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

Abstract

DNA topoisomerase IIa protein (TOP2a) 170 kDa (TOP2a/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 TOP2a/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 TOP2a/170. miRNAsequencing 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 TOP2a/170. Transfecting K562 cells with a reporter plasmid harboring the TOP2a/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 TOP2a/170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2a/170 protein levels without a change in TOP2a/170 mRNA and resulted in attenuated etoposide-induced DNA damage (gain-of-miRNAinhibitory function). Conversely, transfection of miR-9-3p or miR-9-5p inhibitors in K/VP.5 cells (overexpressed miR-9 and low TOP2a/170) led to increased TOP2a/170 protein expression without a change in TOP2a/170 mRNA levels and resulted in enhancement of etoposide-induced DNA damage (loss-ofmiRNA-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.