Transcriptome Sequencing Analysis (RNA-Seq) of Bone Marrow Hematopoietic Stem Cells from Per2-mutant Mice

Circadian rhythm (CR) and environmental low-dose radiation (LDR) evident on Earth’s surface may have coordinated in the evolution of early life; disturbed CR and/or overexposure to radiation are linked with aging and many human diseases including cancer. Although CR affects tumor response and normal tissue radiosensitivity in cancer radiotherapy, it is unclear how CR proteins function in LDR induced radioprotection. Herein, we demonstrated that mice carrying a deficient Period2 gene (C57BL-Per2 def ), a core CR regulator, were highly sensitive to radiation. Compared to Per2 wt mice, the population of bone marrow hemopoietic stem cells (BMHSCs) was reduced and a cluster of genes responsible for DNA damage repair and mitochondrial metabolism was silenced in the Per2 def BMHSCs . Consistently, unlike Per2 wt human and mouse cells capable of inducible radioresistance by LDR, Per2 def cells failed to induce such adaptive protection. Furthermore, LDR enhanced PER2 protein level together with phosphorylated GSK3β (pGSK3β) in the Wnt/β-catenin signaling pathway. Interaction of PER2 with pGSK3β activated β -catenin that in turn transactivated Per2 and other prosurvival genes. These results provide new insights into how CR and LDR communicate in genotoxic stress response. The PER2/pGSK3β/β-catenin/Per2 pathway may serve as a therapeutic approach to reduce normal cell injury in cancer radiotherapy. A total of 2 samples were analyzed in this study. The study included 2 samples of mouse bone marrow hematopoietic stem cells (HMCs) obtained from wild-type (Per2wt) and Per2-deficient (Per2def). The bone marrow HMCs were enriched by cell sorting into culture medium, pelleted by centrifugation, and harvested,. Total RNA was isolated and then submitted for RNA-sequencing analysis.

地球表面普遍存在的昼夜节律（Circadian rhythm, CR）与环境低剂量辐射（Low-dose radiation, LDR）可能在早期生命演化过程中存在协同调控作用；昼夜节律紊乱及/或辐射过量暴露与衰老及包括癌症在内的多种人类疾病密切相关。尽管在癌症放疗中，昼夜节律会影响肿瘤应答与正常组织的辐射敏感性，但目前仍不清楚昼夜节律蛋白如何在低剂量辐射诱导的辐射防护中发挥功能。本研究证实，携带缺陷型周期2（Per2）基因（C57BL-Per2^def，一种核心昼夜节律调控因子）的小鼠对辐射具有极高敏感性。与Per2野生型（Per2 wt）小鼠相比，Per2缺陷型小鼠的骨髓造血干细胞（Bone marrow hemopoietic stem cells, BMHSCs）数量显著减少，且其BMHSCs中负责DNA损伤修复与线粒体代谢的基因簇表达被沉默。与之相一致的是，与可通过低剂量辐射诱导产生辐射抗性的Per2 wt人类及小鼠细胞不同，Per2 def细胞无法诱导出此类适应性防护效应。此外，低剂量辐射可同时上调PER2蛋白与Wnt/β-连环蛋白信号通路中磷酸化糖原合成激酶3β（pGSK3β）的表达水平。PER2与pGSK3β的相互作用可激活β-连环蛋白，后者继而反式激活Per2基因及其他促存活基因。本研究结果为解析昼夜节律与低剂量辐射在遗传毒性应激应答中的交互调控机制提供了全新视角。PER2/pGSK3β/β-连环蛋白/Per2通路有望成为降低癌症放疗中正常细胞损伤的潜在治疗靶点。本研究共分析了2份样本：实验获取了来自野生型（Per2wt）与Per2缺陷型（Per2def）小鼠的骨髓造血干细胞（Hematopoietic stem cells, HMCs）各1份。通过细胞分选将骨髓HMCs富集至培养基中，经离心沉淀后收集细胞，随后提取总RNA并进行RNA测序分析。