MicroRNA Regulation of the Estrogen Receptor Alpha 3'UTR in Canine Mammary Tumor (CMT28) Cells

Reduced estrogen receptor alpha (ERα/ESR1) expression is seen in canine mammary tumors, but the mechanisms involved are not fully understood. This study examined ESR1, miR-18a, and miR-19a expression in canine mammary cell lines. Further CMT28, a canine mammary tumor cell line, was used to test whether these miRNAs can regulate the reporter activity through selected regions of the canine ESR1 3'UTR containing predicted miR-18a and miR-19a binding sites. ESR1 mRNA expression was measured by RT-qPCR, and miR-18a and miR-19a expression was measured using LNA-based assays. ESR1 mRNA expression was much lower in CMT27, CMT28, and CMT47 compared with normal canine mammary epithelial cells, while CMT12 showed higher expression. miR-18a and miR-19a were increased to different levels across the tumor cell lines. Bioinformatic analysis identified predicted miR-18a and miR-19a seed-match sites in the canine ESR1 3'UTR. Luciferase reporter assays showed that both miR-18a and miR-19a significantly reduced activity of the reporter containing selected regions of the 3'UTR of ESR1 encoding predicted miR-18a and miR-19a binding sites. In contrast, neither miRNA significantly reduced activity of the mutant reporter containing deletions in the predicted miR-18a and miR-19a seed-match regions. Anti-miR-18a and anti-miR-19a also significantly increased reporter activity compared with the corresponding miRNA alone. These findings provide functional evidence that miR-18a and miR-19a can regulate reporter activity through selected regions of the canine ESR1 3'UTR. Further studies are needed to determine the role of this regulation in endogenous ESR1 expression and canine mammary tumor biology.