| Summary: | 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). CoCl<sub>2</sub>-induced hypoxia in breast cancer further promotes <i>HIF-1α</i> mRNA and protein expression while reducing <i>VHL</i> 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 (<i>COX-2</i>, <i>EP4</i>, <i>NFκB1</i>, <i>VEGFA</i>) 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 <i>PTEN</i> (a negative regulator of <i>HIF-1α</i>) and <i>NFκB1</i> (positive regulator of <i>COX-2</i> and <i>EP4</i>) expression by downregulation of transcription factors <i>NR2C2</i>, <i>SALL4</i>, and <i>ZNF207</i>. 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, <i>HIF-1α</i> expression is significantly higher in human breast tumors (<i>n</i> = 96) compared to non-cancerous control tissues (<i>n</i> = 20) and is positively correlated with miR526b/miR655 expression. In stratified tumor samples, <i>HIF-1α</i> 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 <i>HIF-1α</i> and miRNA-cluster expression in breast tumors. This study, for the first time, establishes the dynamic roles of miR526b/miR655 in hypoxia.
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