Bta-miR-1296 Regulates The Proliferation,Inflammatory Response And Synthesis Of Extracellular Matrix Of Bovine Mammary Fibroblasts

Bovine mammary gland fibrosis is a common pathological process caused by cow mastitis,which can lead to the decrease of milk production and quality.In severe cases,it will harden the mammary gland and lead to the elimination of cows.The fibrosis process of tissues and organs mainly depends on the deposition and decomposition of extracellular matrix(ECM)and the excessive proliferation of fibroblasts.Micro RNA(miRNA)is a class of short non-coding RNA molecules that negatively regulate gene expression.It has been widely reported that miRNA regulates the heart,liver and lung fibrosis,but the studies on the regulation of miRNA on bovine mammary gland fibrosis are still in the initial stage.Previous studies found that the expression of bta-miR-1296 in the exosomes derived from bovine mammary epithelial cells was significantly decreased in the inflammatory cell model constructed by LPS,suggesting that it may have certain anti-inflammatory effect.Therefore,this study will intend to transfect bta-miR-1296 mimic into bovine mammary fibroblasts(BMFBs)to achieve the overexpression of bta-miR-1296,to explore the effect of bta-miR-1296 on the function of BMFBs.This study will provide a basis for further research on the regulation mechanism of miRNA in bovine mammary gland fibrosis.In this study,BMFBs were isolated and cultured by tissue attachment method.The cell models of miRNA overexpression and inhibition were constructed by liposome transfection technique,and the regulation mechanism of bta-miR-1296 on the proliferation of BMFBs was studied by qRT-PCR,CCK-8,Western Blot and plate colony-forming assay.Then the target genes of bta-miR-1296 were verified by STar Mir software and dual-luciferase reporter assay.After that,qRT-PCR was used to detect the expression of inflammatory cytokines to further verify the regulatory effect of bta-miR-1296 on inflammation.Finally,Western Blot was used to detect the expression of ECM and the effector protein TGF-β1 and p-Smad3 of TGF-β1/Smad3,to explore the mechanism of bta-miR-1296 regulating bovine mammary gland fibrosis.The results of this study showed that the purified BMFBs were mainly in spindle,in radial-shaped growth,with vimentin positive.The results of CCK-8,colony-forming assay and q RT-PCR showed that bta-miR-1296 could significantly inhibit the proliferation of BMFBs.Dual-luciferase reporter assay confirmed that glutamate-ammonia ligase(GLUL)was the direct target gene of bta-miR-1296.Overexpression of bta-miR-1296 could down regulate the m RNA expression of ECM(α-SMA,COL1α1,COL3α1),inflammatory cytokines(IL-6,IL-8,CCL2,TNF-α)and cell growth factors(bFGF,VEGF,PDGF-BB,TGF-β1).Inhibition of bta-miR-1296 could up regulate the expression of the above genes.Overexpression of bta-miR-1296 could inhibit the expression of p-ERK,p-AKT,TGF-β1 and p-Smad3,while inhibition of bta-miR-1296 could promote the expression of p-ERK,p-AKT,TGF-β1 and p-Smad3.In summary,bta-miR-1296 can inhibit the proliferation of BMFBs through the AKT/ERK signaling pathway targeting GLUL.bta-miR-1296 can reduce the transcription level of inflammatory factors in BMFBs,thereby alleviating the inflammatory response of cells.bta-miR-1296 can inhibit the transcription of cell growth factors and inhibit the synthesis of ECM in BMFBs through TGF-β1/Smad3 signal pathway,thereby regulating the occurrence and development of bovine mammary gland fibrosis.This study laid a foundation for further exploring the regulation mechanism of miRNA derived from bovine mammary epithelial cells in bovine mammary gland fibrosis.