Department of Effects and Risks of Ionizing & Non-Ionizing Radiation, Federal Office for Radiation Protection (BfS), Neuherberg, Germany.
Cell Commun Signal. 2024 Oct 2;22(1):471. doi: 10.1186/s12964-024-01845-x.
Adverse effects of ionizing radiation on normal tissues limit the radiation dose in cancer treatment, thereby compromising treatment efficiency. Among the consistently affected non-cancer cells, peripheral blood mononuclear cells (PBMCs) exhibit high radiosensitivity and have the potential to induce systemic effects. PBMC-released extracellular vesicles (EVs), contribute to the communication of such systemic effects. This study aimed to investigate the effects of ionizing radiation on EVs as part of the systemic response of PBMCs in terms of microRNA cargo and biological functions.Therefore, whole blood samples from healthy donors were irradiated ex-vivo (0 Gy, 1 Gy, 2 Gy, 4 Gy) and EVs from PBMCs were isolated after 96 h by PEG precipitation or ultracentrifugation. Candidate microRNAs were examined in PBMC-derived EVs from individual donors. The uptake of membrane-stained fluorescent EVs by different recipient cells was quantified by fluorescence-activated cell sorting analysis. The biological effects of increased miR-34a-5p and of total EVs on recipient cells were assessed.Irradiation of PBMCs induced a dose-dependent upregulation of miR-34a-5p within EVs and PBMCs. However, interindividual differences between donors were noticed in the extent of upregulation, and small EVs displayed more pronounced changes in microRNA levels in comparison to large EVs. Irradiation in presence of the small molecule inhibitor KU-60019 demonstrated that this upregulation is dependent on ATM (Ataxia telangiectasia mutated) activation. Moreover, fibroblasts and keratinocytes were identified as preferred EV recipients. Increased miR-34a-5p levels led to a significant reduction in viability and induction of senescence in keratinocytes but not in fibroblasts, indicating a cell type-specific response.In conclusion, this study further elucidated the complex cellular response of normal tissue after radiation exposure. It confirmed radiation-induced modifications of microRNA expression levels in EVs from PBMCs and identified a robust upregulation of miR-34a-5p in the small EV subfraction, suggesting this microRNA as a potential novel candidate for the development of biomarkers for radiation exposure. Moreover, the different uptake efficiencies observed among specific cell types suggested that EVs induce cell type-specific responses in the intercellular communication of systemic radiation effects.
电离辐射对正常组织的不良影响限制了癌症治疗中的辐射剂量，从而降低了治疗效率。在持续受影响的非癌细胞中，外周血单核细胞 (PBMC) 表现出高放射敏感性，并具有诱导全身效应的潜力。PBMC 释放的细胞外囊泡 (EVs) 有助于这种全身效应的交流。本研究旨在研究电离辐射对 EVs 的影响，作为 PBMC 作为全身反应的一部分，从 miRNA 货物和生物学功能方面进行研究。
因此，从健康供体中采集全血样本进行离体照射 (0 Gy、1 Gy、2 Gy、4 Gy)，并在 96 小时后通过 PEG 沉淀或超速离心从 PBMC 中分离 EVs。从个体供体的 PBMC 衍生 EV 中检查候选 microRNAs。通过荧光激活细胞分选分析定量测量不同受体细胞对膜染色荧光 EV 的摄取。通过增加 miR-34a-5p 和总 EVs 对受体细胞的生物学效应来评估其生物效应。
照射 PBMC 会导致 EVs 和 PBMC 中 miR-34a-5p 的剂量依赖性上调。然而，供体之间的个体差异在上调程度上被注意到，并且与大 EV 相比，小 EV 显示出更明显的 miRNA 水平变化。在小分子抑制剂 KU-60019 的存在下进行照射表明，这种上调依赖于 ATM（共济失调毛细血管扩张突变）的激活。此外，鉴定出成纤维细胞和角质形成细胞为首选的 EV 受体。增加的 miR-34a-5p 水平导致角质形成细胞活力显著降低和衰老诱导，但对成纤维细胞没有影响，表明存在细胞类型特异性反应。
总之，本研究进一步阐明了正常组织在辐射暴露后的复杂细胞反应。它证实了 PBMCs 中的 EV 中辐射诱导的 microRNA 表达水平的修饰，并在小 EV 亚群中鉴定出 miR-34a-5p 的强大上调，表明该 microRNA 作为辐射暴露生物标志物开发的潜在新候选物。此外，在特定细胞类型之间观察到的不同摄取效率表明，EVs 在全身辐射效应的细胞间通讯中诱导细胞类型特异性反应。
