Division of Pharmaceutics and Pharmacology, College of Pharmacy (E.E.K., J.C.-M., V.A.H., A.E., J.L.P., N.S., J.C.Y., T.S.E.) and Department of Biomedical Informatics, College of Medicine (H.G.O., A.S.Y.), The Ohio State University, Columbus, Ohio.
Division of Pharmaceutics and Pharmacology, College of Pharmacy (E.E.K., J.C.-M., V.A.H., A.E., J.L.P., N.S., J.C.Y., T.S.E.) and Department of Biomedical Informatics, College of Medicine (H.G.O., A.S.Y.), The Ohio State University, Columbus, Ohio
Mol Pharmacol. 2020 Mar;97(3):159-170. doi: 10.1124/mol.119.118315. Epub 2019 Dec 13.
DNA topoisomerase II protein (TOP2) 170 kDa (TOP2/170) 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 TOP2/170 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 TOP2/170. 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 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 TOP2/170. Transfecting K562 cells with a reporter plasmid harboring the TOP2/170 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 TOP2/170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2/170 protein levels without a change in TOP2/170 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 TOP2/170) led to increased TOP2/170 protein expression without a change in TOP2/170 mRNA levels 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 DNA topoisomerase II protein 170 kDa expression levels in acquired resistance to etoposide. These findings contribute new information about and potential strategies for circumvention of drug resistance by modulation of microRNA 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 DNA topoisomerase II-targeted therapy.
DNA 拓扑异构酶 II 蛋白 (TOP2) 170 kDa (TOP2/170) 是抗癌药物的重要靶点，其疗效常因耐药性而减弱。我们的实验室已经对人白血病 K562 细胞中甲氨蝶呤的获得性耐药进行了特征描述。与亲本 K562 细胞相比，克隆耐药亚系 K/VP.5 中的 TOP2/170 mRNA 和蛋白水平降低。本研究的目的是确定 miRNA（miRNA）介导的机制是否通过降低 TOP2/170 的表达在耐药中起作用。miRNA 测序显示，与 K562 细胞相比，人 miR-9-3p 和 miR-9-5p 在 K/VP.5 中表达过高的前六种 miRNA 中；通过定量聚合酶链反应验证，两种 miRNA 的表达均升高。TOP2/170 的 3'-非翻译区（UTR）中存在这两种 miRNA 的 miRNA 识别元件（MRE）。用携带 TOP2/170 3'-UTR 的报告质粒转染 K562 细胞，与 miR-9-3p 或 miR-9-5p 模拟物一起转染，可导致荧光素酶表达显著降低。突变 miR-9-3p 或 miR-9-5p MRE 可防止这种降低，表明这些 miRNA 与 TOP2/170 mRNA 之间存在直接相互作用。用 miR-9-3p 或 miR-9-5p 模拟物转染 K562 细胞会导致 TOP2/170 蛋白水平降低，而 TOP2/170 mRNA 不变，并导致依托泊苷诱导的 DNA 损伤减弱（获得 miRNA 抑制功能）。相反，在 K/VP.5 细胞（高表达 miR-9 和低 TOP2/170）中转染 miR-9-3p 或 miR-9-5p 抑制剂会导致 TOP2/170 蛋白表达增加，而 TOP2/170 mRNA 水平不变，并导致依托泊苷诱导的 DNA 损伤增强（失去 miRNA 抑制功能）。综上所述，这些结果强烈表明，这些 miRNA 在获得性依托泊苷耐药中发挥作用，并且是克服耐药的潜在靶点。意义声明：这里呈现的结果表明，miR-9-3p 和 miR-9-5p 降低了获得性依托泊苷耐药中 DNA 拓扑异构酶 II 蛋白 170 kDa 的表达水平。这些发现为通过调节 miRNA 水平来克服耐药性提供了新的信息和潜在策略。此外，化学抗性癌细胞中 miR-9-3p 和 miR-9-5p 的表达增加可能支持它们作为 DNA 拓扑异构酶 II 靶向治疗反应性的生物标志物的验证。
