【摘要】 目的 探讨泛素特异性蛋白酶21(USP21)介导的炎症小体(NLRP3)去泛素化对人鼻咽癌(NPC)细胞放疗敏感性的影响及作用机制。方法 相差显微镜成像评估细胞焦亡。克隆形成实验研究USP21和NLRP3对NPC细胞增殖的影响。qRT-PCR检测NLRP3 mRNA的表达，Western blot检测蛋白分子的表达，免疫共沉淀实验测定泛素化水平，异植瘤模型评价NLRP3在体对细胞焦亡及NPC放疗敏感性的影响。结果 IR引起NPC细胞出现焦亡的形态学特征，且NLRP3、ASC、Cl-caspase-1、IL-1β、IL-18、cl-caspase-3、cl-PARP及Cyto C表达明显升高(P<0.05)。敲低NLRP3减轻IR诱导的细胞焦亡形态学变化。过表达NLRP3诱导更多的焦亡小体出现，并降低细胞集落形成。体内实验表明，在无IR照射时，NLRP3上调对肿瘤荧光信号强度、肿瘤大小及肿瘤体积均无显著影响(P>0.05)。而IR处理后，NLRP3过表达组小鼠体内肿瘤荧光信号更弱，生长速度明显减慢，体积更小，且IR处理后过表达NLRP3的小鼠肿瘤组织中ASC、Cl-caspase-1、IL-1β和IL-18表达水平显著增加(P<0.05)。过表达USP21明显上调NLRP3表达并减少NLRP3的泛素化(P<0.05),而敲低USP21则下调了NLRP3的蛋白水平并增加NLRP3的泛素化(P<0.05)。敲低NLRP3可减弱USP21过表达对焦亡小泡形成的促进作用和集落形成的抑制作用(P<0.05)。结论 USP21介导的NLRP3去泛素化通过诱导焦亡增强人鼻咽癌细胞的放疗敏感性。更多还原

【Abstract】 Objective To explore the influence of ubiquitin-specific protease 21(USP21)-mediated nucleotide-binding domain(NOD)-like receptor(NLR) family member pyrin domain-containing protein 3(NLRP3) deubiquitination on nasopharyngeal carcinoma(NPC) radiosensitivity and its potential mechanism.Methods Pyroptosis was assessed by phase-contrast imaging. The regulatory effects of USP21 and NLRP3 on the proliferation of NPC cells were assessed by colony formation assay. Quantitative reverse-transcription polymerase chain reaction(qRT-PCR) was used to measure the mRNA expression NLRP3. Western blot was performed to analyze protein expressions.Coimmunoprecipitation assay was used to evaluate deubiquitination. A xenograft model was created to investigate the effect of NLRP3 on pyroptosis and radiosensitivity of NPC in vivo.Results Irradiation(IR) induced pyroptosis of NPC cells, and the expressions of NLRP3, ASC, Cl-caspase-1, IL-1β, IL-18, cl-caspase-3, cl-PARP and Cyto C were significantly upregulated(P<0.05). knockdown of NLRP3 relieved IR-induced morphological changes in pyroptosis of NPC cells.Overexpression of NLRP3 promoted the generation of more pyrogens and inhibited colony formation. In vivo experiments showed that NLRP3 overexpression had no significant effect on tumor fluorescence signal intensity, tumor size, and tumor volume without IR irradiation(P>0.05). However, overexpression of NLRP3 in IR-induced xenografts significantly inhibited the tumor fluorescence signal, growth rate, and tumor volume, but upregulated ASC, Cl-caspase-1, IL-1β and IL-18 in the tumor tissue(P<0.05). Overexpression of USP21 significantly upregulated NLRP3 and decreased NLRP3 ubiquitination, while USP21 knockdown downregulated NLRP3 and reduced NLRP3 ubiquitination(P<0.05). Knockdown of NLRP3 reduced the increase in pyrogens and reduction of colony formation induced by USP21 overexpression(P<0.05).Conclusion USP21-mediated NLRP3 deubiquitination enhances radiosensitivity in NPC by inducing pyroptosis.更多还原