脾脏酪氨酸激酶和核因子-κB对新城疫病毒-血凝素-神经氨酸酶上调自然杀伤细胞TRAIL蛋白表达的影响

Impact of spleen tyrosine kinase and nuclear factor-kappaB on Newcastle disease virus-hemagglutinin-neuraminidase upregulating TRAIL protein expression in natural killer cells

目的探讨脾脏酪氨酸激酶(Syk)和核因子-κB(NF-κB)对新城疫病毒(NDV)-血凝素-神经氨酸酶(HN)上调自然杀伤(NK)细胞肿瘤坏死因子相关凋亡诱导配体(TRAIL)蛋白表达的影响。方法将干扰素γ(IFN-γ)受体缺失的NK细胞株(IFNR-/-NK)分为小干扰RNA(siRNA)-Syk1组、siRNA-Syk2组、siRNA-Syk3组、siRNA-p65-1组、siRNA-p65-2组、siRNA-p65-3组、未处理组、脂质体组、siRNA-NC组,未处理组加入磷酸缓冲盐溶液(PBS),脂质体组加入由脂质体3000和Opti-MEM培养基组成的转染复合物,siRNA-NC组加入含非特异性siRNA的转染复合物,其他组加入含相应siRNA的转染复合物。(1)转染36h后,检测各组细胞Syk和(或)p65的mRNA及蛋白的表达水平。(2)转染16h后,将细胞分为siRNA-Syk1+HN组、siRNA-Syk2+HN组、siRNA-Syk3+HN组、siRNA-p65-1+HN组、siRNA-p65-2+HN组、siRNA-p65-3+HN组、脂质体+HN组和siRNA-NC+HN组、未处理组,未处理组细胞加入PBS,其余各组细胞加入NDV-HN。检测各组细胞膜型及分泌型TRAIL蛋白的表达水平,并检测siRNA-Syk1+HN组、siRNA-Syk2+HN组、siRNA-Syk3+HN组、脂质体+HN组、siRNA-NC+HN组和未处理组磷酸化IκBα蛋白的表达水平。结果(1)瞬时转染36h后,与脂质体组比较,siRNA-Syk1、siRNA-Syk2、siRNA-Syk3组的SykmRNA及蛋白表达水平降低,siRNA-p65-1组、siRNA-p65-2组、siRNA-p65-3组p65mRNA及蛋白表达水平亦降低(均P<0.05)。(2)未处理组的膜型TRAIL蛋白及分泌型TRAIL蛋白表达水平均低于其他组,siRNA-Syk1+HN组、siRNA-Syk2+HN组、siRNA-Syk3+HN组及siRNA-p65-1+HN组、siRNA-p65-2+HN组、siRNA-p65-3+HN组的膜型TRAIL蛋白及分泌型TRAIL蛋白表达水平均低于脂质体+HN组(均P<0.05)。未处理组磷酸化IκBα蛋白表达水平均低于其他组,siRNA-Syk1+HN组、siRNA-Syk2+HN组、siRNA-Syk3+HN组磷酸化IκBα蛋白表达水平均低于脂质体+HN组(均P<0.05)。结论敲低Syk或p65可以削弱NDV-HN上调NK细胞TRAIL蛋白的表达。Syk和NF-κB信号通路可能介导NDV-HN直接活化NK细胞表达TRAIL的过程。

Objective To investigate the impact of spleen tyrosine kinase(Syk)and nuclear factor-kappaB(NF-κB)on Newcastle disease virus(NDV)-hemagglutinin-neuraminidase(HN)upregulating tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)protein expression in natural killer(NK)cells.Methods NK cell lines with interferon gamma(INF-γ)receptor deletion(IFN R-/-NK)were divided into small interfering RNA(siRNA)-Syk1 group,siRNA-Syk2 group,siRNA-Syk3 group,siRNA-p65-1 group,siRNA-p65-2 group,siRNA-p65-3 group,treatment-na ve group,liposome group and siRNA-NC group,phosphate-buffered saline(PBS)was added to the cells in the treatment-na ve group,the transfected complex comprised of liposome 3000 and Opti-MEM medium to the liposome group,the transfected complex containing non-specific siRNA to the siRNA-NC group,the transfected complex containing corresponding siRNA to the other groups.(1)After 36 hours of transfection,the expression levels of Syk and/or p65 mRN A and protein were detected in each group.(2)After 16 hours of transfection,cells were divided into siRNA-Syk1+H N group,siRNA-Syk2+HN group,siRNA-Syk3+HN group,siRNA-p65-1+HN group,siRNA-p65-2+HN group,siRNA-p65-3+HN group,liposome+HN group,siRNA-NC+HN group and treatment-na ve group.PBS was added to the cells in the treatment-na ve group,and NDV-HN to the remaining groups.The expression levels of membrane and secretory TRAIL protein were detected in each group,the expression level of phosphorylated IκBαprotein was detected in the siRNA-Syk1+HN group,siRNA-Syk2+HN group,siRNA-Syk3+HN group,liposome+HN group,siRNA-NC+HN group and treatment-na ve group.Results(1)Thirty-six hours after instantaneous transfection,as compared with the liposome group,the siRNA-Syk1,siRNA-Syk2,and siRNA-Syk3 groups had decreased expression levels of Syk mRNA and protein,and the siRNA-p65-1,siRNA-p65-2,siRNA-p65-3 groups had decreased expression levels of p65 mRNA and protein(all P<0.05).(2)The expression levels of membrane and secretory TRAIL protein in the treatment-na ve group were lower than those in the other groups,the expression levels of membrane and secretory TRAIL protein in the siRNA-Syk1+HN group,siRNA-Syk2+HN group,siRNA-Syk3+HN group,siRNA-p65-1+HN group,siRNA-p65-2+HN group and siRNA-p65-3+HN group were lower than those in the liposome+HN group(all P<0.05).The treatment-na ve group had a lower expression level of phosphorylated IκBαprotein than the other groups,and the siRNA-Syk1+HN,siRNA-Syk2+HN,and siRNA-Syk3+HN groups obtained a lower expression level of phosphorylated IκBαprotein compared with the liposome+HN group(all P<0.05).Conclusion Knockdown of Syk or p65 can reduce NDV-HN upregulating TRAIL protein expression in NK cells.Syk and NF-κB signaling pathway might mediate the process of TRAIL expression in NK cells directly activated by NDV-HN.