Identification of bta-miR-15a∼16a cluster expression, localization and regulated target in Holsteins.

Bovine mastitis is an inflammation response of the mammary gland tissues caused mainly by pathogenic bacteria in cows. Previous studies showed that bta-miR-15a and bta-miR-16a modulate immunity and inflammation responses. In this study, we investigated the expression pattern and tissue localization of bta-miR-15a and bta-miR-16a. The expression levels of bta-miR-15a and bta-miR-16a were significantly upregulated in mammary tissues and blood neutrophils of mastitis-infected cows, compared with those of healthy cows (P < 0.05). Through in situ hybridization, we examined the tissue localization of bta-miR-15a and bta-miR-16a and found that they were expressed in the ductal and acinar cells of mammary gland tissues, where they had a stronger expression signal in the mammary tissues of cows with mastitis than that in healthy cows' tissues. Moreover, we identified CD163 as the target gene of bta-miR-15a and bta-miR-16a. Luciferase assay indicated that bta-miR-15a, bta-miR-16a, and bta-miR-15a∼16a cluster led to the significant reduction in the luciferase activity of CD163 3'UTR vector (P < 0.05). Meanwhile, the luciferase activity had a significantly low value compared with that of single bta-miR-15a or bta-miR-16a plasmid (P < 0.05) in the presence of bta-miR-15a∼16a cluster. The bta-miR-15a∼16a cluster may function more effectively in inhibiting luciferase reporter gene activity of target gene CD163 than single miRNA. Our study provides an insight into the relationship between bovine mastitis and gene expression of bta-miR-15a/16a, which suggested that bta-miR-15a∼16a cluster may play a role against mastitis by binding to target CD163 gene in Holstein dairy cattle.