PO.ET08.01 · 实验与分子治疗
靶向替代末端连接可克服miR-21-5p介导的口腔鳞状细胞癌放射抵抗
Targeting alternative end-joining overcomes miR-21-5p -mediated radioresistance in oral squamous cell carcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
放射抵抗仍然是口腔鳞状细胞癌（OSCC）有效放疗的主要障碍，但其分子基础尚未完全阐明。在此，我们揭示了替代末端连接（alt-EJ）DNA修复通路的激活是miR-21-5p介导的OSCC放射抵抗的关键驱动因素。对放射抵抗性OSCC克隆进行的全面miRNA分析揭示了独特的表达特征，其标志为miR-21-5p和miR-486-5p的强烈上调，同时伴有miR-320b和miR-1248的降低。功能实验（包括克隆形成存活实验和同基因小鼠肿瘤模型）表明，强制表达miR-21-5p显著增强了放射抵抗，并伴随持续的DNA损伤应答信号传导和双链断裂（DSB）修复能力的提高。对TCGA-HNSC队列的转录组学和基因组学分析显示，高miR-21-5p表达与经典DNA修复靶点的抑制、不良的放疗结局、升高的肿瘤突变负荷（TMB）以及微同源性介导的插入/缺失频率增加相关，这与alt-EJ活性增强相一致。在独立的OSCC数据集和本地肿瘤样本组中的交叉验证证实，miR-21-5p过表达的肿瘤表现出alt-EJ基因程序的强烈激活，包括PARP1、POLQ及相关辅助因子的上调。对多种癌症细胞系数据库的分析进一步确立了miR-21-5p活性与alt-EJ基因表达呈正相关，且不受微环境影响。在机制上，miR-21-5p过表达使DSB修复的依赖性从同源重组和经典非同源末端连接转向alt-EJ。对alt-EJ组分（PARP1或POLQ）的药理学或遗传学抑制破坏了DSB的修复，并有效恢复了miR-21-5p高表达OSCC细胞的放射敏感性。值得注意的是，在POLQ缺陷细胞中，PARP抑制介导的放射增敏作用被消除，证实了一种POLQ依赖性机制。在体内，miR-21-5p驱动的OSCC肿瘤表现出alt-EJ特征的增强表达、放射抵抗增加和高TMB。在同基因小鼠模型中，照射与PARP阻断的联合治疗抑制了肿瘤生长，降低了alt-EJ基因表达，并延长了生存期。总之，这些发现确立了一条新的miR-21-5p/alt-EJ信号轴，作为OSCC放射抵抗的核心决定因素。靶向alt-EJ代表了一种有前景的治疗策略，可克服miR-21-5p介导的治疗失败，并提高头颈部癌的放疗疗效。
查看英文原文 English abstract
Radiation resistance remains a major barrier to effective radiotherapy in oral squamous cell carcinoma (OSCC), yet its molecular basis is incompletely defined. Here, we uncover activation of the alternative end‑joining (alt‑EJ) DNA repair pathway as a key driver of miR‑21‑5p-mediated radioresistance in OSCC. Comprehensive miRNA profiling of radioresistant OSCC clones revealed distinct expression signatures marked by robust upregulation of miR‑21‑5p and miR‑486‑5p, coupled with reduced miR‑320b and miR‑1248. Functional assays, including clonogenic survival and syngeneic mouse tumor models, demonstrated that enforced expression of miR‑21‑5p markedly enhanced radioresistance, accompanied by sustained DNA damage response signaling and improved double‑strand break (DSB) repair capacity. Transcriptomic and genomic analyses of the TCGA‑HNSC cohort revealed that high miR‑21‑5p expression correlates with suppression of canonical DNA repair targets, poor radiotherapy outcomes, elevated tumor mutational burden (TMB), and an increased frequency of microhomology‑mediated insertions/deletions, consistent with heightened alt‑EJ activity. Cross‑validation in independent OSCC datasets and local tumor panels confirmed that miR‑21‑5p-overexpressing tumors exhibit strong activation of alt‑EJ gene programs, including upregulation of PARP1, POLQ, and associated accessory factors. Analysis of diverse cancer cell line databases further established that miR‑21‑5p activity positively correlates with alt‑EJ gene expression independent of microenvironmental influence. Mechanistically, miR‑21‑5p overexpression shifted DSB repair dependence from homologous recombination and classical non‑homologous end‑joining toward alt‑EJ. Pharmacologic or genetic suppression of alt‑EJ components (PARP1 or POLQ) disrupted DSB resolution and effectively restored radiosensitivity in miR‑21‑5p-high OSCC cells. Notably, radiosensitization by PARP inhibition was abrogated in POLQ‑deficient cells, confirming a POLQ‑dependent mechanism. In vivo, miR‑21‑5p-driven OSCC tumors exhibited enhanced expression of alt‑EJ signatures, increased radioresistance, and high TMB. Combination treatment with irradiation and PARP blockade suppressed tumor growth, diminished alt‑EJ gene expression, and prolonged survival in a syngeneic mouse model. Collectively, these findings establish a novel miR‑21‑5p/alt‑EJ signaling axis as a central determinant of radioresistance in OSCC. Targeting alt‑EJ represents a promising therapeutic strategy to overcome miR‑21‑5p-mediated treatment failure and improve radiotherapy efficacy in head and neck cancers.
利益披露 Disclosure
L. Ma, None..
N. Tayier, None..
W. Hu, None..
J. Chen, None..
Y. Li, None..
L. Wen, None..
Y. Luo, None..
X. Li, None..
Q. Liu, None.