The role of micro RNA ‐27a/b and micro RNA ‐494 in estrogen‐mediated downregulation of tissue factor pathway inhibitor α

Essentials Estrogens are known to influence the expression of microRNAs in breast cancer cells. We looked at microRNAs in estrogenic regulation of tissue factor pathway inhibitor α (TFPIα). Estrogen upregulated microRNA‐27a/b and microRNA‐494 through the estrogen receptor α. MicroRNA‐27a/b and microRNA‐494 are partly involved in estrogenic downregulation of TFPIα.Summary 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 ( MCF 7) through the estrogen receptor α ( ER α). Accumulating evidence also indicates that activation of ER α signaling by estrogens may modulate the expression of target genes indirectly through micro RNA s (mi RNA s). Objectives To examine if mi RNA s are involved in the estrogenic downregulation of TFPI α. 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 mi RNA s was achieved by transfection of mi RNA mimics or inhibitors. Direct targeting of TFPI 3′‐ UTR by miR‐27a/b and miR‐494 was determined by luciferase reporter assay in HEK 293T cells. Effects of 17α‐ethinylestradiol ( EE 2) and fulvestrant on relative miR‐27a/b, miR‐494, and TFPI mRNA levels in MCF 7 cells were determined by qRT ‐ PCR and secreted TFPI α protein by ELISA . Transient knockdown of ER α was achieved by si RNA transfection. Results EE 2 treatment lead to a significant increase in miR‐19a, miR‐27a/b, miR‐494, and miR‐24 mRNA levels in MCF 7 cells through ER α. miR‐27a/b and miR‐494 mimics lead to reduced TFPI mRNA and protein levels. Luciferase assay showed direct targeting of miR‐27a/b and miR‐494 on TFPI mRNA . Impaired estrogen‐mediated downregulation of TFPI mRNA 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 α expression and suggest a possible role of these mi RNA s in the estrogen‐mediated downregulation of TFPI α.