H O Ali1,2,3, A B Arroyo4, R González-Conejero4, B Stavik1,2, N Iversen5, P M Sandset1,2,3, C Martínez4, G Skretting1,2. 1. Department of Haematology, Oslo University Hospital, Oslo, Norway. 2. Research Institute of Internal Medicine, Oslo University Hospital, Oslo, Norway. 3. Institute of Clinical Medicine, University of Oslo, Oslo, Norway. 4. Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, IMIB-Arrixaca, University of Murcia, Murcia, Spain. 5. Department of Medical Genetics, Oslo University Hospital and University of Oslo, Oslo, Norway.
Abstract
UNLABELLED: 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 (MCF7) through the estrogen receptor α (ERα). Accumulating evidence also indicates that activation of ERα 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 TFPIα. 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 17α-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 TFPIα protein by ELISA. Transient knockdown of ERα 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 ERα. 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 TFPIα expression and suggest a possible role of these miRNAs in the estrogen-mediated downregulation of TFPIα.
UNLABELLED: 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 (MCF7) through the estrogen receptor α (ERα). Accumulating evidence also indicates that activation of ERα 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 TFPIα. 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 17α-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 TFPIα protein by ELISA. Transient knockdown of ERα 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 ERα. 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 TFPIα expression and suggest a possible role of these miRNAs in the estrogen-mediated downregulation of TFPIα.
Authors: Robert Gelfand; Dolores Vernet; Kevin W Bruhn; Suren Sarkissyan; David Heber; Jaydutt V Vadgama; Nestor F Gonzalez-Cadavid Journal: Int J Oncol Date: 2016-12-09 Impact factor: 5.650