ZHAO Min,ZHANG Zhenzhen,LIU Quanbo.Effect of miRNA-146a on life cycle of hepatitis B virus in vitro[J].J Third Mil Med Univ,2017,39(17):1702-1708.

miRNA-146a对乙型肝炎病毒生活周期的影响及机制研究(/HTML )




Effect of miRNA-146a on life cycle of hepatitis B virus in vitro
赵敏张祯祯 刘泉波
ZHAO Min ZHANG Zhenzhen LIU Quanbo

Department of Infectious Disease, Key Laboratory of Child Development and Disorders of Ministry of Education, International Science and Technology Cooperation Base of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Children’s Hospital of Chongqing Medical University, Chongqing, 400014, China

hepatitis B virus miR-146a heparan sulfate-glucosamine 3-sulfotransferase 3
R373.21; R39433; R394.3

目的     研究微小RNA-146a(microRNA-146a,miR-146a)对乙型肝炎病毒(hepatitis B virus,HBV)生活周期的影响及其可能机制。方法    利用miRNA芯片比较HepG2.2.15细胞与HepG2细胞 miRNAs表达谱差异,选择miR-146a为研究对象,RT-PCR验证芯片结果。在HepG2.2.15细胞中分别转染miR-146a mimic和inhibitor,RT-PCR检测 HBV复制水平,ELISA和Western blot 检测蛋白水平。双荧光素酶报告系统进一步验证miR-146a与潜在靶点HS3ST3B1相互作用。HepG2.2.15细胞中转染miR-146a mimic,RT-PCR和Western blot分别检测HS3ST3B1 mRNA和蛋白水平。结果     miRNA芯片发现72条miRNAs在HepG2.2.15中表达发生变化,其中27条上调,45条下调, RT-PCR证实,miR-146a在HepG2.2.15细胞(1.55±0.13)中表达水平明显高于HepG2细胞(1.00±0.01) (P<0.05)。HepG2.2.15细胞转染miR-146a mimic后,HBV复制和蛋白水平较对照组明显升高 (P<0.05);转染 miR-146a inhibitor后,HBV复制和蛋白表达水平较对照组明显降低(P<0.05)。生物信息学预测发现HBV抑制因子HS3ST3B1是miR-146a的潜在靶点,双荧光素酶报告系统显示, HS3ST3B1野生型载体的报告荧光较对照组明显下调(P<0.05),突变预测靶位点后, HS3ST3B1突变型载体的报告荧光与对照组差异无统计学意义(P>0.05)。HepG2.2.15细胞中转染miR-146a mimic后,HS3ST3B1 mRNA水平较对照组差异无统计学意义(P>0.05),HS3ST3B1蛋白水平较对照组明显下调。结论    miR-146a能影响HBV生活周期,miR-146a可能通过作用于HBV抑制因子HS3ST3B1 3′UTR抑制其翻译从而影响HBV生活周期。


Objective     To determine the effect of microRNA-146a (miR-146a) on the life cycle of hepatitis B virus (HBV) and investigate the underlying mechanisms. Methods     The miRNA expression profiles were compared by miRNA array between HepG2 and HepG2.2.15 cells. Then miR-146a was chosen as objective, and its expression level was further confirmed by RT-PCR. After miR-146a mimic and inhibitor were transfected into HepG2.2.15 cells respectively, the quantification of HBV replication was determined by RT-PCR, and the levels of HBsAg and HBeAg in the supernatant were measured by ELISA, and the expression of HS3ST3B1 at mRNA and protein levels were tested by RT-PCR and Western blotting.  Dual-luciferase reporter assay was used to detect the interaction between miR-146a and potential target HS3ST3B1. Results     The expression levels of totally 72 miRNAs were changed in HepG2.2.15 cells, with 27 up-regulated and 45 down-regulated. RT-PCR showed the expression level of miR-146a was significantly higher in HepG2.2.15 cells than HepG2 cells (1.55±0.13 vs 1.00±0.01, P<0.05). Transfection of miR-146a mimic into HepG2.2.15 cells resulted in significantly increased HBV replication and levels of HBsAg and HBeAg (P<0.05), while the transfection of its inhibited caused opposite results (P<0.05).Bioinformatic analysis showed that HS3ST3B1 was a potential target of miR-146a. The reporter luciferase reporter system indicated that the reported fluorescence intensity of HS3ST3B1 wild type vector was significantly lower than that of the control group (P<0.05), but showed no significant difference between HS3ST3B1 mutant vector and control group (P>0.05). The mRNA level of HS3ST3B1 was not significantly changed in HepG2.2.15 cells transfected with miR-146a mimic (P>0.05), but its protein level was significantly decreased (P<0.05).  Conclusions    miR-146a affects the life cycle of HBV, which  may be through suppressing the translation of HBV inhibitory factor HS3ST3B1 3′UTR.


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更新日期/Last Update: 2017-09-04