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研究RNF126在EV71感染炎症反应调控中的作用及相关机制。在THP-1诱导分化的巨噬细胞中，以特异性靶向RNF126的干扰RNA降低RNF126表达作为干扰组，以阴性对照干扰RNA作为对照组，以EV71感染不同时间，采用实时荧光定量PCR和ELISA分别检测IL-6、TNF-α、CCL3的mRNA和蛋白质水平表达变化；通过实时荧光定量PCR检测EV71-VP1表达变化；采用空斑形成试验检测病毒滴度变化；以Western blot检测天然免疫NF-κB信号通路相关蛋白的磷酸化和表达变化。通过免疫共沉淀检测RNF126相互作用靶蛋白；观察RNF126对靶蛋白泛素化修饰的影响。结果发现，在EV71感染时，干扰RNF126表达后与对照组相比：IL-6、TNF-α和CCL3的mRNA和蛋白质水平表达显著减少（P<0.05）,EV71-VP1表达增强（P<0.05），病毒滴度增高；NF-κB信号通路中IKKα/β、IκBα和p65磷酸化水平下降，IKKγ、IKKα及p65总蛋白表达无明显变化，IκBα表达增加。RNF126与IKKγ相互作用并介导其K63位泛素化活化。以上结果说明，EV71感染时，RNF126与IKKγ相互作用并介导其K63位泛素化，促进NF-κB信号通路活化及其下游细胞炎症因子产生，抑制病毒复制。
Abstract：To investigate the role of RNF126 in the regulation of inflammation during EV71 infection and related mechanisms. In THP1-induced differentiation of macrophages, interference RNA specifically targeting RNF126to reduce the expression of RNF126 was used as the interference group, and negative interference RNA was used as the control group. Real-time fluorescence quantitative PCR and ELISA were used to detect the mRNA and protein expression of IL-6, TNF-α and CCL3, respectively. The expression of EV71-VP1 was detected by real-time fluorescence quantitative PCR. The change of virus titer was detected by plaque formation test.Phosphorylation and expression of proteins associated with NF-κB signaling pathway were detected by Western Blot. The RNF126 interaction target protein was detected by co-immunoprecipitation. The effect of RNF126 on ubiquitination modification of target protein was observed. Results showed that in EV71 infection, the mRNA and protein levels of IL-6, TNF-α and CCL3 were significantly decreased(P<0.05), the expression of EV71-VP1 was enhanced(P<0.05), and the virus titer was increased compared to the control group after interference with RNF126 expression. The phosphorylation levels of IKKα/β, IκBα and p65 were decreased in NF-κB signaling pathway, while the expressions of IKKγ, IKKα and p65 total proteins were not significantly changed, the expressions of IκBα were increased. RNF126 interacts with IKKγ and mediates its K63 ubiquitination activation. These results suggest that during EV71 infection, RNF126 interacts with IKKγ and mediates its K63ubiquitination, promoting the activation of NF-κB signaling pathway and inducing the production of downstream cellular inflammatory factors, so as to inhibit viral replication.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004628
中图分类号:R373.2
引用信息:
[1]林小嫚,潘倩瑜,何雅青,等.RNF126介导IKKγ的K63位泛素化促进EV71感染炎症反应的机制研究[J].病毒学报,2025,41(01):71-78.DOI:10.13242/j.cnki.bingduxuebao.004628.
基金信息:
深圳市科创委基础研究面上项目（项目号：JCYJ20230807151802006）,题目：HAUSP通过NLRP3/IL-1β调控EV71感染炎症反应的机制研究~~
2024-06-20
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