NLRP3炎性小体在胃癌细胞中的表达及对其焦亡、增殖、侵袭、迁移的影响

Expression of NLRP3 inflammasome in gastric cancer cells and its effect on the pyrophosis,proliferation,invasion and migration

目的:研究胃癌细胞中NLRP3炎性小体的表达及其对增殖、侵袭和迁移的影响。并初步探讨NLRP3介导caspase-1依赖的焦亡信号通路在胃癌发展中的作用。方法:qRT-PCR和Western blotting法分别检测人胃黏膜上皮细胞(GES-1)和人胃癌细胞(MKN45及MGC803)中NLRP3、caspase-1表达;随后下调NLRP3,检测胃癌细胞NLRP3、caspase-1 mRNA和蛋白的表达;Western blotting检测GSDMD、白细胞介素(IL)-18、IL-1β蛋白表达;ELISA法检测细胞培养液上清液中IL-1β、IL-18含量;乳酸脱氢酶(LDH)实验、流式细胞术检测细胞焦亡;CCK-8法及细胞周期试剂盒检测细胞增殖情况;Transwell和划痕实验检测胃癌细胞侵袭和迁移能力。结果:NLRP3、caspase-1在胃癌细胞中较GES-1表达增高(P<0.01)。下调NLRP3后,胃癌细胞LDH释放率及焦亡率显著降低(P<0.01);GSDMD、IL-1β、IL-18表达降低(P<0.01);细胞增殖阻滞在G1期(P<0.01);细胞增殖以及侵袭迁移能力显著减弱。结论:NLRP3在胃癌中高表达,其表达影响胃癌细胞的增殖、侵袭和迁移以及其介导的caspase-1依赖焦亡信号通路关键蛋白的表达。

Objective:To investigate the expression of NLRP3 inflammasome in gastric cancer(GC)and its effects on the proliferation,invasion and migration of cells,and the role of NLRP3-mediated caspase-1-dependent pyroptosis signaling pathway in the development of GC.Methods:The expression levels of NLRP3 and caspase-1 in human gastric epithelial cells(GES-1)and human gastric cancer cells(MKN45 and MGC803)were detected by qRT-PCR and Western blotting.After the expression level of NLRP3 was down-regulated,and the mRNA and protein expression levels of NLRP3 and caspase-1 were detected,the protein expression levels of GSDMD,IL-18 and IL-1βwere detected using Western blotting.The content of IL-1βand IL-18 in supernatant were detected by ELISA,the pyroptosis was detected by LDH and flow cytometry,and the cell proliferation was detected by CCK-8 assay and cell cycle kit.Transwell and wound healing were used to detect the invasion and migration of cells.Results:The expression levels of NLRP3 and caspase-1 in GC cells were higher than those in GES-1(P<0.01).After the expression level of NLRP3 was downregulated,the release LDH rate and pyroptosis rate of LDH significantly decreased(P<0.01),and the expression levels of GSDMD,IL-1βand IL-18 decreased(P<0.01).Compared with the NC group,the cell proliferation was arrested in G1 phase(P<0.01).The proliferation,invasion and migration of siNLRP3 cells were significantly reduced.Conclusions:The expression of NLRP3 in GC cells is high,and its expression affects the proliferation,invasion and migration of GC cells and expression of key proteins in caspase-1-dependent pyroptosis signaling pathway mediated by NLRP3.