Transport Of MiRNA-328-3p By PEDV-derived Exosomes Between IPEC-J2 Cells

Porcine epidemic diarrhea(PED)is an acute,highly contact enteric infection of Porcine mainly caused by Porcine epidemic diarrhea virus(PEDV).All ages pigs are susceptible to disease.Since 2010,highly pathogenic PEDV variants have emerged and circulated widely in various regions in China,resulting in the outbreak of PED and causing huge economic losses to the pig industry in China.As extracellular vesicles actively secreted by cells,exosomes are not only important carriers for material information exchange between cells,but also play an important role in virus transmission and disease.micro RNA(miRNA),as a class of endogenous single-strand non-coding small Rnas,not only has regulatory functions,but also influences the replication,invasion and pathogenic process of viruses through its mediated gene regulation mechanism in host cells.In addition,different viral infections have different effects on the expression levels of certain miRNAs in host cells.More and more studies have shown that some miRNAs can protect themselves from degradation and maintain their stability by being loaded into exosomes or other vesicles.Viruses can affect the expression of miRNA in host cell derived exosomes through their own actions,and with the help of the communication of exosomes between cells,they can modify the immune response of target cells or change their physiological state,creating favorable conditions for viral replication and transmission.Previous laboratory studies have confirmed that miRNA-328-3p is differentially expressed in PEDV-derived exosomes and can act on tight junction proteins.Whether miRNA-328-3p can be transported through exosomes and enter receptor cells to play a role has not been confirmed.Therefore,it is of great significance to study the role of PEDV-derived exosomes in the transmission of miRNA-328-3p between IPEC-J2 cells to further study the molecular mechanism of intestinal mucosal damage caused by PEDV from the perspective of how exosome miRNAs affect the tight junctions between cells.In this study,exosomes from PEDV infected IPEC-J2 cells were extracted by ultrafiltration combined with high efficiency exosome precipitation kit,and the isolated exosomes were purified by immunomagnetic beads.The extracted exosome vesicles were identified by Transmission electron microscopy(TEM)and Western blot identification.Exosomes were labeled with PKH67 fluorescent dye,and the fluorescence signal location of exosomes was observed by uptake and co-culture of exosomes.After transfection with Cy3-miRNA-328-3p mimics,inhibitor and NC control,IPEC-J2 cells with high and low expression of miRNA-328-3p were established.Exosome extraction and fluorescence quantitative PCR were used to detect the transfection efficiency.The Transwell co-culture system was constructed to verify miRNA-328-3p transport.Extractor extractor of IPEC-J2 cells with high expression of miRNA-328-3p was extracted,labeled with PKH67 fluorescent dye,co-cultured with IPEC-J2 cells for different times,and the exosome transport of miRNA-328-3p was verified by fluorescence quantitative PCR,fluorescence microscopy and other methods.The results showed that PEDV-derived exosomes were successfully extracted and labeled by exosomes.After co-culture with cells,it was found that PEDV-derived exosomes could enter IPEC-J2 cells and increase the content of miRNA-328-3p in the cells.At the same time,the content of miRNA-328-3p detected in ventricular cells under Transwell was significantly higher than that in the control group,indicating that miRNA-328-3p could enter the receptor cells.Finally,exosomes transfected with Cy3-labeled miRNA-328-3p mimics were extracted,and the co-localization with cells verified that miRNA-328-3p could be transported through exosomes and enter into IPEC-J2 cells.In conclusion,exosomes derived from IPEC-J2 cells infected with PEDV were successfully extracted in this study,and the exosomes and miRNA-328-3p were labeled by fluorescence,which confirmed that miRNA-328-3p could be transported into IPEC-J2 cells through exosomes.This study provides theoretical basis for further exploring exosome miRNA from the perspective of affecting cell tight junctions,and studying the molecular mechanism of intestinal mucosal injury caused by PEDV.