Functions And Molecular Mechanisms Of LncRNA H19 And Circ0004193 In The Process Of Bovine Mammary Fibrosis

Bovine mammary fibrosis is a disease with a high incidence and infection rate in the dairy industry.Severe fibrosis can lead to mastoscirrhus and even obsolete of bovine,which greatly reduces the economic benefits of the dairy industry.Mastitis caused by different pathogens can induce different degrees of breast fibrosis.Mammary epithelial cell mesenchymal transition(EMT),extracellular matrix protein(ECM)and connective tissue hyperplasia can all participate in the fibrosis of various organs.Long non-coding RNAs and circular RNAs are widely involved in the fibrosis process of various tissues,and it has been confirmed that H19 can participate in epithelial cell EMT,but it has not been reported whether H19 and circular RNA can participate in mammary fibrosis in dairy cows.This study aimed to explore the role of H19 in the process of mammary fibrosis and its mechanism in dairy cows.First,in this study,real-time quantitative PCR(RT-qPCR)detection revealed that the expression level of H19 in normal bovine mammary tissue was significantly lower than that in inflammatory mammary gland tissues,and RT-qPCR showed that H19 expression in inflammatory mammary epithelial cells treated with lipoteichoic acid(LTA)and lipopolysaccharides(LPS)was significantly higher than that of normal mammary epithelium cell.The above indicates that H19 is involved in the process of fibrosis of bovine mammary epithelial cells.To investigate the role of H19 in the process of mammary fibrosis in bovine,we used exogenous TGF-β1 to treat mammary epithelial cells.Western blot was used to detect EMT marker and ECM protein in epithelial cells.The results showed that TGF-β1 could induce mammary epithelial cells EMT and upregulation of ECM protein expression and induce epithelial cell fibrosis.TGF-β1 was found to promote H19 elevation by RT-qPCR.In this study,we established a mammary epithelial cell line that stably overexpressed H19 through molecular cloning techniques.Western blot assays revealed that EMT and ECM proteins were overexpressed in cell lines that stably overexpress H19.Next,the molecular mechanism of H19 inducing EMT in mammary epithelial cells was investigated by western blot and RT-qPCR.The results showed that TGF-β1 can promote the expression of AKT protein in the PI3 K / AKT pathway in epithelial cells,indicating that the PI3 K / AKT pathway is activated in TGF-β1-treated mammary epithelial cells.Using PI3 K / AKT specific inhibitor can inhibit the activation of this pathway,and at the same time the expression of H19 is inhibited,indicating that TGF-β1 could promoted H19 expression through PI3 K / AKT signaling pathway.In the RNA high-throughput sequencing of pathogen-treated bovine mammary epithelial cells,bioinformatics analysis revealed that circRNAs have cellular biological functions such as cell proliferation,enzyme activity,cell migration,and cell transformation.In this study,the reliability of high-throughput results was verified by RT-qPCR and circ0004193 was found to be related to EMT of epithelial cells through bioinformatics analysis.To investigate the relationship between circ0004193 and H19,a cell line stably overexpressing circ0004193 was then established.By RT-qPCR,we found H19 can promote circ0004193 expression and inhibited by it.In conclusion,H19 can mediate TGF-β1-induced EMT through PI3 K / AKT signaling pathway in bovine mammary epithelium and is negatively regulated by circ0004193,thus playing an important role in mammary fibrosis caused by mastitis in cows.