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Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer

Department of Biology, Brandon University, 3rd Floor, John R. Brodie Science Centre, 270-18th Street, Brandon, MB R7A6A9, Canada
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These authors contributed equally to this work.
These authors contributed equally to this work.
Cancers 2020, 12(8), 2008; https://doi.org/10.3390/cancers12082008
Received: 29 June 2020 / Revised: 19 July 2020 / Accepted: 19 July 2020 / Published: 22 July 2020
(This article belongs to the Special Issue MicroRNA and Cancer)
In aggressively growing tumors, hypoxia induces HIF-1α expression promoting angiogenesis. Previously, we have shown that overexpression of oncogenic microRNAs (miRNAs, miRs) miR526b/miR655 in poorly metastatic breast cancer cell lines promotes aggressive cancer phenotypes in vitro and in vivo. Additionally, miR526b/miR655 expression is significantly higher in human breast tumors, and high miR526b/miR655 expression is associated with poor prognosis. However, the roles of miR526b/miR655 in hypoxia are unknown. To test the relationship between miR526b/miR655 and hypoxia, we used various in vitro, in silico, and in situ assays. In normoxia, miRNA-high aggressive breast cancer cell lines show higher HIF-1α expression than miRNA-low poorly metastatic breast cancer cell lines. To test direct involvement of miR526b/miR655 in hypoxia, we analyzed miRNA-high cell lines (MCF7-miR526b, MCF7-miR655, MCF7-COX2, and SKBR3-miR526b) compared to controls (MCF7 and SKBR3). CoCl2-induced hypoxia in breast cancer further promotes HIF-1α mRNA and protein expression while reducing VHL expression (a negative HIF-1α regulator), especially in miRNA-high cell lines. Hypoxia enhances oxidative stress, epithelial to mesenchymal transition, cell migration, and vascular mimicry more prominently in MCF7-miR526b/MCF7-miR655 cell lines compared to MCF7 cells. Hypoxia promotes inflammatory and angiogenesis marker (COX-2, EP4, NFκB1, VEGFA) expression in all miRNA-high cells. Hypoxia upregulates miR526b/miR655 expression in MCF7 cells, thus observed enhancement of hypoxia-induced functions in MCF7 could be attributed to miR526b/miR655 upregulation. In silico bioinformatics analysis shows miR526b/miR655 regulate PTEN (a negative regulator of HIF-1α) and NFκB1 (positive regulator of COX-2 and EP4) expression by downregulation of transcription factors NR2C2, SALL4, and ZNF207. Hypoxia-enhanced functions in miRNA-high cells are inhibited by COX-2 inhibitor (Celecoxib), EP4 antagonist (ONO-AE3-208), and irreversible PI3K/Akt inhibitor (Wortmannin). This establishes that hypoxia enhances miRNA functions following the COX-2/EP4/PI3K/Akt pathways and this pathway can serve as a therapeutic target to abrogate hypoxia and miRNA induced functions in breast cancer. In situ, HIF-1α expression is significantly higher in human breast tumors (n = 96) compared to non-cancerous control tissues (n = 20) and is positively correlated with miR526b/miR655 expression. In stratified tumor samples, HIF-1α expression was significantly higher in ER-positive, PR-positive, and HER2-negative breast tumors. Data extracted from the TCGA database also show a strong correlation between HIF-1α and miRNA-cluster expression in breast tumors. This study, for the first time, establishes the dynamic roles of miR526b/miR655 in hypoxia. View Full-Text
Keywords: Breast cancer; Hypoxia inducible factor 1-alpha (HIF-1α); MicroRNA (miRNA); miR526b; miR655; Oxidative stress; Migration; Cyclooxygenase-2 (COX-2); Prostaglandin E2 receptor 4 (EP4); PI3K/Akt Breast cancer; Hypoxia inducible factor 1-alpha (HIF-1α); MicroRNA (miRNA); miR526b; miR655; Oxidative stress; Migration; Cyclooxygenase-2 (COX-2); Prostaglandin E2 receptor 4 (EP4); PI3K/Akt
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MDPI and ACS Style

Gervin, E.; Shin, B.; Opperman, R.; Cullen, M.; Feser, R.; Maiti, S.; Majumder, M. Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer. Cancers 2020, 12, 2008.

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