Molecular Regulation Of MiR-199a-3p On Inflammatory Response Of Mammary Epithelial Cells In Dairy Cow

Cow mastitis is a disease with extremely high incidence,causing huge economic losses to the pasture.Bovine mammary epithelial cells(bMECs),are the main cells that exert the function of the mammary gland of dairy cows,and as an integral part of the innate immunity of mammary glands,it is the first barrier to protect the mammary gland tissues against the invasion of pathogens.MicroRNAs(miRNAs)are non-coding small RNA that have been demonstrated to regulate gene expression at the post-transcriptional level and to be involved in a variety of biological processes.Previous studies have shown that miR-199a-3p can regulate the functions of multiple cells in mice and humans.However,the molecular role of miR-199a-3p in the inflammatory response of bMECs has not been explored yet.In order to advance the research of the function of miR-199a-3p in dairy mastitis,the present study aimed to investigate the expression pattern and molecular regulation of miR-199a-3p in the bMECs inflammatory response at the molecular and cellular levels,and to explore the effects of miR-199a-3p on cell proliferation and apoptosis in the bMECs inflammatory response.The main research results are as follows:(1)The RNA-seq technology was used to detect the transcriptome in the bMECs of the miR-199a-3p group and the control group,and the differentially expressed mRNA was screened and its function was annotated.The results showed that,compared with the control group,the overexpression group of miR-199a-3p had 140 differentially expressed mRNA(109 up-regulated mRNA and 31 down-regulated mRNA);The results of KEGG and GO analyses indicated that the above differential mRNA might be involved in the regulation of immune and inflammatory responses by regulating PI3K-Akt signaling pathway,TGF-beta signaling pathway,and IL-17 signaling pathway.These results suggested that miR-199a-3p might regulate cow mastitis by affecting the expression of related mRNA and through the above signaling pathways.(2)SP1,MTOR,PTPRF,CD2AP,BCAR3,NET1,and CNOT7 are predicted to be potential target genes of miR-199a-3p,by using bioinformatics methods and surveying literature.In this study,we constructed the recombinant vectors of the above seven potential target genes mRNA 3’UTR double luciferase reporter genes and identified the regulatory relationship between miR-199a-3p and the above potential target genes through double luciferase reporter gene experiments.The results showed that CD2AP was a target gene of miR-199a-3p;There was no targeted regulation relationship between miR-199a-3p and the other six mRNA.(3)qRT-PCR assay was used to detect the expression level of miR-199a-3p in LPS-induced inflammatory response of bMECs.The results showed that compared with 0h,the expression level of miR-199a-3p was up-regulated at 3,6,12 and 24h of LPS-induced inflammatory response,with extremely significant increase at 6h and 12h(P<0.01),and the most increase at 6h,suggesting that miR-199a-3p might have a regulatory role in the bMECs inflammatory response.(4)An inflammatory bMECs model overexpressing and silencing miR-199a-3p was established,and the expression of target genes CD2AP and key genes and proteins of PI3K/AKT/NF-κB signaling pathway,the gene expression and secretion of inflammatory cytokines,as well as cell proliferation and apoptosis were detected.The results showed that overexpression of miR-199a-3p significantly suppressed mRNA expression of target genes CD2AP,signaling pathway key genes(PIK3CA,PIK3R1,AKT1,AKT3,IKBKB,RELA and TLR4),proteins(PI3K,AKT and p-p65),inflammatory factors mRNA(IL-6,IL-1βand TNF-α),and apoptotic genes(CASP3 and CASP9).Meanwhile,it can inhibit the secretion of inflammatory factors,promote the proliferation of bMECs and decrease its apoptosis.When miR-199a-3p was silenced,experimental results opposite to overexpression of miR-199a-3p were obtained.These results suggested that miR-199a-3p inhibited the inflammatory response and apoptosis of bMECs.In summary,miR-199a-3p inhibits LPS-induced inflammation and apoptosis in bMECs by targeting the CD2AP gene and regulating the PI3K/AKT/NF-κB signaling pathway.The research results provide a potential target for molecular treatment of mastitis and also lay a foundation for molecular breeding of anti-mastitis dairy cows.