人巨细胞病毒微小RNA miR-US4-5p靶向抑制PAK2的表达

Human cytomegalovirus miRNA miR-US4-5p inhibits expression of PAK2

目的寻找人巨细胞病毒微小RNA miR-US4-5p调控的靶mRNA,检测miR-US4-5p对靶mRNA PAK2蛋白质表达的调节作用。方法采用Hybrid-PCR方法及Targetscan软件分析从人巨细胞病毒感染的人胚肺成纤维细胞总RNA中筛选候选靶mRNA;应用荧光素酶实验,通过构建PMIR-PAK2报告基因,将其和miR-US4-5p、miRNA阴性对照共转染到HEK293细胞中,验证miR-US4-5p与候选靶mRNA的结合能力;应用Western blot方法,通过将miR-US4-5p、miRNA阴性对照分别转染到HEK293细胞、HELF细胞、THP-1细胞中,验证转染miR-US4-5p对靶mRNA蛋白质表达的调节作用。结果通过HybridPCR方法及Targetscan筛选出12个miR-US4-5p的待选靶mRNA;荧光素酶实验表明:与转染miRNA阴性对照相比,转染miRUS4-5p可以显著地下调PMIR-PAK2的荧光素酶活性,从而验证了miR-US4-5p与PAK2的结合性,在HEK293细胞、HELF、THP-1细胞中PAK2可以被过表达的miR-US4-5p造成不同程度的下调。结论人巨细胞病毒表达的miR-US4-5p在三种不同细胞系中具备抑制PAK2蛋白质表达的能力。

Objective To find the target message RNAs（ mRNA） of human cytomegalovirus（ HCMV） microRNA（ miRNA） miR-US4-5p,and to explore the effects of miR-US4-5p on protein expression of the target mRNA PAK2. Methods Hybrid-PCR and Targetscan were used to screen the candidate target mRNAs in the pool of human embryo lung fibroblast（ HELF） total RNAs. Luciferase report assay was used to validate the specific banding of miR-US4-5p to PAK2 mRNA after PMIR-PAK2 was constructed and contransfected with miR-US4-5p and miRNA negative control to HEK293 cells. Western blot was used to validate the regulation effects of miR-US4-5p on the protein expression of target mRNA after miR-US4-5p,miRNA negative control transfected to HEK293 cells,HELF cells,THP-1cells. Results Twelve mRNAs,including PAK2（ p21-activated Kinase 2）,were identified as the targets of miR-US4-5p. Dual-luciferase assay showed the luciferase activity of PMIR-PAK2 was significantly down-regulated after transfection of miR-US4-5p compared to the miRNA negative control,which validated the binding of miR-US4-5p and PAK2. PAK2 expression was down-regulated by over-expressed miR-US4-5p in HEK293,HELF and THP-1 cells. Conclusion HCMV miR-US4-5p may have the ability to inhibit the protein expression of its target mRNA,PAK2,during infection.