A Study Of PEDV Regulating Cellular Tight Junction Through MiRNA-328-3p

Porcine epidemic diarrhea(PED)is a highly contagious and highly piglet lethal intestinal disease caused by the porcine epidemic diarrhea virus(PEDV)and has been a threat to the world pig industry since its emergence.With the development of research,virology has entered the molecular age,and the molecular mechanism of PEDV pathogenesis has been increasingly studied.Mi RNA is a kind of non-coding small RNA molecules generated by cells and transported to recipient cells by exosomes and other ways,which are ubiquitous in animals,plants and humans,and are popular research objects in the field of molecules in recent years,and miRNAs are regulated in many diseases.Tight junction is the basic structure of the epithelial and endothelial barrier of human and animal bodies,regulating the entry and exit of water,inorganic salts and organic matter in and out of cells,blocking pathogens and harmful substances,maintaining homeostasis,when the tight junction of the intestinal epithelium is damaged,a series of intestinal diseases will occur.The mechanism of PEDV causing intestinal damage in pigs through the regulation of exosomal miRNAs has not been systematically studied at home and abroad.In this study,the effects of PEDV-derived exosome miRNA-328-3p on eight IPEC-J2 cell tight junction proteins and NF-κB pathway were studied by the exosome miRNA-328-3p produced by the target cell of PEDV,porcine small intestinal epithelial cells(IPEC-J2).IPEC-J2 cells were infected by using lentiviral vectors that overexpressed and inhibited the expression of miRNA-328-3p,and the cell lines with stable overexpression and inhibition of miRNA-328-3p were screened.Under the conditions of PEDV infection,eight common tight junction proteins were selected as research objects,compared with normal IPEC-J2 cells,the expression of m RNA and protein expression of eight tight junction proteins were detected,and three tight junction proteins with the most obvious differential expression were screened,namely ZO-1,ZO-3 and claudin-3.Among them,when IPEC-J2 cells overexpressing miRNA-328-3p were infected with PEDV,the expression of ZO-1,ZO-3 and claudin-3 proteins was significantly reduced.When IPEC-J2 cells expressing miRNA-328-3p were inhibited by PEDV infection,the expression of ZO-1 protein and ZO-3 protein decreased,while the expression of cludin-3 protein increased significantly.The NF-κB pathway was selected as the research object,and the overexpressed miRNA-328-3 and ordinary IPEC-J2 cells were treated with pathway inhibitors,and the expression of three differential proteins was detected under the conditions of PEDV infection.The results showed that the expression of three differential proteins ZO-1,ZO-3 and claudin-3 in ordinary and overexpressed cells using pathway inhibitors decreased compared with the reticent cells without pathway inhibitors,and the overexpressed cells decreased to a greater extent.In summary,by constructing lentivirus-infected IPEC-J2 cells that overexpressed and inhibited miRNA-328-3p,we found that PEDV can regulate the expression of IPEC-J2 cell nexus proteins ZO-1,ZO-3 and claudin-3 through miRNA-328-3p.miRNA-328-3p is able to regulate the expression of tightly linked ZO-1,ZO-3 and claudin-3 proteins in IPEC-J2 cells infected with PEDV through the NF-κB pathway.The above results enrich the molecular mechanism of PEDV pathogenesis and provide a theoretical basis for the scientific prevention and control of PED.