miR-769-3p对甲型H1N1流感病毒核蛋白表达的调控

Regulation of Nucleoprotein Gene Expression of H1N1 Influenza A Virus by miR-769-3p

目的:预测靶向甲型流感病毒核蛋白(NP)基因的微小RNA(miRNA),并检测其对NP表达的影响。方法:从miRBase数据库中获取人成熟miRNA序列,利用miRanda软件预测潜在靶向流感病毒A/FM/1/47(H1N1)NP基因的人miRNA;通过双萤光素酶报告基因系统及Western印迹验证所预测的miRNA对NP表达的影响。结果:用miRanda软件在流感病毒A/FM/1/47(H1N1)NP基因上预测得到分值及最小结合自由能均较好的miR-769-3p;双萤光素酶报告基因结果显示miR-769-3p能显著降低报告基因载体萤光素酶的表达;Western印迹结果显示miR-769-3p能明显抑制NP的表达,但突变NP基因上的miR-769-3p结合位点后,miR-769-3p不能抑制NP的表达。结论:miR-769-3p可靶向流感病毒A/FM/1/47(H1N1)NP基因并抑制NP的表达,为抗甲型流感病毒的miRNA药物研发提供了依据和潜在药物靶标。

Objective： To screen the candidate microRNA（miRNA） targeting the nucleoprotein（NP） of HIN1 influ- enza A virus by online tools, and analyze effect of the miRNA on NP expression by methods of biotechnology. Methods： Human mature miRNA sequences were downloaded from miRBase database, and miranda was used to predict the candidate miRNA targeting NP of A/FM/1/47（H1N1） from these sequences. The binding of miRNA with NP gene was tested by using dual-luciferase assays, and the effect of miRNA on NP expression was ana- lyzed by Western blotting. Results： With the excellent miranda score and minimal free energy, miR-769-3p was predicted as the most likely miRNA targeting NP gene. In lnciferase analysis, the NP reporter＇s luciferase activity dramatically decreased, which evidenced the binding of miR-769-3p with NP. Western blotting showed that the ex- pression of wild type NP was significantly suppressed by miR-769-3p, and this suppression did not occur when miR-769-3p targeting sites on NP were mutated. Conclusion： miR-769-3p can directly target NP of A/FM/1/47 （H1N1） and inhibit NP expression. Our study provide data basis and potential drug target for the development of new strategies for anti-influenza A virus intervention.