The role of microRNA-27a/b and microRNA-494 in estrogen-mediated downregulation of tissue factor pathway inhibitor

Background Tissue factor pathway inhibitor (TFPI) has been linked to breast cancer pathogenesis. We have recently reported TFPI mRNA levels to be downregulated by estrogens in a breast cancer cell line (MCF7) through the estrogen receptor (ER). Accumulating evidence also indicates that activation of ER signaling by estrogens may modulate the expression of target genes indirectly through microRNAs (miRNAs). Objectives To examine if miRNAs are involved in the estrogenic downregulation of TFPI. Methods Computational analysis of the TFPI 3-untranslated region (UTR) identified potential binding sites for miR-19a/b, miR-27a/b, miR-494, and miR-24. Transient overexpression or inhibition of the respective miRNAs was achieved by transfection of miRNA mimics or inhibitors. Direct targeting of TFPI 3-UTR by miR-27a/b and miR-494 was determined by luciferase reporter assay in HEK293T cells. Effects of 17-ethinylestradiol (EE2) and fulvestrant on relative miR-27a/b, miR-494, and TFPI mRNA levels in MCF7 cells were determined by qRT-PCR and secreted TFPI protein by ELISA. Transient knockdown of ER was achieved by siRNA transfection. Results EE2 treatment lead to a significant increase in miR-19a, miR-27a/b, miR-494, and miR-24 mRNA levels in MCF7 cells through ER. miR-27a/b and miR-494 mimics lead to reduced TFPImRNA and protein levels. Luciferase assay showed direct targeting of miR-27a/b and miR-494 on TFPImRNA. Impaired estrogen-mediated downregulation of TFPImRNA was detected in anti-miR-27a/b and anti-miR-494 transfected cells. Conclusions Our results provide evidence that miR-27a/b and miR-494 regulate TFPI expression and suggest a possible role of these miRNAs in the estrogen-mediated downregulation of TFPI.