Aberrant overexpression of miR-421 downregulates ATM and leads to a pronounced DSB repair defect and clinical hypersensitivity in SKX squamous cell carcinoma
Aberrant overexpression of miR-421 downregulates ATM and leads to a pronounced DSB repair defect and clinical hypersensitivity in SKX squamous cell carcinoma
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DOI：
10.1016/j.radonc.2012.10.020
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发表时间：
2013-01-01
影响因子：
5.7
通讯作者：
Dahm-Daphi, Jochen
中科院分区：
文献类型：
--
作者：
Mansour, Wael Y.;Bogdanova, Natalia V.;Dahm-Daphi, Jochen
背景：细胞和临床对电离辐射 (IR) 的敏感性由 DNA 双链断裂 (DSB) 修复决定。在此，我们研究了头颈肿瘤病例 (SKX) 对标准放疗极端反应的分子机制。方法：分别使用免疫荧光 (IF) 评估 DSB 修复、蛋白质印迹和实时 PCR 的蛋白质和 mRNA 表达。结果：SKX 细胞表现出明显的放射敏感性，与 IR 后大量残留的 γ-H2AX 病灶相关。这与缺乏典型修复蛋白无关。 SKX细胞不表达任何ATM蛋白。因此，免疫印迹显示对 p-SMC1、p-CHK2 和 p-KAP1 等底物没有 ATM 激酶活性。 ATM全部66个外显子的测序显示没有突变。 ATM mRNA 水平适度降低，可以通过 5'-Aza-C 处理来恢复，但不会恢复蛋白质水平。重要的是，我们通过 6 倍增强的 miR-421 水平证明了 SKX 细胞中的转录后调节，miR-421 靶向 ATM mRNA 的 3'-UTR。用抗 miR-421 抑制剂或 microRNA 不敏感 ATM 载体转染 SKX 细胞可恢复 ATM 表达并消除超放射敏感性。 结论：这是第一份描述 microRNA 介导的 ATM 下调导致临床上明显的肿瘤放射敏感性的报告。 (c) 2012 Elsevier Ireland Ltd. 保留所有权利。放射治疗和肿瘤学106（2013）147-154
Background: Cellular and clinical sensitivity to ionizing radiation (IR) is determined by DNA double-strand breaks (DSB) repair. Here, we investigate the molecular mechanism underlying the extreme response of a head and neck tumor case (SKX) to standard radiotherapy.Methods: Immunofluorescence (IF) was used for the assessment of DSB repair, Western blot and real-time PCR for protein and mRNA expression, respectively.Results: SKX cells exhibited a pronounced radiosensitivity associated with numerous residual gamma-H2AX foci after IR. This was not associated with lacking canonical repair proteins. SKX cells did not express any ATM protein. Accordingly, immunoblotting revealed no ATM kinase activity toward substrates such as p-SMC1, p-CHK2 and p-KAP1. Sequencing of all 66 exons of ATM showed no mutation. ATM mRNA level was moderately reduced, which could be reverted by 5'-Aza-C treatment but without restoring protein levels. Importantly, we demonstrated a post-transcriptional regulation in SKX cells via 6-fold enhanced levels of miR-421, which targets the 3'-UTR of ATM mRNA. Transfection of SKX cells with either anti-miR-421 inhibitor or a microRNA-insensitive ATM vector recovered ATM expression and abrogated the hyper-radiosensitivity.Conclusion: This is the first report describing microRNA-mediated down-regulation of ATM leading to clinically manifest tumor radiosensitivity. (c) 2012 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 106 (2013) 147-154