Mei Zijie, Su Tingshi, Ye Junjie, Yang Chunxu, Zhang Shimin, Xie Conghua
Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan, Hubei, 430071, China.
Radiat Res. 2015 Feb;183(2):196-207. doi: 10.1667/RR13784.1. Epub 2015 Jan 16.
Enhancing radiosensitivity is an important area of investigation for improving breast cancer therapy outcomes. The aim of this study was to assess the role of the miR-15 family in the radiosensitivity of breast cancer cells. MicroRNAs (miRNAs) encoded by the miR-15 cluster are known to induce G1 arrest and apoptosis by targeting G1 checkpoints and the anti-apoptotic B cell lymphoma 2 (BCL-2) gene. However, the effect of the miR-15 family on G2/M arrest and radiosensitivity remains poorly understood. In the current study, cells transfected with miR-15a/15b/16 mimic or inhibitor were irradiated and examined by: clonogenic assays, phosphorylated H2AX assay, flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), real-time PCR and Western blot. Real-time PCR was also used to monitor time-dependent changes of miR-15a/15b/16 expression after irradiation. A putative target site for miR-15a/15b/16 within the Chk1 and Wee1 3' UTRs was confirmed using luciferase reporter assays. Additionally, siRNA was used to validate the effect of Chk1 and Wee1 on radiosensitivity in breast cancer cells. In our study, we investigated the effects of radiation on the miR-15 family and found a time-dependent change in the expression of miR-15a/15b/16 in breast cancer cells postirradiation, as well as an increase in miR-15 family-mediated sensitization of breast cancer cells to radiation. The increase in radiosensitivity induced by the miR-15 family was associated with persistent unrepaired DNA damage, abrogation of radiation-induced G2 arrest and suppressed cell proliferation, and appear to involve both the checkpoint kinase 1 (Chk1) and Wee1. In addition, we found that inhibition of the miR-15 family could not induce cell resistance to radiation. These findings suggest that the expression of the miR-15 family contributes to increased radiosensitivity of breast cancer cells by influencing G2/M checkpoint proteins.
提高放射敏感性是改善乳腺癌治疗效果的一个重要研究领域。本研究的目的是评估miR-15家族在乳腺癌细胞放射敏感性中的作用。已知由miR-15簇编码的微小RNA（miRNA）通过靶向G1检查点和抗凋亡的B细胞淋巴瘤2（BCL-2）基因诱导G1期阻滞和凋亡。然而，miR-15家族对G2/M期阻滞和放射敏感性的影响仍知之甚少。在本研究中，用miR-15a/15b/16模拟物或抑制剂转染的细胞接受照射，并通过以下方法进行检测：克隆形成试验、磷酸化H2AX试验、流式细胞术、3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）、实时定量聚合酶链反应（PCR）和蛋白质免疫印迹法。实时定量PCR还用于监测照射后miR-15a/15b/16表达的时间依赖性变化。使用荧光素酶报告基因试验证实了Chk1和Wee1 3'非翻译区（UTR）内miR-15a/15b/16的假定靶位点。此外，使用小干扰RNA（siRNA）验证Chk1和Wee1对乳腺癌细胞放射敏感性的影响。在我们的研究中，我们研究了辐射对miR-15家族的影响，发现照射后乳腺癌细胞中miR-15a/15b/16的表达存在时间依赖性变化，以及miR-15家族介导的乳腺癌细胞对辐射的敏感性增加。miR-15家族诱导的放射敏感性增加与持续未修复的DNA损伤、辐射诱导的G2期阻滞的消除和细胞增殖的抑制有关, 并且似乎涉及检查点激酶1（Chk1）和Wee1两者。此外，我们发现抑制miR-15家族不能诱导细胞对辐射产生抗性。这些发现表明，miR-15家族的表达通过影响G2/M检查点蛋白有助于提高乳腺癌细胞的放射敏感性。
