Characterization of microRNAs as Post-Transcriptional Modulators of DNA Topoisomerase IIα in Etoposide (VP-16) Resistant K562 Cells.

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The Ohio State University

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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.



TOP2A, miRNA, Chemoresistance, hsa-miR-9-3p, hsa-miR-9-5p