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The enzyme DNA topoisomerase IIα (TOP2α) induces covalent complexes with DNA and produces transient double-strand DNA breaks crucial for processes such as replication and normal chromosomal dysjunction at mitosis. TOP2α is an important target for clinically effective anticancer agents, such as etoposide (VP-16), since these drugs stabilize the otherwise short-lived enzyme-DNA covalent complexes, thereby inducing cytotoxic DNA damage. However, the efficacy of these agents is limited by chemoresistance. Our lab has characterized acquired resistance to VP-16 in human leukemia K562 cells. The cloned resistant cell line, K/VP.5, contains reduced levels of TOP2α compared to parental K562 cells. The goal of this project is to test the hypothesis that TOP2α levels are decreased in K/VP.5 cells, in part, through miRNA-mediated mechanisms.

Pooled miRNA qPCR profiling experiments were performed to investigate the expression levels of ~500 miRNAs in K562 and K/VP.5 cells. hsa-miR-9-3p and -5p (miR-9-3p and -5p) were overexpressed in K/VP.5 cells compared to K562 cells. The TOP2α 3ʹ-UTR harbors putative miRNA recognition elements (MRE) for these miRNAs. Therefore, these miRNAs were chosen for further study. To assess post-transcriptional regulation of TOP2α by miRNAs, a dual luciferase reporter plasmid harboring the entire 3ʹ-UTR of TOP2α mRNA (998 bp) was constructed (psiTOP2α/UTR). Transfection with psiTOP2α/UTR demonstrated decreased luciferase expression in K/VP.5 compared with K562 cells, suggesting altered post-transcriptional regulation in resistant cells. K562 cells that were co-transfected with psiTOP2α/UTR and miR-9-3p or -5p mimic resulted in a statistically significant decrease in luciferase expression only for miR-9-5p. Mutating the putative miR-9-5p seed sequence prevented the decrease in luciferase activity, demonstrating a direct interaction of this miRNA with the MRE of TOP2α. Immunoblotting for TOP2α in K562 cells transfected with miR-9-3p or -5p mimic resulted in decreased TOP2α protein compared to mock transfected K562 cells. Conversely, immunoblotting for TOP2α in K/VP.5 cells transfected with miR-9-3p or -5p inhibitor resulted in an increase of TOP2α protein, strongly suggesting a role for both miRNAs in acquired resistance to VP-16.

Our findings indicate that miR-9-3p and -5p regulate TOP2α expression levels. In addition, results presented here contribute to the elucidation of chemoresistance mechanisms and have the potential for circumvention of drug resistance by modulation of miRNA concentrations.