RNA sequencing analysis of gene expression changes during the development of radiation proctitis in a mouse model

Background: Radiation proctitis (RP) is the most common complication of radiotherapy for pelvic tumor. Currently there is a lack of effective clinical treatment and its underlying mechanism is poorly understood. In this study, we aimed to dynamically reveal the mechanism of RP progression from the perspective of RNomics using a mouse model, so as to help develop reasonable therapeutic strategies for RP. Results: Mice were delivered a single dose of 25Gy rectal irradiation, and the rectal tissues were removed at 4 hours, 1 day, 3 days, 2 weeks and 8 weeks post-irradiation (PI) for both histopathological assessment and RNA-seq analysis. According to the histopathological characteristics, we divided the development process of our RP animal model into three stages: acute (4 hours, 1 day and 3 days PI), subacute (2 weeks PI) and chronic (8 weeks PI), which could recapitulate the features of different stages of human RP. Bioinformatics analysis of the RNA-seq data showed that in the acute injury period after radiation, the altered genes were mainly enriched in DNA damage response, p53 signaling pathway and metabolic changes; while in the subacute and chronic stages of tissue reconstruction, genes involved in the biological processes of vessel development, extracellular matrix organization, inflammatory and immune responses were dysregulated. We further identified the hub genes in the most significant biological process at each time point using protein-protein interaction (PPI) analysis and verified the differential expression of these genes by quantitative real-time-PCR (qRT-PCR) analysis. Conclusions: Our study revealed the molecular events sequentially occurred during the course of RP development and might provide molecular basis for designing drugs targeting different stages of RP development. Overall design: A total of 27 RNA samples from the rectal tissues of mice in both irradiated and control group at 4 hours, 1 day, 3 days, 2 weeks and 8 weeks post-irradiation were collected and subjected to high-throughput RNA sequencing. Three biological replicates of the RNA sample were prepared for each time point in both groups. The same control samples were shared for 4 hours and 1 day.

背景：放射性直肠炎（Radiation proctitis, RP）是盆腔肿瘤放疗最常见的并发症。目前临床尚缺乏有效的治疗手段，其潜在发病机制仍未明确。本研究以小鼠为模型，从RNA组学（RNomics）视角动态揭示RP的进展机制，以期为RP合理治疗策略的开发提供理论参考。
结果：对小鼠单次给予25Gy直肠照射，分别于照射后（post-irradiation, PI）4小时、1天、3天、2周及8周采集直肠组织，开展组织病理学评估与RNA测序（RNA-seq）分析。根据组织病理学特征，本研究将RP动物模型的病程划分为三个阶段：急性期（照射后4小时、1天及3天）、亚急性期（照射后2周）与慢性期（照射后8周），该模型可复现人类RP不同阶段的病理特征。对RNA测序数据的生物信息学分析显示，辐射后急性损伤期的差异表达基因主要富集于DNA损伤应答、p53信号通路及代谢改变过程；而在组织重构的亚急性与慢性阶段，参与血管发育、细胞外基质组织、炎症与免疫应答等生物学过程的基因表达失调。本研究进一步通过蛋白质相互作用（protein-protein interaction, PPI）分析筛选出各时间点最显著生物学过程的核心基因（hub genes），并采用实时定量聚合酶链反应（quantitative real-time-PCR, qRT-PCR）验证了这些基因的差异表达情况。
结论：本研究揭示了RP病程中依次发生的分子事件，可为针对RP不同发展阶段的靶向药物研发提供分子基础。
整体实验设计：本研究共收集照射组与对照组小鼠在照射后4小时、1天、3天、2周及8周的直肠组织RNA样本共计27份，进行高通量RNA测序。两组各时间点均设置3次生物学重复，其中照射后4小时与1天的对照样本共享。