Effects Of Silencing RIG-I Gene On The Expression Of TRAIL,TNF-?,IL-6 Genes And Production Of Nitric Oxide In NDV-activated Mouse Macrophages

Background and objective:Oncolytic virus therapy has been applied as a supplementary way in the clinic treatment of some cancers.The oncolytic Newcastle disease virus(NDV)possesses anti-tumor effect that is not only related to apoptosis and autophagy,but also related to its ability to stimulate immune cells.In recent years,more and more attention about oncolytic NDV has been paid to its regulatory role on the anti-tumor effect of immune cells.It was found that tumoricidal activity in macrophage was enhanced after NDV stimulation.That was mediated not only by up-expression of secreting cytotoxic factors such as IFN(interferon),TNF(tumor necrosis factor)as well as TRAIL(TNF-related apoptosis-inducing ligand),but also by releasing cytotoxic factors such as nitric oxide(NO).However,the pathways involved in the up-expression of these factors described as above are yet to be identified.It was widely known that retinoic acid inducible gene I(RIG-I)functions as a cytosolic pattern recognition receptor that initiates innate antiviral immunity by detecting exogenous viral RNAs.Recent studies showed that RIG-I participates in various cellular activities by sensing exogenous viral RNAs under different circumstances.However,it is not clear whether RIG-I participates in activationof macrophages by NDV.Therefore,the effects of RNAi silencing RIG-I gene on the expression of TRAIL,TNF-?,IL-6 genes and NO production in NDV-activated mouse macrophage were investigated.Our results will provide insights into tumor immunotherapy based on NDV-macrophage stimulation.Methods:1.The mouse macrophage strainRAW264.7was used in this study.The effects of NDV stimulation on the mRNA level of TRAIL,TNF-? and IL-6 in macrophage strain RAW264.7 were investigated using real-time fluorescence quantitative PCR(qPCR).The effects of NDVstimulation on the expression of RIG-I in macrophage strain RAW264.7 wereinvestigated using qPCR and western blotting,respectively.The concentration of NO released by RAW264.7 after NDVactivation was detected by Griess method.2.Blocking experiments using siRNA interference were conducted to investigate whether NDV-induced activation of macrophage was dependent on RIG-I pathway.The effects of silencing RIG-I gene on then expression of TRAIL,TNF-?,IL-6 gene in NDV-activated mouse macrophage strain RAW264.7 were investigated using qPCR.The effects of silencing RIG-I gene on the concentration of NO released was detected by Griess method.Results:1.The marked increase mRNA levels of TRAIL,TNF-aand IL-6 in the mouse macrophage RAW264.7 cells were observed when NDV or UV-NDV was used as stimuliin vitro(p < 0.05).The upregulated RIG-I expression wasobserved when NDV was used as stimuli in vitro(p< 0.05).The concentration of NO was significantly increased when NDV or UV-NDV was used as stimuli in vitro(p < 0.01).2.SiRNA-1101 and siRNA-1943 were used to interfere with RIG-I gene in macrophages,respectively.The marked decrease mRNA levels of RIG-I were observedin the interference group(p < 0.01).Silence of RIG-I in macrophages partly impair NDV-induced TRAIL expression and NDV-induced IL-6 expression,respectively.However,silence of RIG-I in macrophages did not impair the TNF-? mRNA level and the NO concentration.Conclusion:We studied the effects of RNAi silencing RIG-I gene on the expression of TRAIL,TNF-?,IL-6 genes and NO production in NDV-activated mouse macrophage.We found that the up-regulation of TRAIL and IL-6 were activated by NDV in macrophage via an RIG-I pathway.This finding indicates the potential immune-stimulatory capabilities of NDV and provide evidences in tumor immunotherapy based on NDV-macrophage stimulation.