Analysis of miRNA and mRNA Expression Profiles Highlights Alterations in Ionizing Radiation Response of Human Lymphocytes under Modeled Microgravity

BackgroundIonizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. Methodology/Principal FindingsWe analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of “Response to DNA damage” is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. Conclusions/SignificanceOn the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL.**背景** 电离辐射（Ionizing Radiation, IR）对人体细胞具有极强危害，因为机体对IR产生的DNA损伤应答（DNA-damage response, DDR）异常可诱发癌变起始。DDR通路的紊乱可源于微小RNA（microRNAs, miRNAs）介导的基因调控功能异常，而miRNAs是一类小型非编码RNA，可作为基因表达的转录后调控因子。本研究旨在阐明微重力条件下miRNAs在IR诱导DDR调控中的作用：微重力是宇航员在太空任务中经历的失重环境，该环境可对细胞产生协同效应，进一步增加辐射暴露的风险。 **方法与主要结果** 我们对经0.2 Gy与2 Gy γ射线照射后的人外周血淋巴细胞（peripheral blood lymphocytes, PBL）进行体外培养，分别在常重力（1 g）与模拟微重力（modeled microgravity, MMG）环境中孵育4小时与24小时，并分析其miRNA表达谱。结果显示，模拟微重力可通过减少辐射应答性miRNA的数量，改变受辐照PBL的miRNA表达特征。此外，let-7i*、miR-7、miR-7-1*、miR-27a、miR-144、miR-200a、miR-598、miR-650的表达均受辐射与模拟微重力的联合作用而失调。对同一供体PBL的miRNA与mRNA表达谱进行整合分析后，我们发现DDR通路存在显著的miRNA-mRNA负相关关系。基因本体（Gene Ontology, GO）富集分析显示，当PBL在常重力环境中孵育时，"Response to DNA damage"这一生物过程显著富集，而在模拟微重力环境中则无此富集现象。此外，p53通路的部分负相关基因在常重力与模拟微重力环境中的表达水平存在显著差异。利用荧光素酶报告基因构建体开展的功能验证实验，证实了通过靶基因预测分析得到的miRNA-mRNA互作关系。 **结论与意义** 总体而言，通过整合转录组（transcriptome）与microRNA组（microRNome）数据，我们证实模拟微重力可影响人PBL对IR的DNA损伤应答。