摘要
目的探讨细胞中ADAR1对锌指蛋白ZNF655的作用及其对乙肝病毒(HBV)复制的影响。方法在人肝癌细胞系HepG2细胞中,利用桑格测序验证ZNF655 3'UTR存在ADAR1 RNA编辑位点;RT-qPCR检测ADAR1和ZNF655 mRNA的表达及HBV total RNA和HBV 3.5 kb RNA的表达;双荧光素酶报告基因检测荧光素酶的相对表达;Western blot检测ADAR1和ZNF655蛋白的表达;ELISA检测ZNF655对HBV标志物HBs Ag和HBe Ag的影响。结果 ZNF655基因3'UTR上chr7:99575277位点在DNA水平为纯合型,在RNA水平为杂合型;ZNF655基因3'UTR上chr7:99575277位点的编辑型G比正常型A的荧光素酶活性显著升高(P<0.001);在转录和翻译水平,ADAR1都显著增加了ZNF655的表达(P<0.001);ZNF655对HBV的表达起到促进的作用。结论 ADAR1通过编辑ZNF655的3'UTR上的chr7:99575277位点,使编辑位点A转换成G,上调了基因的表达,进而起到对HBV复制的促进作用。
Objective To explore the effect of ADAR1 on ZNF655 and the regulation of ZNF655 on the expression of HBV. Methods Sanger sequencing was used to validate the 3' UTR region of ZNF655 in ADAR1. The expression of ADAR1 and ZNF655 mRNA as well as HBV RNA were detected by RT-qPCR. Dual luciferase report plasmid assay was used to detect the expression of luciferase. To detect the expression of ADAR1 and ZNF655 protein by Western blot. HBs Ag and HBe Ag was detected by ELISA. Results The chr7: 99575277 loci on ZNF655 3'UTR was homozygous in DNA level and hybrid in RNA level. On the 3'UTR editing site of ZNF655,the luciferase activity of the edited G allele was significantly higher than that of the normal A allele( P<0. 001). The expression of ZNF655 was upregulated by ADAR1 in the level of transcription and translation( P<0. 01).ZNF655 significantly promoted the expression of HBV. Conclusions The chr7: 99575277 loci on ZNF655 3'UTR is edited by ADAR1,promoting the expression of ZNF655,which upregulated the expression of HBV.
引文
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