Long-term gene expression changes in irradiated male and female rhesus macaque

Purpose: Long-term transcriptome changes can be expected in survivors after lethal irradiation. We aimed to characterize these changes in males and females and after different cytokine treatments. Material and Methods: Peripheral blood was drawn in male and female rhesus macaque (n=142), which survived whole-body exposure with 700 cGy (LD66/60). Peripheral whole blood was drawn pre-exposure and before sacrificing the surviving animals after 60 days. Results: We evaluated gene expression in a three-phase study design where phase I was a whole-genome screening (NGS) for mRNAs using five pre- and post-exposure RNA samples (n=20). Differential gene expression (DGE) was calculated separately for each sex between samples of survivors and pre-exposure samples, utilized as the reference. Altogether 1,243 up- and down-regulated genes were identified. Altogether 37 candidate mRNAs were chosen for validation in phase II using the remaining samples (n=117) and utilizing qRT-PCR. Altogether 17 genes showed significant or borderline significant (t-test) DGE in groups of untreated or treated males and females, but nine most promising genes (CD248, EDAR, FAM19A5, GAL3ST4, GCNT4, HBG2/1, LRRN1, NOG, SYT14). For phase III, we validated our 41 rhesus macaque candidate genes with results from an earlier conducted study on male baboons. Altogether 34% (14 out of 41) genes showed a concordantly DGE in males of both species (DGE defined as (FC) > |1.5|). Conclusions: 60 days after radiation exposure, we identified (1) cytokine treatment independent long-term transcriptional changes, (2) females with almost twice as much deregulated genes appeared more radio-responsive than males on gene expression level but interestingly did not show a correlation between hematopoietic nadirs and sex in corresponding clinical data and (3) Panther analysis revealed a strong association with immunological processes as well as the WNT pathway for both sexes, highlighting a possible correlation of secondary malignancy development due to immunological alterations.研究目的：致死剂量辐射后存活个体可出现长期转录组变化，本研究旨在解析雌雄个体经不同细胞因子处理后的此类变化特征。材料与方法：选取142只经700 cGy（LD66/60）全身照射后存活的雌雄恒河猴，分别于照射前及照射后60天处死存活动物前采集外周全血样本。研究结果：本研究采用三阶段实验设计开展基因表达分析：第一阶段为全基因组mRNA测序（NGS）筛选，使用5组照射前后的RNA样本，总样本量为20；以照射前样本作为对照，分别按雌雄性别计算存活个体与对照样本间的差异基因表达（Differential Gene Expression, DGE），共鉴定出1243个上调及下调基因。第二阶段选取37个候选mRNA，使用剩余的117份样本通过实时荧光定量PCR（qRT-PCR）进行验证；最终在未处理或经细胞因子处理的雌雄个体组中，共鉴定出17个具有显著或临界显著（t检验）差异的表达基因，并从中筛选出9个最具研究价值的靶基因：CD248、EDAR、FAM19A5、GAL3ST4、GCNT4、HBG2/1、LRRN1、NOG、SYT14。第三阶段，我们利用早前开展的雄性狒狒研究数据，对本研究中的41个恒河猴候选基因进行跨物种验证；结果显示，以折叠变化（Fold Change, FC）> |1.5|作为差异表达判定标准，共有34%（41个中的14个）基因在两种灵长类的雄性个体中呈现一致的差异表达趋势。研究结论：辐射暴露后60天，本研究明确了三类核心发现：（1）存在不依赖细胞因子处理的长期转录组变化；（2）雌性个体的差异表达基因数量约为雄性的两倍，在基因表达层面表现出比雄性更强的辐射应答敏感性，但对应临床数据中并未观察到造血功能最低点与性别存在相关性；（3）Panther富集分析显示，雌雄个体的差异表达基因均显著富集于免疫过程及WNT信号通路，提示辐射诱导的免疫功能异常可能与继发性恶性肿瘤发生存在潜在关联。