表达慢病毒质粒,以最佳感染复数MOI值侵染HBV全基因组1.3倍体HBV稳转优势单克隆株HepGA14(对照组),分别命名为HepGA14-VL1044(miR122过表达组)和HepGA14-VL1043(miR122干扰组)。qPCR检测HepG2和HepGA14中的中miR-122的表达情况。ELISA分别检测3组细胞中上清HBsAg和HBeAg表达量,qPCR检测3组细胞的HBV DNA和miR-122表达水平。结果:最佳MOI值为30。与对照组比较,过表达miR-122慢病毒质粒侵染HepGA14后可上调优势细胞株内miR-122表达量,下调HBV DNA复制活性及HBsAg、HBeAg的表达量;干扰miR-122表达后,可上调HepGA14的HBV DNA复制活性及HBsAg、HBeAg的表达量,差异均有统计学意义(均P<0.0001)。结论:成功构建miR-122慢病毒过表达/干扰载体,并建立稳定过表达/干扰miR-122的HepGA14细胞系;miR-122可调控HBsAg、HBeAg、HBV-DNA的表达水平。

Objective:To observe the changes of hepatitis B surface antigen(HBsAg),hepatitis B e antigen(HBeAg)and hepatitis B virus DNA(HBV DNA)expression function by using miR-122 overexpression/interference expression of lentiviral plasmid to intervene HBV stable dominant HepGA14.Methods:miR-122 overexpression and interference expression lentiviral plasmids were constructed to infect 1.3-fold HBV stable and dominant monoclonal strain HepGA14(control group)of HBV whole genome at an MOI value of optimal multiplicity of infection,which were named HepGA14-VL1044(miR-122 overexpression group)and HepGA14-VL1043(miR-122 interference group).qPCR was used to detect the expression of miR-122 in HepG2 and HepGA14.ELISA was employed to detect the supernatant HBsAg and HBeAg expression in the three groups of cells.HBV DNA and miR-122 expression levels of the cells in the three groups were detected by qPCR.Results:The best MOI value was 30.Compared with the blank control group,the expression of miR-122 in the dominant cell line was up-regulated after HepGA14 was infected by overexpression of miR-122 lentivirus plasmid,while the replication activity of HBV DNA and the expression level of HBsAg and HBeAg were down-regulated.Interfering with the expression of miR-122 could up-regulate the HBV DNA replication activity of HepGA14 and the expression of HBsAg and HBeAg(P<0.0001).Conclusion:The miR-122 lentivirus overexpression/interference vector was successfully constructed,and the HepGA14 cell line with stable overexpression/interference of miR-122 was established.miR-122 can regulate the expression levels of HBsAg,HBeAg and HBV-DNA.