Expression Profiles Of Genes And MicroRNAs From Bovine Mammary Glands In Response To Streptococcus Agalatiae-induced Mastitis And Regulation Of MiR-122 On EPO And JAK-STAT Pathway

Mastitis is the most common disease in dairy cows.It casues serious economic losses as well as troubls long-term developments of the dairy industry all over the world.Mastitis was mainly caused by pathogenic bacteria invaded into the mammary gland tissue.Streptococcus is one of the main pathogenic bacteria.Streptococcus is Gram-positive bacterium with shape of spherical or oval and it grows in chains or pairs.Streptococcus agalactiae(S.agalactiae)causes subclinical mastitis from 11%to 43%in dairy cows.Dariy cows have low resistance and self-cure ration to this infection.Antibiotics are often used to treat mastitis.However S.agalactiae has drug resistance from 50%to 100%for Penicillin G,Amoxicillin,Ampicillin,and so on.Understanding the pathophysiological process and the host immune response to mastitis in mammary tissue are very important for developing new strategies to control or prevent mastitis.MicroRNA(miRNA)is a kind of single stranded non-coding RNA about 22 oligonucleotides.MiRNA can regulate more than 60%of the encoding protein genes involved in cell growth,proliferation,apoptosis,and immune response and so on.The characteristics of miRNAs expression are tissue specificity and time-series.Studying the differentially expressed miRNAs in mammary gland tissue during the process of immune response will help to understand the mechanism of the body’s immune response on the molecular level and provid new methods to control mastitis.In this study,we constructed dairy cows’ S.agalactiae mastitis model,used Affymetrix gene chip to get differentially expressed genes and then analyzed pathways related to immune.At the same time,we used Solexa sequenceing technology to establish two small RNA libraries and analyze differentially expressed miNRAs.Potential target genes of these differentially expressed miRNAs were analyzed by GO and KEGG pathways.According to the analytical results of the two expression profiles,we adopted double luciferase reporter gene system to validate the relationship of miR-122 and EPO.Then we transfected miR-122 mimic and miR-122 inhibitor into mammary epithelial cells and measured the expreesion of EPO and genes involved in JAK-STAT signal pathway by real-time fluorescence quantitative technique.The research results were shown as following:(1)Induction of S.agalactiae-type mastitisTwenty-four hours following the S.agalactiae challenge,the test udders exhibited clinical symptoms including redness,swelling,pyrexia,hardness and pain.The milk SCC of the test quarters increased rapidly(>2,000,000 cells/ml).In contrast,the SCC from the control udders was nomal.The bacteria detected in the test milk samples were only S.agalactiae.Furthermore,the biopsy of the infected mammary gland tissues stained with haematoxylin and eosin was demonstrated pathological changes compared with the control.Obvious lesions appeared in the infected udders,the mesenchyme was swollen,parts of the lumen were atrophied,the mammary epithelial cells were loosely connected,and the intercellular gap was increased.Desquamated mammary epithelial cells,polymorphonuclear neutrophils,macrophages,lymphocytes and other inflammatory cells were concentrated in the lumen.All these results showed that S.agalactiae mastitis was induced successfully.(2)Expression profiles of genes from bovine mammary glands in response to S.agalactiae-induced mastitisAffymetrix gene microarray results showed a total of 136 significant differentially expressed genes were identified in the test group compared to the control group,including 78 up-regulated genes and 58 down-regulated genes.Among these genes,18 up-regulated genes including CD34,SDC4,CDH5,APLNR,HTR2B,ACTN2,CACNA1A,CD55,DCP1A,ELMOl,GALNTL4,GAN01,HTR2B,LOC514818,NOTCH2,VWF,PROS1,ITGB4 and 11 down-regulated genes including EPO,CYPEF3,ABCA12,CCL17,LIPG,JAG1,RPS15,TAF4B,C4BPB,XRCC3 and FST were involved in 38 pathways,including 13 immune related pathways such as JAK-STAT,Cytokine-cytokine receptor interaction,Complement and coagulation cascades,Hematopoietic cell lineage,Notch signal pathway and so on.(3)Expression profiles of miRNAs from bovine mammary glands in response to S.agalactiae-induced mastitisThe miRNA expression profile resluts showed that a total of 18,535,913 and 20,847,000 effective reads were obtained from the test and control groups,respectively.In total,373 known and 399 novel miRNAs were detected in the test group,and 358 known and 232 novel miRNAs were uncovered in the control group.A total of 35 differentially expressed miRNAs were identified in the test group compared to the control group,including 10 up-regulated miRNAs(miR-122,miR-223,miR-16a,miR-2284k,miR-2484,miR-451,miR-383,miR-486,miR-2332,miR-326)and 25 down-regulated miRNAs(miR-378b,miR-145,miR-136,miR-26a,miR-33a,miR-335,miR-3660,miR-146a and miR-206,etc).The RNAhybrid software predicted 18,801 targets genes of these 35 miRNAs.Furthermore,GO analysis demonstrated that target genes’ moleculre functions were focused on stimulate response,signal,biological regulation,cell killing and immune.KEGG analysis demonstrated that the predicted targets were enriched in several immune response and signal transduction pathways,including the RIG-I-like receptor signalling pathway,cytosolic DNA sensing pathway and Notch signal pathway.(4)Validation of miR-122 targeting EPO 3’UTRAccording to the result that EPO was the predicted target gene of miR-122,wild and mutant seqences of bovine EPO gene 3’UTR were inserted into the pmiR-RB-REPORTTM vector.miR-122 mimic with wild or mutant veter were transfected into 293T cells.The two reporter genes expression levels were tested using the ELISA instrument.The results showed that EPO gene was the target gene of miR-122 and the targeted sequence was ACACTCC.(5)Construction of Bovine Primary Mammary Epithelial Cells Culture and determined transfection conditionsPurified primary mammary gland epithelial cells formed island-like and grew with typical type of slabstone and cobblestone.Primary mammary gland epithelial cells growth curve was"S"shape and consistened with general rule of cell growth.Mammary epithelial cells expressedβ-casein mRNA successfully.The final transfection conditition were 100nM FM-siRNA with 1μL transfection reagent for 96-well plates.(6)Effect of miR-122 on EPO and JAK-STAT pathway genes expressionTransfection of the miR-122 mimic into the bovine mammary epithelial cells increased miR-122 expression significantly while reduced EPO expression significantly.The expression of EPOR,JAK2,STAT5A,STAT5B,involved in JAK-STAT pathway,were down-regulated.Bcl-2,as the down-stream gene of STAT5,its expression was down-regulated significantly.However,transfection of miR-122 inhibitor into the bovine mammary epithelial cells,STAT5B expression increased significantly and EPO,EPOR,JAK2,STAT5A expression increased.