The Preliminary Discussion Of Molecular Function Of MiR-145 Targeting FSCN1 To Regulate The S.aureus Mastitis Of Dairy Cows

With the improvement of people's living standard,milk has become a very important nutrient food,and people's demand for the quality of milk is getting higher and higher.Dairy cow mastitis is an important bottleneck restricting milk quality.Dairy cow mastitis usually refers to the inflammation caused by the infection of pathogenic microorganisms.The occurrence of mastitis can lead to a significant decline in milk yield and dairy quality,which can cause severe economic loss to the dairy farming industry.In this study,the miRNA expression profile and transcriptome sequencing of staphylococcus aureus challenged mammary gland tissue and attacking PBS tissue were used to screen out the down-regulated miRNAs and the target genes whose expression was up-regulated,to verify the targeting relationship between candidate miRNAs and immune genes,as well as its related functions in mammary epithelial cell lines.1 Functional Study of miR-145 in Mac-TTo investigate the function of miR-145 in Mac-T,we over-expressed and interfered with miR-145 in Mac-T,and tested its overexpression and interference effects.Elisa and EdU were used to detect changes in the secretion of several Mac-T cytokines and changes in cell proliferation.The results showed that miR-145 can reduce the secretion of IL-12 and the secretion of TNF-a in the overexpression of bovine mammary epithelial cell lines,and increase the secretion of IFN-y significantly.The proliferation of bovine mammary epithelial cells after overexpression of miR-145 was inhibited at the same time.2 Prediction of miR-145 target genes and identification of target gene FSCN1The transcriptome data of mammary gland tissues challenged by Staphylococcus aureus,combined with TargetScan,miRanda,and PicTar microRNA target gene prediction software predicted several candidate target genes.The target genes of miR-145 were screened by real-time PCR(qRT-PCR),Western Blot,and Luciferase multiplex verification.The results showed that the mRNA expression of FSCN1 changed significantly after miR-145 overexpression or down-regulation,and over-expression of miR-145 could result in a significant down-regulation of FSCN1 protein expression.The 3'UTR of the FSCN1 gene mRNA has a definite target binding site for miR-145 and down-regulates the activity of FSCN1.Therefore,miR-145 can target and regulate FSCN1.3 Functional Study of FSCN1 in Mac-TRNAi method was used to interfere with the expression of FSCN1 in Mac-T cell line.Fluorescence quantitative PCR validated that the knockdown effect of siRNA designed was significant,and Elisa was used to detect the secretion of cytokines in Mac-T cell line and the status of the proliferation of cells in EdU proliferation test.Thus,the effect of FSCN1 on the two functions of Mac-T cell line was elucidated.The results showed that the knockdown of FSCN1 significantly increased the secretion of IL-12,while the secretion of TNF-? was significantly downregulated.4 conclusionStaphylococcus aureus-infected mammary gland tissue,body starts self-protection and produces inflammation,Bta-miR-145 expression is down-regulated,thus releasing its interference effect on FSCN1 gene,FSCN1 expression up-regulation promotes mammary epithelial cell proliferation to recover after bacterial invasion damage Breast cell tissue.