miRNA-146a对乙型肝炎病毒生活周期的影响及机制研究

Effect of miRNA-146a on life cycle of hepatitis B virus in vitro

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Hep G2.2.15细胞与Hep G2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Hep G2.2.15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。Hep G2.2.15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Hep G2.2.15中表达发生变化,其中27条上调,45条下调,RT-PCR证实,miR-146a在Hep G2.2.15细胞(1.55±0.13)中表达水平明显高于Hep G2细胞(1.00±0.01)(P<0.05)。Hep G2.2.15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)。Hep G2.2.15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 superuatant were measured by ELISA, and the expression of HS3ST3B1 at mRNA and protein levels were tested by RT-PCR and Western blotting. Dualluciferase 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 ceils than HepG2 ceils （ 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 transfeeted 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.