miR-133a-3p高表达对ZIKV复制的影响及其潜在机制

Effect of miR-133a-3p over expression on ZIKV replication and its underlying mechanism

目的探究寨卡病毒(ZIKV)感染A549细胞后诱导的小分子非编码RNA-133a-3p(miR-133a-3p)对ZIKV复制的影响及其潜在机制。方法在1×10^5/mL腺癌人类肺泡基底上皮细胞系A549中感染ZIKV,感染复数(MOI)为0.5,感染后在不同时间点收取RNA样品,以荧光定量PCR(RT-qPCR)法检测miR-133a-3p相对表达变化;同时在1×10^5/mL的A549中,转染3μL的miR-133a-3p模拟物(miR-133a-3p mimic)使miR-133a-3p高表达,转染后24 h,接种MOI=0.5的ZIKV或用终浓度为100μg/L的polyⅠ∶C处理细胞,感染后48 h收取细胞RNA或蛋白样品,通过RT-qPCR和蛋白质印迹法(Western blots)检测ZIKV的RNA的复制水平及宿主抗病毒先天免疫相关基因表达水平;将1μg含干扰素刺激应答元件(ISRE)的萤火虫荧光报告质粒(ISRE-luc)和50 ng海肾荧光报告质粒(pRL-TK)与3μL的miR-133a-3p共转入1×10^5/mL的A549中,转染后24 h,接种MOI=0.5的ZIKV或用终浓度为100μg/L的polyⅠ∶C处理细胞,在处理48 h后通过双荧光报告实验检测含ISRE序列的萤火虫荧光蛋白表达情况。结果ZIKV感染可影响A549细胞内miR-133a-3p表达,相对于0 h,在8、16、24、36、48和72 h,miR-133a-3p值分别为:0.45±0.048、0.4±0.084、0.52±0.004、0.82±0.229、1.41±0.167、2.86±0.219;miR-133a-3p过表达使miR-133a-3p mimic组的ZIKV NS5 RNA相对于mimic control组值为:0.3355±0.031,使IFN-α/β、ISG15、IFIT1(按图A→H的顺序)相对值为:3.46±1.10^5、1.329±0.198、2.723±0.966、1.48±0.0595、3.3±0.4、2.83±0.442、5.79±0.277、1.98±0.362、5.96±0.796、5.94±1.007、7.07±0.1115、5.96±0.7957、5.96±0.796、7.16±0.42、2.2±0.491、1.92±0.189。结论ZIKV感染A549细胞影响miR-133a-3p表达;miR-133a-3p过表达抑制ZIKV复制并激活Jak/STAT信号通路。

Objective To explore the effect and potential mechanism of small molecule non-coding RNA-133a-3p(miR-133a-3p)on ZIKV replication in A549 cells spiked with Zika virus(ZIKV).Methods ZIKV was spiked into 1×10^5/mL A549(adenocarcinoma human alveolar basal epithelial cell line)with a multiplicity of infection(MOI)at 0.5.After the infection,RNA samples were collected at different time points,and the relative expression changes of miR-133a-3p was determined by RT-qPCR.In A549 of 1×10^5/mL,3μL of miR-133a-3p mimic was transfected to make miR-133a-3p reach a high expression.24 h after transfection,ZIKV(MOI=0.5)or polyⅠ∶C at a final concentration of 100μg/L was used to treat cells.Cellular RNA or protein samples was collected 48 hours after infection.The replication level of ZIKV RNA and expression level of host anti-virus innate immunity related gene were detected by RT-qPCR and Western blots.1μg of firefly fluorescent reporter plasmid(ISRE-luc)containing interferon-stimulated response element(ISRE)and 50 ng of Renilla fluorescent reporter plasmid(pRL-TK)were co-transformed with 3μL of miR-133a-3p into A549 of 1×10^5/mL.24h after transfection,we inoculated ZIKV(MOI=0.5)or treated cells with polyI∶C with a final concentration of 100μg/L.and 48 h after the treatment,the ISRE sequence containing Firefly fluorescent protein expression was detected.Results ZIKV infection can affect the expression of miR-133a-3p in A549 cells.Compared to 0h,the relative expression of miR-133a-3p at 8,16,24,36,48 and 72 h was 0.45±0.048,0.4±0.084,0.52±0.004,0.82±0.229,1.41±0.167,and 2.86±0.219,respectively.Overexpression of miR-133a-3p maked the ZIKV NS5 RNA expression in miR-133a-3p mimic group relative to the mimic control group was 0.3355±0.031,and maked the relative expression levels of IFN-α/β,ISG15 and IFIT1(in the order of Figure A to H)was 3.46±1.10^5,1.329±0.198,2.723±0.966,1.48±0.0595,3.3±0.4,2.83±0.442,5.79±0.277,1.98±0.362,5.96±0.796,5.94±1.007,7.07±0.1115,5.96±0.7957,5.96±0.796,7.16±0.42,2.2±0.491,and 1.92±0.189.Conclusion The miR-133a-3p expression was affected in A549 cells spiked with ZIKV;miR-133a-3p overexpression inhibits ZIKV replication;miR-133a-3p overexpression activates the Jak/STAT signaling pathway.