Study Of The Molecular Mechanisms Of MiR-148a Targeting CCR10 To Regulate S.aureus Mastitis Of Dairy Cows

Mastitis is one of the most important diseases in dairy farms.Clinical and severe subclinical mastitis not only damages the udder tissue,but also reduces the yield and quality of dairy products.Therefore,revealing the pathogenesis of dairy cow mastitis is particularly important for the effective prevention and control of dairy cow mastitis.As a class of non-coding RNA molecules,miRNAs are widely involved in the occurrence and development of diseases including cow mastitis.CCR10 is a member of the CC subfamily of chemokines and plays an important role in the occurrence and development of inflammation and cancer.important role.Based on the results of previous high-throughput sequencing analysis in the laboratory,this experimental study screened out the differentially expressed miR-148 a and CCR10 in the mammary gland tissue of healthy dairy cows and inflamed dairy cow mammary gland tissues,using bioinformatics software,fluorescence quantitative PCR,RTCA,ELISA And flow cytometry and other methods,we finally revealed the molecular mechanism of miR-148 a involved in the proliferation and apoptosis of MAC-T cells under inflammatory conditions by targeting CCR10.The main findings of the test are as follows:1.CCR10 gene cloning and bioinformatics analysisIn this experiment,the known bovine CCR10 gene mRNA sequence in the NCBI database was used as a template to design specific primers,and the full-length sequence of the CDS region was cloned by RT-PCR technology.Bioinformatics software was used to analyze the sequence characteristics and homology of bovine CCR10 protein gene.As well as the physicochemical properties of the encoded product and other biological properties.The results showed that the CDS region of the bovine CCR10 gene was successfully cloned in the experiment.The protein mainly exists in the endoplasmic reticulum and is a hydrophilic transmembrane protein.The homology analysis and phylogenetic tree results of bovine CCR10 found the relationship between cattle and sheep.It has the closest relationship and the furthest relative to chicken;in the prediction of secondary structure,the protein is mainly composed of α-helix and random coil.The above results provide a theoretical basis for further analysis of bovine CCR10 gene in the future.2.Study on the molecular mechanism of miR-148 a targeting CCR10Using bioinformatics software Targetscan7.0 and Hybrid prediction,it was found that the 3’UTR region of CCR10 contains the potential binding site of miR-148 a.In order to verify whether there is targeted regulation between the two,we transiently co-transfected the successfully constructed PMIR-CCR10-3’UTR and PEZX-miR-148 a vectors into 293 T cells,and then measured the changes in dual luciferase activity.The results showed that compared with the control group,the dual luciferase activity expressed in the miR-148a+CCR10 group was significantly decreased（P<0.01）.MAC-T cells were transfected with miR-148 a mimic,miR-148 a inhibitor and NC,respectively,and the expression changes of CCR10 at mRNA and protein levels were detected by real-time quantitative PCR and ELISA,respectively.The results showed that transfection of miR-148 a mimic could significantly reduce the expression of CCR10 mRNA and protein（P<0.01;P<0.05）,while transfection of miR-148 a inhibitor could up-regulate the expression of CCR10 mRNA and protein（P<0.01;P<0.05）,indicating that miR-148 a can target and regulate CCR10 and affect its expression.3.Functional study of miR-148 a in dairy cow MAC-T cells under inflammatory conditionsBased on the LTA inflammatory cell model successfully constructed in the previous laboratory,the gain and loss of function of miR-148 a in MAC-T cells under inflammatory conditions were studied.MACT was transfected with miR-148 a mimic and miR-148 a inhibitor,and NC control group,respectively,and the proliferation and apoptosis of MAC-T cells were detected by RTCA（real-time label-free dynamic cell analysis）and flow cytometry.Changes in CCR10 levels were detected in cell supernatants by ELISA.The results of RTCA assay showed that overexpression of miR-148 a could promote the proliferation of MAC-T cells,and inhibiting the expression of miR-148 a could inhibit cell proliferation.The results of flow cytometry showed that transfection of miR-148 a mimic could significantly reduce the apoptosis of MAC-T cells（P<0.05）,on the contrary,transfection of miR-148 a inhibitor could significantly promote the apoptosis of MAC-T cells death.4.The function of CCR10 in dairy cow MAC-T cells under inflammatory conditionsTo further study the effect of CCR10 on MAC-T under inflammatory conditions,the si RNA mimic（bta-CCR10-si-1）of the CCR10 gene was synthesized in vitro by a biological company,and bta-CCR10-si-1 was stained in mammary epithelial cells.The proliferation and apoptosis of cow mammary epithelial cells were detected by flow cytometry and RTCA,and the expression of CCR10 in the cell supernatant after btaCCR10-si-1 interference was detected by ELISA kit.Using flow cytometry and RTCA tests,it was found that compared with the NC group,CCR10 interference could significantly promote the proliferation of mammary epithelial cells;RTCA results showed that CCR10 interference had a promoting effect on cell proliferation;ELISA results showed that CCR10 after interference Protein expression was significantly reduced.In summary,under inflammatory conditions,miR-148 a can inhibit the expression of cow mammary epithelial cells by binding to the 3’UTR of CCR10 gene;miR-148 a promotes the proliferation of mammary epithelial cells by regulating the target gene CCR10,and then participates in cow mammary glands.The pathogenesis of inflammation.