HSV-2miR-H4-5p负调节CDKL2基因表达并抑制ActD引起的Vero细胞凋亡

HSV-2 miR-H4-5p Negatively Regulates CDKL2 Gene Expression,Blocking Actinomycin D(ActD)-induced Apoptosis in Vero Cells

单纯疱疹病毒Ⅱ型(herpes simplex virusⅡ,HSV-2)编码的microRNA-H4-5p(miR-H4-5p)可与病毒神经毒力蛋白ICP34.5mRNA互补结合并抑制其表达,从而降低病毒感染对细胞的毒力作用,以减弱细胞对病毒的免疫作用.但miR-H4-5p是否可靶向作用于宿主细胞基因目前仍不十分清楚.本研究证明,miR-H4-5p可通过靶向细胞周期依赖性激酶(cyclin-dependent kinase-like 2,CDKL2)基因表达抗防线菌素D(actinomycin D,Act D)诱导的非洲绿猴肾上皮细胞(African green monkey kidney cells,Vero)细胞凋亡.生物信息学方法在线预测miR-H4-5p潜在靶基因CDKL2的结合位点,并构建双萤光素酶报告系统检测miR-H4-5p靶向作用.数据表明,miR-H4-5p可有效抑制萤光素酶的表达.q PCR和Western印迹数据表明,miR-H4-5p可在mRNA水平和蛋白水平抑制CDKL2表达.构建过表达载体pmR-mcherry/miR-H4-5p(cherry/H4-5p),将cherry/H4-5p与pmRmcherry空质粒转染至Vero细胞,转染24 h后加入终浓度为1μg/m L放线菌素D诱导细胞凋亡.MTT法、流式细胞术和Western印迹检测Bax、Bcl-2表达.数据表明,miR-H4-5p可抑制Act D诱导的Vero细胞活性降低.本实验提示,miR-H4-5p可通过靶向调节宿主细胞基因抑制细胞凋亡,可能以此方式共同参与HSV-2潜伏感染的建立.但是这种抑制凋亡作用的通路及其机制目前仍不清楚,miR-H4-5p是否可靶向更多的宿主基因仍需要进一步实验验证.

miRNA-H4 encoded by herpes simplex virus Ⅱ shares perfect complementarity with infected cell protein ICP34. 5 mRNA, a viral PKR inhibitor, and regulates its expression to attenuate the innate immune response. It remains unknown whether HSV-2 encoded miRNA-H4 can target host genes. Data in our study suggested that miR-H4-5p may prevent actinomycin D （ActD）-induced apoptosis of African green monkey kidney cells （ Vero cell） via targeting cyclin-dependent kinase-like 2 （CDKL2） gene.Bioinformatics analyzed the possible binding sites between miR-H4-5p and 3＇UTR of CDKL2 mRNA, then dual luciferase assay was carried out to determine the targeting role of miR-H4-5p. Compared to negative control, miR-H4-5p could significantly reduce the relative luciferase activity. Expression of CDKL2 was greatly reduced at mRNA and protein leve via qPCR and Western blot assay. Recombinant plasmid pmR-mcherry/miR-H4-5p （cherry/H4-5p） and empty vector （pmR-eherry） were transfected into Vero cells following treatment of 1 μg/rnL AetD for 36 h. MTT assay, flow cytometry assay and detection of Bcl-2, Bax expression by Western blot indicated that miR-H4-5p over-expression could effectively prevent depression of cell viability. Our findings suggested that miR-H4-5p could prevent cell apoptosis via inhibiting related gene expression, of which might involve in establishment of HSV latent infection but little is known about the mechanism, more potential cellular targets of miR-H4-5p need to be further experimentally validated.