摘要
目的探究Runt相关转录因子1(Runt-related transcription factor 1,RUNX1)在鼻息肉(nasal polyps,NPs)组织中的表达差异,并分析RUNX1在NPs原代人鼻黏膜上皮细胞(primary human nasal epithelial cells,pHNECs)凋亡中的作用及调控机制。方法以免疫蛋白印迹(WB)和免疫组织化学染色检测组织层面RUNX1的表达水平。采用肿瘤坏死因子α(TNF-α,20 ng/ml)体外诱导pHNECs发生凋亡,进行Hoechst染核观察细胞凋亡。随后,通过实时荧光定量反转录聚合酶链反应(qRT-PCR)和WB检测凋亡相关蛋白B淋巴细胞瘤基因2(B-cell lymphoma-2,BCL-2);BCL2相关蛋白质X(BCL2-associated X,BAX);含半胱氨酸的天冬氨酸蛋白水解酶3(cysteinyl aspartate specific proteinase-3,Caspase-3)的表达,评估凋亡水平。将sh-RUNX1-6质粒转染至pHNECs凋亡模型,用WB和流式细胞术评估RUNX1沉默对凋亡的影响。通过SPSS 19.0软件和GraphPad Prism 5软件进行统计学分析。结果NPs组织中RUNX1的表达明显高于正常下鼻甲组织,差异具有统计学意义(0.274±0.042比0.110±0.027,t=9.675,P<0.05)。与下鼻甲组织相比,NPs中BAX和Caspase-3高表达,而BCL-2低表达,差异具有统计学意义(BAX 0.346±0.032比0.302±0.037,Caspase-30.228±0.061比0.158±0.065,BCL-20.090±0.047比0.276±0.057,t值分别为2.680、2.361、7.575,P值均<0.05)。TNF-α诱导后pHNECs中RUNX1的蛋白、mRNA表达和凋亡水平均呈时间依赖性升高,差异具有统计学意义(P值均<0.05)。sh-RUNX1-6质粒转染能够下调TNF-α刺激下pHNECs中RUNX1的表达。沉默TNF-α刺激下pHNECs中的RUNX1后,细胞中BAX和Caspase-3的蛋白水平降低,而BCL-2表达增高,细胞凋亡率降低,差异有统计学意义(P值均<0.05)。结论RUNX1在NPs组织中高表达,沉默RUNX1能够抑制TNF-α诱导的pHNECs凋亡,从而减轻细胞炎症损伤。
Objective To explore the expression of Runt-related transcription factor 1(RUNX1)in nasal polyps(NPs)tissues and the potential role on apoptosis of primary human nasal epithelial cells(pHNECs)in NPs.Methods The expression level of RUNX1 in NPs tissues was determined by Western blot(WB)and immunohistochemical staining(IHC).In vitro,TNF-α(20 ng/ml)was used to stimulate pHNECs to establish the apoptosis injury model.Hoechst staining was performed to observe pHNECs apoptosis by kit.Subsequently,quantitative real-time PCR(qRT-PCR)and WB were utilized to detect the expression of apoptosis-related proteins B-cell lymphoma-2(BCL-2),BCL2-associated X(BAX)and cysteinyl aspartate specific proteinase-3(Caspase-3)to assess the level of apoptosis.The plasmid of sh-RUNX1-6 was transfected into the pHNECs apoptosis model,then the effect of RUNX1 silence on apoptosis was evaluated by WB and flow cytometry.Statistical analysis was performed by the SPSS 19.0 and GraphPad Prism5 software.Results The expression of RUNX1 in NPs tissue was significantly higher than that in inferior turbinates,and the difference was statistically significant(0.274±0.042 vs 0.110±0.027,t=9.675,P<0.05).Compared with the inferior turbinates,BAX and Caspase-3 expressions were increased whereas BCL-2 was decreased in NPs,and the differences were statistically significant(BAX 0.346±0.032 vs 0.302±0.037,Caspase-30.228±0.061 vs 0.158±0.065,BCL-20.090±0.047 vs 0.276±0.057,t value was 2.680,2.361 and 7.575,respectively,all P<0.05).The expression levels of RUNX1 and apoptosis in pHNECs increased in a time-dependent manner after TNF-αexposure(P<0.05).Plasmid of sh-RUNX1-6 transfected silenced the expression of RUNX1 in pHNECs treated by TNF-α.After silencing RUNX1 in pHNECs apoptosis model,the protein levels of BAX and Caspase-3 were decreased,while the expression of BCL-2 was increased,the rate of apoptosis was decreased(P<0.05).Conclusions RUNX1 is increased in NPs.Silencing RUNX1 can inhibit the apoptosis and reduce cell inflammatory damage of pHNECs
作者
裴银银
黄丹怡
张婷
张薇
张洁
张少聪
类云
周勇
程雷
陈静
Pei Yinyin;Huang Danyi;Zhang Ting;Zhang Wei;Zhang Jie;Zhang Shaocong;Lei Yun;Zhou Yong;Cheng Lei;Chen Jing(Department of Otorhinolaryngology Head and Neck Surgery,Affiliated Hospital of Nantong University,Institute of Otorhinolaryngology Head and Neck Surgery,Affiliated Hospital of Nantong University,Nantong 226001,Jiangsu Province,China;Clinical College,Medical School of Nantong University,Nantong 226001,Jiangsu Province,China;Department of Otorhinolaryngology,The First Affiliated Hospital,Nanjing Medical University,Clinical Allergy Center,The First Affiliated Hospital,Nanjing Medical University,International Center for Allergy Research,Nanjing Medical University,Nanjing 210029,China)
出处
《中华耳鼻咽喉头颈外科杂志》
CSCD
北大核心
2021年第12期1328-1335,共8页
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
基金
吴阶平医学基金会临床研究项目(320.6750.18272)。
