hsa-miR-9-3p and hsa-miR-9-5p as Post-Transcriptional Modulators of DNA Topoisomerase II in Human Leukemia K562 Cells with Acquired Resistance to Etoposide.

DNA topoisomerase II protein (TOP2) 170 kDa (TOP2/170) is an important target for anticancer agents whose efficacy is often attenuated by chemoresistance. Our laboratory has characterized acquired resistance to etoposide in human leukemia K562 cells. The clonal resistant subline K/VP.5 contains reduced TOP2/170 mRNA and protein levels compared with parental K562 cells. The aim of this study was to determine whether microRNA (miRNA)-mediated mechanisms play a role in drug resistance via decreased expression of TOP2/170. miRNA-sequencing revealed that human miR-9-3p and miR-9-5p were among the top six of those overexpressed in K/VP.5 compared with K562 cells; validation by quantitative polymerase chain reaction demonstrated overexpression of both miRNAs. miRNA recognition elements (MREs) for both miRNAs are present in the 3'-untranslated region (UTR) of TOP2/170. Transfecting K562 cells with a reporter plasmid harboring the TOP2/170 3'-UTR together with either miR-9-3p or miR-9-5p mimics resulted in a statistically significant decrease in luciferase expression. Mutating the miR-9-3p or miR-9-5p MREs prevented this decrease, demonstrating direct interaction between these miRNAs and TOP2/170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2/170 protein levels without a change in TOP2/170 mRNA and resulted in attenuated etoposide-induced DNA damage (gain-of-miRNA-inhibitory function). Conversely, transfection of miR-9-3p or miR-9-5p inhibitors in K/VP.5 cells (overexpressed miR-9 and low TOP2/170) led to increased TOP2/170 protein expression without a change in TOP2/170 mRNA levels and resulted in enhancement of etoposide-induced DNA damage (loss-of-miRNA-inhibitory function). Taken together, these results strongly suggest that these miRNAs play a role in and are potential targets for circumvention of acquired resistance to etoposide. SIGNIFICANCE STATEMENT: Results presented here indicate that miR-9-3p and miR-9-5p decrease DNA topoisomerase II protein 170 kDa expression levels in acquired resistance to etoposide. These findings contribute new information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. Furthermore, increased expression of miR-9-3p and miR-9-5p in chemoresistant cancer cells may support their validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy.