The Roles Of Non-coding RNAs On PCV2-induced Inflammation Response In Porcine Epithelial Intestinal Cell

Porcine circovirus type 2(Porcine circovirus type 2,PCV2)is the causative agent of porcine circovirus associated disease(Porcine circovirus associated disease,PCVAD).Enteritis is an important clinical manifestation of PCV2.meanwhile,Intestinal infection is considered to be an important route of PCV2 infection.Non-coding RNAs refers to RNA that does not encode proteins.Recent studies have found that non-coding RNA(miRNA,lncRNA,circRNA)plays an important role in regulating apoptosis and inflammation.However,the regulatory mechanism of non-coding RNAs in host inflammatory response induced by PCV2 infection has not been reported.Therefore,In this study,IPEC-J2 cells were selected as cell model,based on high-throughput sequencing,we analyzed the differential expression of microRNAs,lncRNAs and circRNAs in IPEC-J2 cells infected with PCV2,and then takes apoptosis and cytokine secretion as the cut-in poin,The regulation mechanism of non-coding RNA in inflammatory response induced by PCV2 infection was revealed.The main results were as follows:1.PCV2 infection induces apoptosis and changes of cytokine expression in IPEC-J2 cells.To investigate the effect of PCV2 infection on apoptosis and cytokine expression of IPEC-J2 cells.IPEC-J2 cells were infected with PCV2 at different time points(0 h,12 h,24 h,36 h,48 h).Flow cytometry and TUNEL assay were used to detect the effect of PCV2 infection on apoptosis of IPEC-J2 cells.The effects of PCV2 infection on the expression of IPEC-J2 cytokines(IL-7,IL-8,IL-6,TNF-?,INF-?,IL-4,IL-10,TGF-?)were detected by ELISA.The results showed that with the prolongation of PCV2 infection time,the apoptotic rate of IPEC-J2 cells increased gradually.The expression of cytokines was also significantly affected.After a comprehensive review on apoptosis and cytokines,IPEC-J2 cells infected with PCV2 for 36 hours were selected for the RNA sequencing(RNA-Seq).2.PCV2 infection affects the expression profiles of microRNAs,lncRNA and circRNA in IPEC-J2 cells.The expression profiles of noncoding RNAs in IPEC-J2 cells infected with PCV2 were analyzed by high throughput sequencing and bioinformatics.Differential analysis showed that there were 93 differentially expressed microRNAs,210 circRNAs and 197 lncRNAs.There were 24 up-regulated and 69 down-regulated micRNA.102 of circRNA were up-regulated and 108 down-regulated,129 of the lncRNA were up-regulated and 68 down-regulated(fold change>1.5,p<0.01).Real-time PCR was used to validate the sequencing results,which was consistent with the results of high-throughput sequencing.Target Scan and microRNAanda were used to predict the target genes of differentially expressed microRNAs,lncRNA and circRNA.GO annotation and KEGG cluster analysis were performed for the predicted target genes.3.Regulation of ssc-miR-429-3p on apoptosis and cytokine expression in IPEC-J2 cells infected with PCV2.According to the results of bioinformatics analysis,we screened the differentially expressed ssc-miR-429-3p,which are related to apoptosis and cytokine secretion,as the research object.Apoptosis and cytokine secretion of IPEC-J2 cells were detected by over-expression of ssc-miR-429-3p.It was found that over-expression of ssc-miR-429-3p resulted in the decrease of apoptosis and the change of cytokine secretion in IPEC-J2 cells.These results suggest that ssc-miR-429-3p plays a regulatory role in IPEC-J2 apoptosis and cytokine secretion induced by PCV2.Predicting and validating the target gene of ssc-miR-429-3p,we found that the activity of double luciferase in cells containing IL1 A 3'UTR recombinant plasmid decreased significantly after overexpression of ssc-miR-429-3p.It indicated that IL1 A was the target gene of ssc-miR-429-3p.4.Regulation of apoptosis and cytokine expression in IPEC-J2 cells infected with PCV2 by circRNA 4410 and lncRNA MSTRG.19762.1.The software predicted that there was a binding site of ssc-miR-429-3p in the sequence of circRNA 4410 and lncRNA MSTRG.19762.1.RAP validation showed that circRNA 4410,lncRNA MSTRG.19762.1 were targeted to ssc-miR-429-3p.After interfering with the expression of circRNA 4410 and lncRNA MSTRG.19762.1(siRNA),it was found that the apoptosis and cytokine secretion of IPEC-J2 cells were affected.At the same time,circRNA 4410 and lncRNA MSTRG.19762.1 can enhance the expression level of IL1 A,the target gene of ssc-miR-429-3p.The results showed that circRNA 4410 and lncRNA MSTRG.19762.1 could inhibit the expression of IL1 A gene by binding to ssc-miR-429-3p,and then regulate the apoptosis and cytokine secretion of IPEC-J2 cells.This study explored the interaction mechanism between PCV2 and host from different perspectives;To lay a foundation for elucidating the molecular mechanism of PCV2 infection and pathogenesis;Provide new ideas for further research of PCV2.