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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.
The miRNA-H4-5p (miR-H4-5p) encoded by herpes simplex virusⅡ (HSV-2) can complementarily inhibit and inhibit the expression of the virion virulence protein ICP34.5mRNA to reduce the viral infection However, whether miR-H4-5p can target gene in host cells is still not clear at present.This study demonstrates that miR-H4-5p can target cells Cyclin-dependent kinase-like 2 (CDKL2) gene was expressed in Actinomycin-induced apoptosis in African green monkey kidney cells (Vero). Bioinformatics method to predict the binding site of CDKL2, a potential target gene of miR-H4-5p, and to establish a dual luciferase reporter system to detect the targeting effect of miR-H4-5p.The data show that miR-H4-5p can effectively inhibit Luciferase expression.q PCR and Western blot data show that miR-H4-5p can inhibit CDKL2 expression at the mRNA level and protein level.Overexpression vector pmR-mcherry / miR-H4-5p (cherry / H4-5p ), The empty vector of cherry / H4-5p and pmRmcherry was transfected into Vero cells and transfected for 24 h The concentration of 1μg / ml L Actinomycin D induced apoptosis.MTT method, flow cytometry and Western blot detection of Bax, Bcl-2 data.The data show that miR-H4-5p can inhibit Act D-induced Vero cells This study suggests that miR-H4-5p may be involved in the establishment of latent infection with HSV-2 by targeting the gene suppression of host cell apoptosis in this way.However, this pathway of inhibiting apoptosis and its Mechanism remains unclear whether miR-H4-5p can target more host genes still need further experimental verification.