Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Peking Union Medical College and Chinese Academy of Medical Sciences, Tianjin 300192, P.R. China.
Oncol Rep. 2018 Mar;39(3):977-984. doi: 10.3892/or.2017.6156. Epub 2017 Dec 15.
Radioresistance remains a major challenge in the treatment of glioma, and the response of patients to radio-therapy varies considerably. MicroRNAs (miRNAs) are involved in various biological processes. The purpose of the present study was to investigate miRNAs involved in the response to radiation in glioma cell lines. Total RNA was isolated from human glioma U251 cells 30 min after γ-ray exposure and hybridized to an miRNA chip array. miRNA expression profiles were analyzed by quantitative real-time PCR. pcDNA3/EGFP-miR-212 mimic transfection was used to verify the function of miR-212 in colony formation tests, and the effect of miR-212 overexpression on U251 cells was examined by western blot analysis of apoptosis-related proteins (Bcl-2, Bax, caspase-3 and cytochrome c). The target genes of miR-212 were predicted using bioinformatic tools including miRNA databases, and breast cancer susceptibility gene 1 (BRCA1) was selected for further confirmation by EGFP fluorescence reporter and loss- and gain-of-function assays. Of the 16 candidate miRNAs showing altered expression, five were assessed by real-time PCR; miR-212 was identified as contributing to the radioresistance of glioma cells and was shown to attenuate radiation-induced apoptosis. miR-212 negatively regulated BRCA1 expression by interacting with its 3'-untranslated region, suggesting a correlation between BRCA1 expression and radiosensitivity in glioma cells. U-118MG and SHG-44 cell lines were used to confirm these observations. The response of glioma cells to radiation involves the miR-212-mediated modulation of BRCA1 gene expression, suggesting that the miR-212/BRCA1 axis may play a potential role in the radiotherapy of gliomas.
放射抗拒仍是胶质瘤治疗的一大挑战，患者对放疗的反应差异很大。微小 RNA（miRNA）参与多种生物学过程。本研究旨在探讨与胶质瘤细胞系对辐射反应相关的 miRNA。γ射线照射后 30min 从人胶质瘤 U251 细胞中分离总 RNA，并与 miRNA 芯片杂交。采用实时定量 PCR 分析 miRNA 表达谱。pcDNA3/EGFP-miR-212 模拟物转染用于验证 miR-212 在集落形成试验中的功能，通过 Western blot 分析凋亡相关蛋白（Bcl-2、Bax、caspase-3 和细胞色素 c）检测 miR-212 过表达对 U251 细胞的影响。采用 miRNA 数据库等生物信息学工具预测 miR-212 的靶基因，并通过 EGFP 荧光报告和功能丧失与获得实验选择乳腺癌易感基因 1（BRCA1）进行进一步确认。在 16 个候选 miRNA 中，有 5 个通过实时 PCR 进行评估；miR-212 被鉴定为导致胶质瘤细胞放射抗性，并被证明可减弱辐射诱导的细胞凋亡。miR-212 通过与其 3'-非翻译区相互作用负调控 BRCA1 表达，表明 BRCA1 表达与胶质瘤细胞的放射敏感性之间存在相关性。U-118MG 和 SHG-44 细胞系用于证实这些观察结果。胶质瘤细胞对辐射的反应涉及 miR-212 介导的 BRCA1 基因表达的调节，表明 miR-212/BRCA1 轴可能在胶质瘤的放疗中发挥潜在作用。
