The Expression And Function Of LncRNAs Associated With ETEC Infected Porcine Intestinal Epithelial Cells

Enterotoxigenic Escherichia coli(ETEC)is one of the main pathogens causing diarrhea in piglets.It can cause yellow dysentery,white dysentery,and edema disease in piglets.The main clinical symptoms of sick piglets are diarrhea,dehydration and emaciation,which can cause death in severe cases and cause huge economic losses to the pig industry.Long noncoding RNA(lncRNA)is a kind of noncoding RNA longer than 200 nt.Previous Studies have found that bacterial infection can cause abnormal expression of lncRNA in host cells,which is involved in the regulation of the diseases.In this study,high-throughput sequencing was used to analyze the expression of lncRNA in porcine intestinal epithelial cells during ETEC infection,to screen lncRNA related to ETEC infection and study its function and explore the molecular mechanism of ETEC infection.This study provides theoretical basis for revealing the pathogenesis of piglet diarrhea and finding new prevention and treatment targets.Firstly,ETEC F41 strain was used to infect IPEC-J2 cells.High throughput sequencing of lncRNA was performed before infection,at early infection and at late infection to screen the differentially expressed lncRNA at different infection stages.qRT-PCR was used to verify the reliability of sequencing data.The results showed that 10150 novel lncRNAs were identified in IPEC-J2 cells.Among them,100 differentially expressed lncRNAs were found at early infection,40 of which were up-regulated and 60 down-regulated;161 differentially expressed lncRNAs were found at late infection,65 of which were up-regulated and 96 of which were down-regulated.Secondly,the target genes of differentially expressed lncRNA were predicted by analyzing the location relationship and expression correlation between lncRNA and protein coding genes.Through the GO function enrichment analysis and KEGG pathway enrichment analysis of the target genes of differential expression of lncRNA.Therefore,during ETEC infection in IPEC-J2 cells,lncRNA plays an important role in cell biosynthesis,metabolic regulation,signal transduction,cell cycle and immune function.Based on differential expression analysis,we found that the expression of candidate lncRNA MPHOSPH9-OT1 was significantly increased at early and late infection.The log2 fold change value was 14.08(P<0.01)at early infection and 16.14(P<0.01)at late infection,indicating that it may play a key role in regulating ETEC infection.At the same time,in order to understand the distribution of MPHOSPH9-OT1 in pig,we detected the expression of MPHOSPH9-OT1 in different porcine organs,the results showed that MPHOSPH9-OT1 was expressed in intestine,heart and liver,and the expression level was higher in intestine.By using shRNA interference and lentivirus transfection technology,the MPHOSPH9-OT1 knockdown and overexpression IPEC-J2 cell lines were established.Through the characterization of the monolayer cell transmembrane resistance(TEER),the expression of tight junction proteins Occludin,Claudin-1 and ZO-1,as well as the ETEC adhesion rate ofof MPHOSPH9-OT1 knockdown and overexpression IPEC-J2 cells,we found that the normal expression of MPHOSPH9-OT1 plays an important role in maintaining the homeostasis of intestinalbarrier.The expression changes of 13 predicted target genes were detected using qRT-PCR,which showed that the expression of CXCL8 mRNA was significantly correlated with MPHOSPH9-OT1 expression level.Meanwhile,in the early and late infection,MPHOSPH9-OT1 targets and regulates CXCL8 expression.Therefore,CXCL8 was preliminarily identified as its target.The differentially expressed lncRNAs are widely involved in cell integrity,proliferation,apoptosis and immune related biological processes and molecular pathways,indicating that lncRNAs play an important regulatory role in the process of ETEC infection.We found that the differentially expressed lncRNA MPHOSPH9-OT1 may regulate the intestinal barrier function and integrity by regulating the expression of CXCL8.This study provides a theoretical basis for further elucidating the interaction mechanism between host cells and ETEC infection,and provides a new idea for the prevention and treatment of piglet diarrhea caused by ETEC infection.