Objective: To investigate the expressions of Nod-like receptor protein 3 (NLRP3), cysteine-aspartic acid protease 1 (Caspase-1), and Gasdermin D (GSDMD) in nasopharyngeal carcinoma (NPC), and their relationships with the recurrence and metastasis of NPC. Methods: A retrospective study was conducted on 421 patients diagnosed with NPC between December 2014 and January 2020. The expressions of NLRP3, Caspase-1, and GSDMD in pathological specimens were examined with immunohistochemistry and multiplex immunofluorescence staining. Univariate and multivariate Cox regression analyses were applied to identify the factors influencing NPC recurrence and metastasis. In vitro experiments with NPC cell line HNE-2 were used to explore the functional mechanisms of NLRP3, Caspase-1, and GSDMD. Results: Multivariate Cox analysis revealed that tumor staging of Ⅲ-Ⅳ(HRrecurrence=2.74, 95%CIrecurrence: 1.61-4.65; HRmetastasis=1.90, 95%CImetastasis: 1.04-3.49) and pre-treatment plasma EBV-DNA levels≥1 500 copies/ml (HRrecurrence=1.91, 95%CIrecurrence: 1.13-3.23; HRmetastasis=2.07, 95%CImetastasis: 1.23-3.50)were independent risk factors for NPC recurrence and metastasis, while positive expression of NLRP3(HRrecurrence=0.17, 95%CIrecurrence: 0.08-0.35; HRmetastasis=0.30, 95%CImetastasis: 0.15-0.59), Caspase-1(HRrecurrence=0.32, 95%CIrecurrence: 0.18-0.59; HRmetastasis=0.43, 95%CImetastasis: 0.25-0.76), and GSDMD(HRrecurrence=0.48, 95%CIrecurrence: 0.25-0.91; HRmetastasis=0.96, 95%CImetastasis: 0.53-1.74) served as independent protective factors. Age (HR=1.02, 95%CI: 1.01-1.04) and intensity-modulated radiotherapy (HR=0.51, 95%CI: 0.30-0.88) were independent factors for NPC recurrence, whereas chemotherapy (HR=0.50, 95%CI: 0.29-0.88) acted as an independent protective factor for NPC metastasis (all P<0.05). NPC patients with positive expressions of the three proteins had higher locoregional recurrence-free survival, distant metastasis-free survival, and overall survival compared to those with negative expressions (all P<0.05). In vitro experiments revealed that the overexpression of NLRP3 activated the NLRP3/Caspase-1/GSDMD signaling pathway, as evidenced by Western Blot analysis. Enzyme-linked immunosorbent assay and scanning electron microscopy demonstrated that overexpression of NLRP3 promoted pyroptosis in HNE-2 cells. Cellular functional assays further confirmed that overexpression of NLRP3 significantly inhibited the proliferation, invasion, and migration of HNE-2 cells. Conclusion: Positive expressions of NLRP3, Caspase-1, and GSDMD serves as independent protective factors for recurrence and metastasis of NPC, potentially by promoting cell pyroptosis and thus inhibiting NPC cell proliferation, invasion, and migration.
目的： 探究NOD样受体热蛋白结构域相关蛋白3（Nod-like receptor protein 3，NLRP3）、半胱天冬氨酸蛋白酶1（cysteine-aspartic acid protease 1，Caspase-1）和消皮素D（Gasdermin D，GSDMD）在鼻咽癌（nasopharyngeal carcinoma，NPC）中的表达以及其同NPC复发转移的关系。 方法： 回顾性分析2014年12月至2020年1月黄石市中心医院确诊为NPC的421例病例资料，其中男262例，女159例，年龄18~88岁。通过免疫组化和多重免疫荧光染色法观察NLRP3、Caspase-1和GSDMD在病理标本中的表达。结合随访资料，应用单因素和多因素Cox回归分析NPC复发转移的影响因素。此外，使用NPC细胞系HNE-2进行体外实验，探究NLRP3、Caspase-1和GSDMD的功能机制。 结果： 多因素分析显示，Ⅲ~Ⅳ期肿瘤分期（HR复发=2.74，95%CI复发：1.61~4.65；HR转移=1.90，95%CI转移：1.04~3.49）和治疗前血浆EBV-DNA≥1 500 copies/ml（HR复发=1.91，95%CI复发：1.13~3.23；HR转移=2.07，95%CI转移：1.23~3.50）是NPC患者发生复发和转移的独立危险因子，而NLRP3（HR复发=0.17，95%CI复发：0.08~0.35；HR转移=0.30，95%CI转移：0.15~0.59）、Caspase-1（HR复发=0.32，95%CI复发：0.18~0.59；HR转移=0.43，95%CI转移：0.25~0.76）和GSDMD（HR复发=0.48，95%CI复发：0.25~0.91；HR转移=0.96，95%CI转移：0.53~1.74）的阳性表达则是独立保护因子；年龄（HR=1.02，95%CI：1.01~1.04）和调强放疗（HR=0.51，95%CI：0.30~0.88）是NPC患者复发独立影响因子，化疗（HR=0.50，95%CI：0.29~0.88）则是NPC转移的独立保护因子（P值均<0.05）。三者阳性表达的NPC患者局部-区域性无复发生存期、远处无转移生存期以及总生存期均高于三者阴性表达者（P值均<0.05）。体外实验中，蛋白免疫印迹法（WB）分析揭示，NLRP3的过表达可以激活NLRP3/Caspase-1/GSDMD信号通路。酶联免疫吸附试验和扫描电镜显示，NLRP3的过表达能促进HNE-2细胞的焦亡。细胞功能分析进一步证实，NLRP3的过表达能显著抑制HNE-2细胞的增殖、侵袭和转移能力。 结论： NLRP3、Caspase-1和GSDMD表达阳性是NPC患者复发转移的独立保护因子，能够通过促进细胞焦亡发生而抑制NPC增殖、侵袭和转移。.