USP39抑制肿瘤细胞放射敏感性的机制

Mechanism of USP39 inhibiting radiation sensitivity of tumor cells

目的探讨泛素特异性肽酶39(ubiquitin-specific peptidase 39,USP39)对肿瘤细胞DNA损伤应答通路的生物学作用。方法将不同肿瘤细胞系(293T,HeLa,U2OS,T47D)分别在含有100 mL/L FBS的DMEM或RPMI-1640培养基中,在37℃含有50 mL/L CO 2的培养箱中培养;用MTS[3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-etrazolium,inner salt]方法检测敲低USP39对肿瘤细胞放射敏感性的影响;流式细胞术检测同源重组(homologous recombination,HR)和非同源末端连接(non-homologous end joining,NHEJ)修复效率;Western blot检测敲低USP39对DNA损伤应答相关分子的表达情况;用免疫共沉淀、蛋白纯化及质谱技术,检测与USP39相互作用的蛋白并且进行基因本体论(gene ontology,GO)分析;通过微辐射诱导DNA损伤并利用激光共聚焦显微镜检测其募集至DNA损伤位点的情况。结果敲低USP39导致肿瘤细胞的放射敏感性显著增加(P<0.05);敲低USP39显著抑制肿瘤细胞的HR与NHEJ修复效率(P<0.05);敲低USP39能够促进DNA损伤应答因子蛋白的表达;USP39能够聚集至DNA损伤位点;USP39相互作用蛋白与多个DNA损伤应答相关的信号通路有关。结论USP39对DNA损伤应答过程具有重要作用。

Objective To investigate the biological effects of ubiquitin-specific peptidase 39(USP39)on the DNA damage response pathway of tumor cells.Methods Tumor cells(293T,HeLa,U2OS,T47D)were cultured in DMEM medium or RPMI-1640 containing 100 mL/L FBS in a humidified atmosphere containing 50 mL/L CO 2 at 37℃.The effect of knockdown of USP39 on the radiosensitivity of tumor cells was detected by MTS[3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-etrazolium,inner salt].The efficiency of HR repair and NHEJ repair was detected by cytometry.The expression of DNA damage-responsive proteins by knockdown of USP39 was examined by Western blot.The proteins interacting with USP39 were detected by co-immunoprecipitation,protein purification and mass spectrometry,and then gene ontology analysis was performed.DNA damage was induced by micro-irradiation and its recruitment to DNA damage sites was detected by agonistic confocal microscopy.Results Knockdown of USP39 resulted in increased radiosensitivity of tumor cells(P<0.05).Knockdown of USP39 inhibited homologous recombination and non-homologous end joining repair efficiency of tumor cells(P<0.05).Knockdown of USP39 promoted the expression of DNA damage response protein.USP39 aggregated to DNA damage sites;USP39 interacting proteins were involved in multiple signaling pathways associated with DNA damage response.Conclusion USP39 plays an important role in the DNA damage response.