The Study Of Differentially Expressed Genes In NLRC5 Knockdown Model Of IPEC-J2 Cells Infected By PSV

NLRC5(NLR family,CARD domain containing 5)is the newly discovered innate immune receptor,being a member of Nod-like receptors family.By now,it has been testified among multiple cell types of human and mice,it has been proved that NLRC5 has remarkable regulatory effect upon producing pathway to I type IFN.Moreover,NLRC5 not only up-regulates the transcription and expression of the classical MHC type I molecule gene,but also plays a key role in the formation process of inflammasome.It plays a significant role in antiviral and antibacterial innate immune process.Mucous immune has a very important influence on body immune,but the effect of NLRC5 on the innate immune on intestinal epithelium and intestinal mucous membrane is not known at present.The study intend to build a knockdown model of IPEC-J2 cells by taking porcine sapelovirus(PSV)as the model virus,trying to probe into the function and related mechanism of NlRC5 in intestinal mucous membrane innate immune.The experimental methods and results of the study are as follows.1.NLRC5 knockdown model of IPEC-J2 cells is successfully built,the reducing rate of gene expression reaches 94%,which provides a reliable basis for the experiment afterwards.2.Infecting PSV on NLRC5 knockdown model of IPEC-J2 cells model(MOI=1)and control cell group transfected of scramble sequence.The three points of time: 6th hour,12 th hour and 24 th hour are chosen as the research subject,three samples are set in each point-of-time in 2 groups,then the cells are collected in the three points of time to extract RNA.3.Transcriptome sequencing on RNA samples is done with RNA-Seq technology,differentially expressed genes are screened from the experimental groups and control groups which are analyzed by GO and KEGG.Besides,it is verified by qRT-PCR.After being infected by PSV in the 6th hour,12 th hour and 24 th hour,differentially expressed genes are screened from the experimental groups and control groups in these three point-of time.We find out that 61 differentially expressed genes are gained in the 6th hour,among which 41 genes are down-regulated and 20 genes are up-regulated.As in the 12 th hour,117 differentially expressed genes are gained,among which 85 genes are down-regulated and 32 genes are up-regulated.For the 24 th hour,22 differentially expressed genes are gained,among which 17 genes are down-regulated and 5 genes are up-regulated.The results of qRT-PCR and transcriptome sequencing are basically consistent,confirming the high reliability and credibility of the sequence data.GO and KEGG analysis on differentially expressed genes results of the three point-of-time are summarized as followed.In the 6th hour,GO analysis tested 3 genera including biological process,cellular component and molecular function and 32 subclasses,among which behavior,biological adhesion,cell and binding are the main subclasses.The KEGG analysis of this point-of-time includes 80 pathways,Hepatitis C,Herpes simplex infection,RIG-I-like receptor signaling pathway and Ubiquitin mediated proteolysis.In the 12 th hour,GO analysis on differentially expressed genes involves 38 subclasses,biological adhesion and catalytic activity are the main ones.The KEGG analysis of this point-of-time includes 139 pathways,rich in Cytokine-cytokine receptor interaction,Influenza A,Hepatitis C,Rheumatoid arthritis,NOD-like receptor signaling pathway and Intestinal immune network for IgA production.In the 24 th hour,GO analysis on differentially expressed genes involves 28 subclasses,regulation of biological process,cells and binding are the main classes.The KEGG analysis of this point-of-time includes 29 pathways,rich in Huntington's disease,Herpes simplex infection,Cytokine-cytokine receptor interaction and RIG-I-like receptor signaling pathway.The study on differentially expressed genes on NLRC5 knockdown IPEC-J2 cells model(MOI=1)it is found that the key genes can be grouped into three categories,IFN related genes?receptor related genes and inflammasome related genes.The study has further found out that NLRC5 shows positive regulation to the expression of?type IFN and ?type IFN its related genes.NLRC5 shows positive regulation to the expression of inflammasome,and it plays a role in the inflammatory reaction of the body.Besides,Intestinal immune network for IgA production shows significantly-rich during the medium-term of infection and the involved key genes in this pathway are down-regulated,which indicates that NLRC5 plays an important role in Intestinal mucosal immunity,but the specific regulatory mechanism needs to be further studied.