miRNA93和miRNA192在H3N2亚型SIV病毒复制及宿主抗病毒免疫中的作用

目的探讨分离于广西猪流感病毒SW/Guangxi/NS2783/2010和SW/Guangxi/NS650/2012跨种属感染人肺腺癌A549细胞的miRNA93和miRNA192对病毒复制及宿主抗病毒免疫的影响。方法通过测定不同稀释浓度的病毒感染细胞HA滴度值,确定病毒最佳稀释浓度。荧光定量PCR检测病毒感染细胞后miRNA93和miRNA192表达,Western blot检测病毒NP和HA蛋白表达水平;转染miRNA93和miRNA192抑制剂后,重新检测miRNA93、miRNA192、IFN-β及病毒NP、HA蛋白表达水平。结果不同稀释度病毒感染人A549细胞后HA滴度的结果显示病毒SW2783的最佳稀释度为10-3,而SW650的HA滴度无明显变化趋势,提示病毒SW2783对人A549细胞具有较好的适应性和感染能力。荧光定量PCR和Western blot结果提示两株病毒感染细胞后病毒NP和HA蛋白表达均先升高后降低,加入miRNA93抑制剂,两株病毒NP和HA蛋白的表达均上调;加入miRNA192抑制剂,病毒SW2783的HA蛋白表达下调(P=2.10×10-4),而SW650的HA蛋白表达上调(P=5.45×10-5),NP蛋白反而下调(P=0.034);ELISA结果提示病毒SW2783和SW650感染细胞后炎症因子IFN-β表达水平随感染时间延长而升高。结论研究表明miRNA93和miRNA192的表达与SIV病毒增殖及宿主抗病毒免疫有关,可作为干预猪流感病毒跨种属感染的新靶点。

Effects of miRNA93 and miRNA192 on the replication and antiviral immune of host for H3N2 subtype SIV

Objective To study the productions of miRNA93 and miRNA192 and their effects on virus replication and host anti-infection immunity of human lung adenocarcinoma A549 cells infected by SW/Guangxi/NS2783/2010 and SW/Guangxi/NS650/2012.Methods By measuring the HA titer value of the infected cells at different dilutions of the virus,we determined the optimal dilution concentration of the virus.The expression levels of miRNA93,miRNA192,virus NP and HA were detected by qRT-PCR and Western blot.After transfecting miRNA93 and miRNA192 inhibitors,the expression levels of miRNA93,miRNA192,IFN-βand viral NP and HA proteins were redetected.Results The results of HA titer showed that the optimal dilution of virus SW2783 was 10-3 when A549 cells were infected with different dilutions of virus,while the HA titer of SW650 showed that there was no obvious change trend.The results suggested that virus SW2783 has better adaptability and infection ability for human A549 cells.qRT-PCR and Western blot results showed that the expressions of viral NP and HA protein increased first and then decreased when two viruses infected cell.When adding the miRNA93 inhibitor,the expression of NP and HA proteins were up-regulated.The expression of HA protein about virus SW2783 was down-regulated by adding miRNA192 inhibitor(P=2.10×10-4),while the expression of HA protein about SW650 was up-regulated(P=5.45×10-5),and NP protein was down-regulated(P=0.034).ELISA result showed that the expression level of inflammatory factor IFN-βincreased with time extension when SW2783 and SW650 viruses infected cell.Conclusion The research has shown that the expressions of miRNA93 and miRNA192 are related to viral proliferation and anti-infection immunity of swine influenza virus(SIV),which may be a new target for cross-species infection intervention of SIV.