The Analysis Of Non-coding RNA Expression And Regulatory Networks In The Mice Brain During Japanese Encephalitis Virus Infection

Japanese encephalitis virus(JEV)is an arbovirus that belongs to the genus Flavivirus of Flaviviridae which can cause Japanese encephalitis(JE)in humans and animals.It has an estimated worldwide annual incidence of 45,000 human cases and 10,000 deaths.Approximately 50% of the survivors develop permanent neurologic or psychiatric sequelae.Japanese encephalitis also can pose a huge threat to the pig industry.It will cause orchitis in boars and reproductive failure in sows.Japanese encephalitis virus can pass through the blood-brain barrier and enter the central nervous system to target neurons,causing neuronal death and a serious inflammatory storm in the brain.However,the molecular mechanism of Japanese encephalitis remains to be clarified.Since the hypothesis of ceRNA(competing endogenous RNA,ceRNA)was proposed in 2011,it has been found that non-coding RNAs such as lncRNA and circRNA can also competitively bind to miRNA through miRNA response element with mRNA,thus regulating the target gene of miRNA.Although the ceRNA network has been reported to be involved in the infection of various viruses,the molecular mechanism of lncRNA,circRNA,and ceRNA network during the JEV infection still needs to be clarified.In this study,we analyzed the expression of mRNA,lncRNA,miRNA,and circRNA by using JEV-infected mouse brain tissue samples and construct the ceRNA network to explore the relationship between ceRNA and JEV infection.The main research contents are as follows:1.Construction and analysis of mRNA expression profiles in JEV-infected mice brain tissueWe identified 995 differentially expressed mRNA a by the microarray analysis using the JEV-infected mice brain tissue which contains 796 were up-regulated and 199 were down-regulated.The GO and KEGG enrichment analysis showed that the differentially expressed genes mainly focused on the inflammatory response,innate immunity,antiviral response,synaptic transmission,ion transport et al.2.Construction and analysis of miRNA expression profiles in JEV-infected mice brain tissue.We identified 58 differentially expressed miRNA by high-throughput sequencing technology in JEV-infected mice brain which contains 37 were up-regulated and 21 were down-regulated.The GO and KEGG enrichment analysis showed that the target genes of up-regulated miRNA were mainly related to the dopaminergic synapse,circadian entrainment,c AMP signaling pathway,and the target genes of down-regulated miRNA were mainly involved in TNF signaling pathway,Apoptosis,NF-κB signaling pathway,JAK-STAT signaling pathway et al.3.Construction and analysis of lncRNA expression profiles in of JEV-infected mice brain tissueWe identified 1114 lncRNA by the microarray analysis using the JEV-infected mice brain tissue which contains 131 were up-regulated and 983 were down-regulated.Then we constructed the lncRNA-mRNA and lncRNA-lncRNA co-expression network,and we found that lncRNA E52329 and N54010 can regulate JEV-mediated inflammatory response through MKK4/7-JNK signaling pathway in microglia cells through real-time quantitative PCR and immunoblotting experiments.4.Construction and analysis of circRNA expression profiles in of JEV-infected mice brain tissueWe identified 180 differentially expressed circRNA by high-throughput sequencing technology in JEV-infected mouse brain which contains 125 were up-regulated and 55 were down-regulated and verified by real-time quantitative PCR in mouse brain tissue and BV2 cells.Then we analyzed the expression characteristics of circRNA,such as length,species,chromosome distribution,and GC content.The GO and KEGG enrichment analysis of circRNA-derived linear genes revealed that differently expressed circRNA-derived linear genes are mainly involved in histone methylation,transcriptional misregulation in cancer,neurotransmitter secretion,cation channel activity et al.5.Construction and analysis of ceRNA regulatory network in JEV-infected mice brain tissueWe constructed the ceRNA network through miRanda software by using the differentially expressed mRNA,lncRNA,miRNA,and circRNA data of JEV-infected mice brain tissue.The ceRNA subnetwork associated with inflammation was constructed by selecting mRNA associated with inflammatory responses.We verified the function of key RNA molecules in ceRNA networks by real-time PCR and dual-luciferase assays and found that circ＿0000220 could regulate the expression of BCL3,MK2,and TRIM25 through serves as miR-326-3p sponges and regulate JEV-induced inflammation.In this study,the expression profiles of mRNA,lncRNA,miRNA,and circRNA of JEV-infected mice brain tissue were detected by microarray and high-throughput sequencing techniques.Then we predicted the function of RNA and verified it through experiments.Finally,we explore the key RNA molecules by constructing the ceRNA network.Moreover,this work provided new insight into the molecular mechanisms underlying JEV-infected hosts and JEV-mediated inflammatory responses.