Crucial Role for Early Growth Response-1 in the Transcriptional Regulation of miR-20b in Breast Cancer
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Dongping Li1,2, Yaroslav Ilnytskyy1, Anna Kovalchuk1, Levon M. Khachigian3, Roderick T. Bronson4, Bo Wang1,2 and Olga Kovalchuk1
1 Department of Biological Sciences, University of Lethbridge, Lethbridge, Canada;
2 Department of Biochemistry, Qiqihar Medical University, Qiqihar, P.R. China;
3 Centre for Vascular Research, University of New South Wales, Sydney, Australia;
4 The Dana Farber/Harvard Comprehensive Cancer Center, Boston, Massachusetts, USA.
Olga Kovalchuk, email:
Bo Wang, email:
Keywords: EGR1, miR-20b, transcription, PTEN, BRCA1, breast cancer, proliferation, migration, cell cycle arrest
Received: July 8, 2013 Accepted: July 26, 2013 Published: July 28, 2013
Transcriptional regulation of miRNAs that control the pathogenesis of breast cancer remains largely unknown. Here, we showed that ionizing radiation, a known breast carcinogen, triggered the differential expression of miR-20b in mammary tissues. We identified several GC-rich consensus binding motifs for the zinc finger transcription factor early growth response-1 (EGR1) in miR-20b promoter. miR-20b was upregulated by IR and its upregulation correlated with EGR1 expression in the breast cancer cell line HCC1806. Therefore, we used HCC1806 cells as a model system to explore the role of EGR1 in miR-20b transcription. siRNA knockdown of EGR1 attenuated miR-20b expression. Luciferase assays showed that whereas EGR1 stimulated luciferase activity driven by the wild-type miR-20b promoter, this induction was abolished in the mutant miR-20 promoter construct. We noted significant enrichment of EGR1 at miR-20b promoter in HCC1806 cells compared with normal human mammary epithelial cells. Suppression of miR-20b significantly inhibited HCC1806 cell proliferation and migration, and led to G0/G1 and S phase arrest. In vitro RNA-pull down assays indicated that miR-20b targets numerous tumor suppressors, including PTEN and BRCA1, which were downregulated in HCC1806. Conversely, suppression of miR-20b increased PTEN and BRCA1 levels. Moreover, immunohistochemical and FISH analyses showed that the miR-20b expression correlated significantly with EGR1 levels in breast cancer tissues. Our findings thus demonstrate for the first time that EGR1 is a key player in the transcriptional control of miR-20b, and miR-20b may in turn function as an oncogene by contributing to breast tumorigenesis via tumor suppressor targeting.
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