Preliminary Study on 53BP1-Mediated DNA Double-Strand Break Response in Spermatogonial Stem Cells
Preliminary Study on 53BP1-Mediated DNA Double-Strand Break Response in Spermatogonial Stem Cells
复制标题
DOI：
10.1007/s43032-022-01122-7
复制
发表时间：
2022-11
影响因子：
2.9
通讯作者：
W. Le;Fan Zhou;J. Xiang;Yiming Weng;Denglong Wu;Jun Xu;Jinfu Zhang
中科院分区：
文献类型：
--
作者：
W. Le;Fan Zhou;J. Xiang;Yiming Weng;Denglong Wu;Jun Xu;Jinfu Zhang
53BP1在体细胞中介导DNA修复过程，但53BP1在生殖系干细胞中的功能仍不清楚。本研究通过辐射和HU处理建立动物和细胞DNA损伤修复(DDR)模型，用免疫荧光染色和激光共聚焦显微镜检测53BP1、p-Chk2和p-p53在mSSCs损伤修复过程中的表达。建立Trp53bp1-shRNA下调表达53BP1的mSSCs细胞系，EDU染色分析细胞周期和细胞增殖情况。此外，应用NHEJ报告载体检测Trp53bp1表达下调(KD)后的修复效果。结果表明，53BP1在体内和体外的DDR过程中均可在小鼠骨髓间充质干细胞内形成焦点信号，且不依赖于γ-H_2AX。53BP1下游蛋白、p-p53和p-Chk2参与并动态表达于DDR反应中。下调mSSCs中Trp53bp1的表达不能显著抑制细胞的增殖，但可能增加细胞对HU的敏感性。流式细胞仪分析显示Trp53BP-KD SSCs的NHEJ修复效率显著降低。我们揭示了53BP1在SSCS DDR过程中的具体机制，有望为男性不育的诊断和治疗提供新的理论依据和见解。
53BP1 mediates DNA repair process in somatic cells; however, the function of 53BP1 in germline stem cells still remains unclear. In the present study, animals and cells DNA damage repair (DDR) model was established by irradiation and HU treatment; immunofluorescence staining and laser confocal microscopy were used to detect the expression of 53BP1, p-CHK2, and p-P53 in the DDR process of mSSCs. 53BP1 knockdown expression mSSCs cell line conducted byTrp53bp1-shRNA was established and EdU staining was adopted to analyze cell cycle and cell proliferation. Moreover, NHEJ reporter vector was applied to detect the repair efficacy afterTrp53bp1knocked-down (KD) expression. Results showed that 53BP1 could form foci signals in mSSCs during DDR process both in vivo and in vitro, which was independent of γH2AX. 53BP1 downstream protein, p-P53, and p-CHK2 were involved and dynamically expressed in DDR response. Knocking down ofTrp53bp1expression in mSSCs could not dramatically inhibit cell proliferation, but may increase cell sensitivity to HU. The NHEJ repair efficacy was sharply decreased inTrp53bp-KD SSCs via flow cytometry analysis. We revealed the specific mechanism of 53BP1 in SSCs DDR process, which is expected to provide a new theoretical basis and insights for the diagnosis and treatment of male infertility.