沉默A549细胞中的CD46和DSG2可抑制人3型和7型腺病毒的侵入及IL-8的释放

Silencing CD46 and DSG2 in host A549 cells inhibits entry of human adenovirus type 3 and type 7 and reduces interleukin-8 release

目的探讨沉默受体CD46、桥粒芯蛋白(DSG2)对人3型腺病毒(HAdV-3)和7型腺病毒(HAdV-7)的侵入及炎症因子释放的影响。方法通过RNA干扰技术沉默A549细胞中CD46、DSG2的表达。实验分为HAdV-3组、HAdV-3+siRNA-NC组(siRNA无关序列对照组)、HAdV-3+siRNA-CD46组(转染siRNA-CD46)、HAdV-3+siRNA-DSG2组(转染siRNA-DSG2);HAdV-7组、HAdV-7+siRNA-NC组(siRNA无关序列对照组)、HAdV-7+siRNA-CD46组(转染siRNA-CD46)、HAdV-7+siRNA-DSG2组(转染siRNA-DSG2)。HAdV-3、7感染A549细胞后0.5、2 h,通过激光共聚焦显微镜观察两种腺病毒与受体CD46、DSG2结合水平;体外转染siRNA-CD46、siRNA-DSG2后不同时间点,qRT-PCR技术检测HAdV-3、7拷贝数,ELISA检测转染后IL-8表达情况。结果腺病毒感染A549细胞后0.5、2 h,HAdV-3、7与其受体CD46、DSG2结合共定位;腺病毒入胞后,随时间延长,病毒拷贝数增加,转染siRNA-CD46后2、6、12、24 h,HAdV+siRNA-CD46组的病毒拷贝数较HAdV组降低(HAdV-3+siRNA-CD46 vs HAdV-3:6 h,P<0.05;12、24 h,P<0.0001;2 h,降低趋势无统计学差异;HAdV-7+siRNA-CD46 vs HAdV-7:2 h,P<0.01;6 h,P<0.001;12、24 h,P<0.0001)。转染siRNA-DSG2后2、6、12、24 h,同样明显降低了病毒载量(HAdV-3+siRNA-DSG2 vs HAdV-3,HAdV-7+siRNA-DSG2 vs HAdV-7,P<0.0001)。HAdV-3、7感染A549细胞后,炎症因子IL-8释放增多(P<0.0001),沉默CD46和DSG2的表达后2、6 h,炎症因子IL-8水平降低(P<0.0001)。结论HAdV-3、7与其受体CD46、DSG2结合后入胞,病毒拷贝数随时间延长而增加,沉默CD46、DSG2后,抑制HAdV-3、7型腺病毒的侵入及IL-8释放。

Objective To investigate the effect of silencing CD46 and desmoglein 2(DSG2)in host A549 cells on the entry of human adenovirus type 3(HAdV-3)and type 7(HAdV-7)and host cell secretion of inflammatory cytokines.Methods RNA interference technique was use to silence the expression of CD46 or DSG2 in human epithelial alveolar A549 cells as the host cells of HAdV-3 or HAdV-7.The binding of the viruses with CD46 and DSG2 were observed with immunofluorescence staining at 0.5 and 1 h after viral infection.The viral load in the host cells was determined with qRT-PCR,and IL-8 secretion level was measured using ELISA.Results In infected A549 cells,immunofluorescent staining revealed colocalization of HAdV-3 and HAdV-37 with their receptors CD46 and DSG2 at 0.5 h and 2 h after infection,and the copy number of the viruses increased progressively after the infection in a time-dependent manner.In A549 cells with CD46 silencing,the virus titers were significantly lower at 2,6,12 and 24 h postinfection in comparison with the cells without gene silencing;the virus titers were also significantly decreased in the cells with DSG2 silencing.The secretion level of IL-8 increased significantly in A549 cells without siRNA transfection following infection with HAdV-3 and HAdV-7(P<0.0001),but decreased significantly in cells with CD46 and DSG2 silencing(P<0.0001).Conclusion HAdV-3 and HAdV-7 enter host cells by binding to their receptors CD46 and DSG2,and virus titer and cytokines release increase with infection time.Silencing CD46 and DSG2 can inhibit virus entry and cytokine IL-8 production in host cells.