@article {Kaniamol.119.118315, author = {Evan E. Kania and Jessika Carvajal-Moreno and Victor A. Hernandez and Anthony English and Jonathan L. Papa and Nicholas Shkolnikov and Hatice Gulcin Ozer and Ayse Selen Yilmaz and Jack C. Yalowich and Terry S. Elton}, title = {hsa-miR-9-3p and hsa-miR-9-5p as Post-Transcriptional Modulators of DNA Topoisomerase IIα in Human Leukemia K562 Cells with Acquired Resistance to Etoposide}, elocation-id = {mol.119.118315}, year = {2019}, doi = {10.1124/mol.119.118315}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {DNA topoisomerase IIα (TOP2α/170) is an important target for anticancer agents whose efficacy is often attenuated by chemoresistance. Our lab has characterized acquired resistance to etoposide in human leukemia K562 cells. The clonal resistant subline, K/VP.5, contains reduced TOP2α/170 mRNA and protein levels compared to parental K562 cells. The aim of this study was to determine whether miRNA-mediated mechanisms play a role in drug resistance via decreased expression of TOP2α/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 to K562 cells; validation by qPCR demonstrated overexpression of both miRNAs. miRNA recognition elements (MREs) for both miRNAs are present in the 3'-UTR of TOP2α/170. Transfecting K562 cells with a reporter plasmid harboring the TOP2α/170 3'-UTR (psiTOP2α/170/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 TOP2α/170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2α/170 protein levels without a change in TOP2α/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 TOP2α/170) led to increased TOP2α/170 protein expression without a change in TOP2α/170 mRNA, 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 TOP2α/170 expression levels in acquired resistance to etoposide. These findings contribute new information about and potential strategies for circumvention of drug resistance; by modulation of miRNA 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 TOP2-targeted therapy.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2019/12/13/mol.119.118315}, eprint = {https://molpharm.aspetjournals.org/content/early/2019/12/13/mol.119.118315.full.pdf}, journal = {Molecular Pharmacology} }