Porcine Delta Coronavirus Inhibits NHE3 Activity Of Porcine Intestinal Epithelial Cells Through MiR-361-3p-SLC9A3 Regulatory Axis

Porcine Deltacoronavirus(PDCo V)is an enveloped single-stranded positive-sense RNA virus,which belongs to the genus Deltacoronavirus under the Coronavirus family.The main symptoms of PDCo V infection are acute vomiting,severe diarrhea and weight loss.Severe weight loss greatly increases the culling rate and mortality of suckling piglets,so the harm to piglets is particularly serious.So far,PDCo V has broken out and circulating in many countries including China,the United States,Canada,Thailand and South Korea,and has caused huge economic losses to the global pig industry.However,little is known about the pathogenic mechanism and molecular mechanism of PDCo V causing severe diarrhea in piglets.Solute carrier family 9 subfamily A member 3(SLC9A3)belongs to the solute carrier family and is the core gene encoding Sodium-hydrogen exchanger member 3(NHE3).NHE3 is the small intestine The main transporter involved in the Na+/H+exchange on the apical membrane of surface epithelial cells can cause diarrhea and even death when the NHE3 activity is in a long-term inhibitory state.In the early stage,the laboratory used PDCo V CHN-GD16-05 isolates to conduct a challenge test on 2-day-old piglets,and took samples of intestinal tissues and sent them to related companies for high-throughput sequencing of transcriptomics,and performed software analysis and visualization of some results Relationship network diagram.The results show that the differentially expressed and biologically significant mi RNA-m RNA regulation is mainly enriched in the protein digestion signal pathway of the digestive system.Related gene m RNA is significantly down-regulated,and related mi RNA is significantly up-regulated.Among them,SLC9A3 gene has the highest enrichment.In this study,PDCo V was used as the research object to explore the relationship among candidate mi RNAs,SLC9A3 gene and NHE3 protein expression and NHE3 activity in IPI-2I cells infected by PDCo V.First,IPEC-J2 cells were subjected to trypsin tolerance test,optimal multiplicity of infection screening,and virus infection ability test of different porcine cell lines,and successfully used IPI-2I cells to establish a PDCo V cell model in vitro.In order to verify the credibility of the high-throughput sequencing results,and collect cell lysates at different time points of virus infection,the q RT-PCR test results show that the 10 gene m RNAs and 12 mi RNAs involved in the correlation network diagram can all be different after PDCo V infection of the cells Sexual expression indicates that the high-throughput sequencing results of the previous trials are true and credible.Then this study explored the function of SLC9A3/NHE3.In order to verify that PDCo V infection affects the expression of SLC9A3 gene and NHE3 protein and NHE3 activity after infection of IPI-2I cells,samples from different time points of virus infection were collected for q RT-PCR,western blotting and NHE3 activity detection.The results showed that the expression of SLC9A3 gene was down-regulated and the expression of NHE3 protein was down-regulated.Down-regulation and down-regulation of NHE3 activity indicate that PDCo V infection can inhibit NHE3 activity.In order to verify that SLC9A3 gene expression affects NHE3 protein expression and NHE3 activity,the SLC9A3 gene was over/lost expression.The results showed that SLC9A3 gene positively regulates NHE3 protein expression and NHE3 activity.After screening 7 mi RNAs related to SLC9A3 gene using online target gene prediction software,4 candidate mi RNAs(ssc-mi R-133a-3p,ssc-mi R-326,ssc-mi R-133a-3p,ssc-mi R-326,ssc-mi R-338,ssc-mi R-361-3p).In order to explore the influence of each candidate mi RNAs on SLC9A3/NHE3,mi RNA mimics/inhibitors were used to perform over/loss expression of mi RNA.q RT-PCR and western blotting showed that each candidate mi RNAs can negatively regulate the expression of SLC9A3 gene and NHE3 protein to varying degrees.The dual luciferase activity verification test confirmed that there is a targeted binding relationship between ssc-mi R-361-3p and SLC9A3 gene.Simultaneous transfection of ssc-mi R-361-3p mimics can down-regulate NHE3 activity,while transfection of ssc-mi R-361-3p inhibitors can up-regulate NHE3 activity,indicating that SLC9A3 gene is the target gene of ssc-mi R-361-3p,and ssc-mi R-361-3p negatively regulates NHE3 activity.Finally,the NHE3 activity was detected by inhibiting ssc-mi R-361-3p and infected with PDCo V.The results showed no significant difference from the control group,indicating that ssc-mi R-361-3p inhibitors reversed the inhibitory effect of PDCo V on NHE3 activity.In summary,this study confirmed that PDCo V inhibits the NHE3 activity of IPI-2I cells through the mi R-361-3p-SLC9A3 regulatory axis,which provides a theoretical basis for analyzing the pathogenic mechanism of severe diarrhea caused by PDCo V in piglets.