The Role And Mechanism Of RNA Helicase DHX35 In Regulating The Production Of Type Ⅰ Interferon In Respiratory Epithelial Cells

Background:As a place for gas exchange,the respiratory tract is constantly exposed to the air and is easy to be infected with bacteria,viruses and parasites,which poses a serious threat to human health.According to the World Health Organization,3.5 million people die each year due to respiratory infections,which are also the leading cause of death in children under the age of 5 worldwide.Studies have shown that when virus infection occurs,on the one hand,the respiratory epithelium acts as a natural physical barrier to isolate the virus directly outside the cell.On the other hand,when the virus enters the body,cells clear the virus by producing antiviral cytokines.Type I interferon（including IFN-α and IFN-β）is the most important antiviral cytokine and can be produced by most cells in the body.The body recognizes "non-self" pathogen-associated molecular patterns（PAMPs）through a series of pattern recognition receptors（PRRs）,the most famous PRRs include Toll-like receptors,c GAS,and RIG-I like receptors.In addition,there are other PRRs in the cytoplasm that can recognize pathogen-associated molecular patterns to mediate the production of type I interferons,many of which are members of the RNA helicase family of RIG-I-like receptors.DHX35 is a member of the DEx D/H-Box subfamily of RNA helicases.Gene Ontology（GO）analysis showed that DHX35 had molecular functions related to nucleic acid binding and helicase activity.However,the role of DHX35 in viral infection and its mechanism is not clear.Aim:Through the research of this topic,we will clarify the role of DHX35 in the antiviral infection,and discover the mechanism of DHX35 mediating the secretion of type I interferon in respiratory epithelial cells.This study provides new ideas and targets for the design of drugs and vaccines related to respiratory diseases caused by virus infection.Methods:1.Total RNA and protein were extracted from different types of respiratory epithelial cell lines,B cells,T cells,DCs,p DCs and monocytes,and the m RNA and protein expression levels of DHX35 were detected by real-time quantitative PCR（qPCR）and western blot2.Total RNA and total proteins were extracted from respiratory epithelial cells transfected with poly（I:C）and poly（dA:dT）,and the expression of DHX35 was detected by qPCR and western blot.3.The expression of DHX35 was silenced by siRNA（unrelated sequence as control）or the recombinant plasmid pCMV-HA-DHX35 was overexpressed in respiratory epithelial cells,and then transfected with poly（I:C）or poly（dA:dT）.qPCR and ELISA were used to detect the expression levels of IFN-β,IL-6,CXCL-10 in cell and culture supernatants to determine the function of DHX35 in inducing natural immune response in respiratory epithelial cells.4.The recombinant plasmid pCMV-HA-DHX35 was constructed and transfected into HEK293 T cells.After 48 hours,the cells were lysed and incubated with biotin-labeled poly（I:C）or poly（dA:dT）.Then,neutral magnetic beads were added to pull down the complex,and the expression of DHX35 was detected by western blot to determine the interaction between DHX35 and poly（I:C）or poly（dA:dT）.The interaction of DHX35 with poly（I:C）or poly（dA:dT）was further determined by competitive inhibition experiments.5.The plasmids pCMV-HA-DHX35 and pCMV-MYC-MAVS were co-expressed in HEK 293 T cells,and the cells co-expressed pCMV-HA-DHX35 and empty vector pCMV-MYC were used as control.A MYC labeled complex is pulled down using beads bound to MYC antibodies.Finally,Western blot was used to detect the expression of HA-DHX35 in the complex to determine the interaction between DHX35 and MAVS.6.Silenced the expression of DHX35 by siRNA to explore its effects on downstream signaling pathways mediated by molecules such as IRF3,p65,and ERK.Results:1.DHX35 was constitutively expressed in respiratory epithelial cells,and entered the cytoplasm from the nucleus after stimulation2.Knockdown of DHX35 inhibited the secretion of IFN-β,IL-6 and CXCL-10 in respiratory epithelial cells,while DHX35 overexpression promoted the secretion of these antiviral related cytokines.3.DHX35 could recognize and bind poly（I:C）and poly（dA:dT）.4.DHX35 binded to MAVS to regulate the secretion of type I interferon.5.Knockdown DHX35 inhibited the activation of IRF3,P65,ERK mediated signaling pathways.Conclusion:In this study,we found that DHX35 is a novel pattern recognition receptor,which could directly recognize and bind to the viral nucleic acid mimics poly（I:C）and poly（dA:dT）,and regulated the secretion of downstream type I interferon and inflammatory factors through the adaptor protein MAVS in respiratory epithelial cells.This study provides new ideas and targets for the design of drugs and vaccines related to respiratory diseases caused by virus infection.