Identification And Analysis Of The MiRNA Expression Profiles In The Primary Neurons Of Mice Infected With Japanese Encephalitis Virus

ObjectiveJapanese encephalitis(JE)is a central nervous system disease,caused by Japanese encephalitis virus(JEV)infection producing severe neuroinflammation in the central nervous system.MicroRNAs(miRNAs)are endogenous non-coding small RNA molecules with a length of about 20-24 nucleotides.They can degrade or inhibit the expression of target genes through complementarily binding with target genes,and play important roles in the post-transcriptional regulation of gene expression networks.In this study,miRNAs were taken as the entry point,and the interaction between JEV and the host at the miRNAs level was preliminarily explored by studying the miRNAs expression profiles of primary neurons in mice infected with JEV,so as to provide direction and theoretical support for further studying the mechanism of neural dysfunction induced by JEV.Method1.Primary neurons of the suckling mice within 24 h of birth were cultured in vitro to detect the state of neuron culture;2.JEV(NJ2008 strain)was infected with primary neurons of mice.Plaque assay was used to detect virus titer at different time.High-throughput sequencing was performed on cell samples of the infected and control groups with the highest titer and the significantly differentially expressed miRNAs were screened out by data analysis.Then the quantitative real-time PCR was used to verify differentially expressed miRNAs and the changes of miRNAs expression profiles in JEV-infected primary neurons of suckling mice.3.MiRNAs with significant expression differences were selected and transfected miRNA mimics into the primary neurons of suckling mice for overexpression.Quantitative real-time PCR was used to detect the expression of JEV-E gene and the function of miRNAs was preliminarily analyzed.Result1.Immunofluorescence staining of microtubule-associated protein-2(MAP-2)in neurons showed the vitro culture system of primary neurons in suckling mice was established,and it can be used as the target cell for JEV infection;2.The results of the plaque test showed that the titer of JEV were the highest after 48 h of infection.The results of high-throughput sequencing showed that the numbers of known miRNAs in the samples of the infected and control groups were 1,348 and 1,297,respectively.Through bioinformatic analysis,26 miRNAs with significant expression differences were screened out,among which 18 miRNAs were up-regulated and 8 miRNAs were down-regulated.After quantitative real-time PCR verification,the expression profiles of 26 miRNAs were basically consistent with the results of high-throughput sequencing;3.The results of quantitative real-time PCR of the JEV-E gene indicated that the expressions of mir-21a-3p mir-223-5p mir-147-3p mir-155-5p and mir-146a-5p could promote the expression of JEV-E gene in neurons,while the expression of mir-301 a was just on the contrast.ConclusionIn this study,miRNAs expression profiles changes of JEV(NJ2008 strain)infected mice primary neurons were researched at the cellular level.There were 26 miRNAs with significant expression differences determined by high-throughput sequencing and quantitative real-time PCR after JEV infected.The function of these miRNAs with significant expression differences was preliminarily researched and these results confirmed that the expression of miRNAs in primary neurons could affect the replication of JEV.This study provided the theoretical basis and direction for further studies on the regulatory function of miRNAs in the mechanism of neural dysfunction induced by JEV.