Effects of hsa‐miR‐9‐3p and hsa‐miR‐9‐5p on Topoisomerase IIβ Expression in Human Leukemia K562 Cells with Acquired Resistance to Etoposide

Abstract

DNA topoisomerase IIα (TOP2α/170; 170kDa) and Topoisomerase IIβ (TOP2β/180; 180kDa) are important targets for DNA damage‐stabilizing anticancer drugs, whose clinical efficacy is often attenuated by chemoresistance. Our laboratory has characterized an etoposide‐resistant human leukemia K562 clonal subline, designated K/VP.5. These cells show a decrease in both TOP2α/170 and TOP2β/180 expression. We recently demonstrated that a microRNA (miRNA)‐mediated post transcriptional mechanism plays a role in drug resistance via reduced TOP2α/170 protein levels in K/VP.5 cells. We hypothesize that a miRNA mechanism is similarly responsible for decreased TOP2β/180 levels in K/VP.5 cells. Both miR‐9‐3p and miR‐9‐5p are overexpressed in K/VP.5 compared with K562 cells, demonstrated by miRNA sequencing, and validated by quantitative polymerase chain reaction (qPCR). The 3’‐untranslated region (UTR) of TOP2β/180 contains a well‐defined miRNA recognition element (MRE) for miR‐9‐5p and putative MREs for miR‐9‐3p. Co‐transfection of K562 cells with a luciferase reporter plasmid harboring TOP2β/180‐3’UTR plus miR‐9‐3p and/or miR‐9‐5p mimics resulted in a statistically significant decrease in luciferase expression. miR‐9‐5p MRE mutation prevented this decrease validating direct interaction between this miRNA and TOP2β/180 mRNA. Transfection of K562 cells with miR‐9‐3p or miR‐9‐5p mimics led to decreased TOP2β protein levels without a change in TOP2β/180 mRNA. Inversely, K/VP.5 cells transfected with miR‐9‐3p or miR‐9‐5p inhibitors led to increased TOP2β/180 protein and no change in TOP2β/180 mRNA. Taken together, these results strongly suggest that TOP2β/180 mRNA is translationally repressed by miR‐9‐3p and miR‐9‐5p, and that these miRNAs play a role in acquired resistance to etoposide via regulation of TOP2β/180. Further experiments are underway to characterize miR‐9‐3p and miR‐9‐5p effects on TOP2β/180‐specific cytotoxicity.