| Enzootic bovine leukemia(EBL)is an infectious neoplastic disease caused by bovine leukemia virus(BLV).At present,the seroprevalence of this disease in some herds of China has approached 50%,which has been ignored because of the absence of obvious clinical symptoms,and this phenomenon causes a serious global public health problem.BLV can infect the MAC-T by the blood and lymphatic system,which can lead to the decrease of antibacterial ability of cells.A pre-experimental period found that BLV infection promoted an increased number of adhesions of S.aureus on the MAC-T,but the specific mechanism was still unclear.The mammary epithelium of dairy cattle is a defensive barrier on the mammary gland.It’s decreased antimicrobial capacity can cause the infection of mastitis pathogenic microorganisms such as S.aureus.The S.aureus is able to colonize the mammary epithelium for a long time,inducing apoptosis of the MAC-T and the expression of pro-inflammatory factors,thereby promoting the occurrence and development of mammary inflammation.BLV encoded miRNAs(BLV miRNAs),are functional elements necessary for BLV replication and pathogenicity.Our target gene prediction in the pre experimental period indicated.BLV-miR-B1-5p may target and inhibit bovine MUC1 expression and transcription,leading to increased susceptibility of MAC-T to bacteria.From this,we speculated that BLV may promote the adhesion of S.aureus on MAC-T through target inhibition of MUC1 by BLV-miR-B1-5p.To define the effect of BLV-miR-B1-5p on MAC-T adherent bacteria,the bacteria were cocultured with the treated MAC-T.Differences in bacterial numbers were compared.To clarify whether BLV-miR-B1-5p targets MUC1,target site prediction analysis of binding sites was performed using the STar Mir and UCSC Genome Broeser.We next clarified the targeting relationship by dual luciferase assay to verify the target gene prediction results.Finally,the effect of BLV-miR-B1-5p on MUC1 was clarified by quantitative PCR,WB,and immunofluorescence of cells.To determine whether BLV-miR-B1-5p promotes bacterial adhesion to MAC-T by targeting MUC1,MUC1 small interfering RNA(siRNA)was designed.Interfering with MUC1 expression in MAC-T,utilizing S.aureus infects perturbed cells,to determine whether differential MUC1 expression in MAC-T affects bacterial adhesion.To further determine the effect of blv-mir-b1-5p on epithelial cells.Endometrial epithelial cells from cows were used to repeat the above experiments.The results of the bacterial adhesion cell assay showed that BLV-miR-B1-5p with different loadings could significantly increase the number of adherenting S.aureus;Target site prediction showed that BLV-miR-B1-5p can fully target the mucin family gene MUC1(nucleotides 57 to 84 from the 3’UTR end).And the dual luciferase reporter assays validated it.The results of q PCR,WB,and cell immunofluorescence experiments showed that transfection with different concentrations of BLV-miR-B1-5p significantly inhibited MUC1 gene and protein expression levels in bovine mammary epithelial cells.When MUC1 expression was disrupted in the MAC-T,the rate of S.aureus adherence to the cells increased significantly.Meanwhile,transfection of different concentrations of BLV-miR-B1-5p similarly significantly increased the number of S.aureus in the experiment on bovine endometrial epithelial cells.Furthermore,the results of q PCR,WB,immunofluorescence of cells as well as MUC1 interference experiments were consistent with those obtained on MAC-T.It is thus established that BLV-miR-B1-5p promotes S.aureus adhesion on MAC-T through target repression of MUC1 and that this function of BLV-miR-B1-5p may have similar functions on different epithelial cells.This conclusion is beneficial for purifying leukemia in scaleup pasture cattle and protecting breast and uterus health in dairy cattle. |
