| Summary: | In eukaryotes, overproduction of reactive oxygen species (ROS) causes oxidative stress, which contributes to chronic inflammation and cancer. MicroRNAs (miRNAs) are small, endogenously produced RNAs that play a major role in cancer progression. We established that overexpression of miR526b/miR655 promotes aggressive breast cancer phenotypes. Here, we investigated the roles of miR526b/miR655 in oxidative stress in breast cancer using in vitro and in silico assays. miRNA-overexpression in MCF7 cells directly enhances ROS and superoxide (SO) production, detected with fluorescence assays. We found that cell-free conditioned media contain extracellular miR526b/miR655 and treatment with these miRNA-conditioned media causes overproduction of ROS/SO in MCF7 and primary cells (HUVECs). Thioredoxin Reductase 1 (TXNRD1) is an oxidoreductase that maintains ROS/SO concentration. Overexpression of <i>TXNRD1</i> is associated with breast cancer progression. We observed that miR526b/miR655 overexpression upregulates <i>TXNRD1</i> expression in MCF7 cells, and treatment with miRNA-conditioned media upregulates <i>TXNRD1</i> in both MCF7 and HUVECs. Bioinformatic analysis identifies two negative regulators of TXNRD1, TCF21 and PBRM1, as direct targets of miR526b/miR655. We validated that <i>TCF21</i> and <i>PBRM1</i> were significantly downregulated with miRNA upregulation, establishing a link between miR526b/miR655 and TXNRD1. Finally, treatments with oxidative stress inducers such as H<sub>2</sub>O<sub>2</sub> or miRNA-conditioned media showed an upregulation of miR526b/miR655 expression in MCF7 cells, indicating that oxidative stress also induces miRNA overexpression. This study establishes the dynamic functions of miR526b/miR655 in oxidative stress induction in breast cancer.
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