Department of Urology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China.
Int J Mol Med. 2018 Mar;41(3):1765-1773. doi: 10.3892/ijmm.2018.3374. Epub 2018 Jan 10.
miR‑214 has been reported to be downregulated in several cancer types, such as bladder cancer. However, its involvement in apoptosis and chemoresistance has not been investigated. The present study aimed to clarify the biological function of miR‑214 and potential mechanisms in chemoresistance of bladder cancer cells. Reverse transcription‑quantitative polymerase chain reaction demonstrated that miR‑214 was downregulated in bladder cancer tissues compared with the level in normal tissues. miR‑214 was downregulated in bladder cancer cell lines compared with the level in the normal cell line SV‑HUC‑1. miR‑214 mimics were transfected into T24 and J82 cell lines to restore its expression. The results indicated that miR‑214 mimic inhibited proliferation and invasion in these cell lines. In addition, miR‑214 mimic reduced cisplatin resistance in T24 and J82 cells, indicated by the inhibition of cell viability and upregulation of cell apoptosis. Western blotting demonstrated that miR‑214 mimic was able to upregulate cleaved caspase‑3 and cleaved poly (ADP‑ribose) polymerase (PARP), while downregulate caspase‑3 and PARP expression, and AKT phosphorylation. Using prediction software, it was revealed that the netrin‑1 oncoprotein is on the target list of miR‑214. miR‑214 also downregulated netrin‑1 protein and mRNA expression levels in the T24 and J82 cell lines. Luciferase reporter assays demonstrated that netrin‑1 acted as a direct target of miR‑214. A negative correlation between netrin‑1 and miR‑214 expression in bladder cancer tissues was also observed. In addition, cisplatin treatment could induce netrin‑1 protein expression in bladder cancer cells and miR‑214 mimic partly blocked this phenomenon. Netrin‑1 plasmid transfection inhibited cisplatin‑induced apoptosis, upregulated AKT phosphorylation, and downregulated caspase‑3 and PARP cleavage. Netrin‑1 was restored in cells transfected with miR‑214 mimic using plasmid transfection. Netrin‑1 transfection restored AKT phosphorylation and blocked caspase/PARP cleavage in the T24 and J82 cell lines. In conclusion, the present study demonstrated that miR‑214 is downregulated in bladder cancer tissues and cell lines. miR‑214 reduces chemoresistance by targeting netrin‑1 in bladder cancer cell lines.
miR-214 在几种癌症类型中被报道下调，例如膀胱癌。然而，其在细胞凋亡和化疗耐药中的作用尚未被研究。本研究旨在阐明 miR-214 在膀胱癌细胞化疗耐药中的生物学功能及其潜在机制。逆转录定量聚合酶链反应显示，miR-214 在膀胱癌组织中下调，与正常组织水平相比。miR-214 在膀胱癌细胞系中下调，与正常细胞系 SV-HUC-1 相比。将 miR-214 模拟物转染至 T24 和 J82 细胞系以恢复其表达。结果表明，miR-214 模拟物抑制这些细胞系的增殖和侵袭。此外，miR-214 模拟物降低了 T24 和 J82 细胞中的顺铂耐药性，表现为细胞活力抑制和细胞凋亡增加。Western blot 分析表明，miR-214 模拟物能够上调裂解的半胱天冬酶-3 和裂解的多聚（ADP-核糖）聚合酶（PARP），同时下调半胱天冬酶-3 和 PARP 表达以及 AKT 磷酸化。使用预测软件表明，轴突导向因子 1 癌蛋白是 miR-214 的靶标列表之一。miR-214 还下调了 T24 和 J82 细胞系中的 netrin-1 蛋白和 mRNA 表达水平。荧光素酶报告基因检测表明，netrin-1 是 miR-214 的直接靶标。还观察到膀胱癌组织中 netrin-1 和 miR-214 表达之间存在负相关。此外，顺铂处理可诱导膀胱癌细胞中 netrin-1 蛋白表达，miR-214 模拟物部分阻断了这种现象。netrin-1 质粒转染抑制顺铂诱导的细胞凋亡，上调 AKT 磷酸化，并下调半胱天冬酶/ PARP 裂解。使用质粒转染恢复 miR-214 模拟物转染的细胞中的 netrin-1。netrin-1 转染恢复了 T24 和 J82 细胞系中的 AKT 磷酸化并阻断了 caspase/PARP 裂解。总之，本研究表明 miR-214 在膀胱癌组织和细胞系中下调。miR-214 通过靶向膀胱癌细胞系中的 netrin-1 降低化疗耐药性。
