Verification Of Differential MiRNA Functions In Exosomes After LPS Treatment Of Cow Mammary Epithelial Cells

Cow mastitis is an inflammatory manifestation of mammary tissue caused by physical element,chemical factor and biological threat.After the pathogen invades the mammary duct,the mammary epithelial cells act as the first defense barrier of mammary tissue,and start the immune recognition and response to the pathogen.Micro RNAs(miRNAs)are a class of short non-coding RNA molecules that negatively regulate gene expression.Studies have shown that miRNAs perform important functions in the inflammatory process of cow mastitis.Exosomes are small vesicles with a diameter of 40-200 nm,which are excluded from the cell membrane after the fusion of MVB and cell membrane.The main substances in exosomes are nucleic acid,protein and lipid.Previous studies have shown that exosomes are important intercellular communication media.The secretion and contents of exosomes are greatly affected by the physiological state of the cells from which they originate.When the pathogenic bacteria stimulate the mammary epithelial cells of dairy cows,the expression pattern of miRNA in the exosomes secreted by them may be changed,which may have a certain impact on the occurrence and development of mastitis in dairy cows.In this study,lipopolysaccharide(LPS)was used to treat dairy cow mammary epithelial cells to simulate the physiological state of E.coli invading dairy cow mammary tissue.We collected the exosomes secreted by cells.The miRNAs that may perform regulatory functions in exosomes secreted by cells were further screened and identified in the inflammatory process of breast tissue.In this study,differential ultracentrifugation was used to extract exosomes from bovine mammary epithelial cells(normal cells and cells treated with LPS for 6 h).Transmission electron microscopy was used to observe the morphological trait of the obtained exosomes,and the existence of surface marker proteins was detected by Western Blot.The exosome miRNAs were sequenced by high-throughput sequencing technology to screen the differentially expressed miRNAs that may play a regulatory role in bovine mastitis.The differentially expressed miRNAs from the obtained exosomes were identified by q RT-PCR.To verify the regulatory effect of differentially expressed miRNA on inflammation,we constructed cell models of over expression and inhibition of differentially expressed miRNA.The results of this study showed that the diameter of cell supernatant extract particles was 40-120 nm under transmission electron microscope,and the size was uniform.It was a typical cup like structure,which was resemblance to the classical structure of exosomes.CD9,CD63 and TSG101 proteins were measured in the obtained exosome samples by Western Blot.Seven differentially expressed miRNAs were found in our miRNA sequencing data(P < 0.05).Two of them were up regulated(miR-193b-5p and miR-193b＿R2).Five of them were down regulated(miR-1225-p5,miR-1296＿R-2,miR-126-5p,miR-141-5p＿1ss11TC and PC-5p-57291＿49).The target genes of differentially expressed miRNAs were analyzed by go and KEGG bioinformatics.The target genes of differentially expressed miRNAs were enriched in signal transduction,redox process,protein phosphorylation,protein transport,protein hydrolysis and other functions.They were mainly involved in lysosome,leukocyte migration across endothelial cells,regulation of inflammatory mediators of TRP channel and other biological reaction processes.After bovine mammary epithelial cells were treated with LPS,the seven miRNAs that had been screened were verified by qRTPCR.The variation trend of four miRNAs was consistent with the sequencing results(miR-193b-5p、miR-1296＿R-2、miR-126-5p、miR-141-5p＿1ss11TC).The expression of miR-126-5p and miR-193b-5p were significantly higher than that of the other two miRNAs.Furthermore,the cell models of miR-126-5p and miR-193b-5p overexpression and inhibition were constructed.Overexpression of miR-126-5p up regulated IL-6 and down regulated IL-1β and TGF-β1.Inhibition of miR-126-5p up regulated TGF-β1 and TNF-α,and down regulated IL-6.Overexpression of miR-193b-5p up regulated IL-6 and down regulated IL-1β,TNF-α and TGF-β1.Inhibition of miR-193b-5p down regulated IL-1β,TNF-α and TGF-β1.Overexpression of miR-126-5p and miR-193b-5p increased the phosphorylation level of p65 and IκBα.In this study,we successfully isolated and identified exosomes from bovine mammary epithelial cells.The miRNA expression profiles of exosomes(secreted by normal cells and cells treated with LPS for 6 h)were successfully constructed.After LPS treatment,miR-126-5p was down regulated and miR-193b-5p was up regulated.Overexpression of miR-126-5p and miR-193b-5p can promote the transcription of IL-6 and the activation of NF-κB signaling pathway.This study laid a foundation for further exploring the inflammatory regulation mechanism of miRNA derived from bovine mammary epithelial cells.