摘要
目的:探究Zeste同源物2增强子(EZH2)对非小细胞肺癌(NSCLC)细胞生长和细胞因子表达的影响及机制。方法:免疫组化染色观察NSCLC组织及肿瘤边缘正常组织内EZH2蛋白表达,实时荧光定量聚合酶链式反应(qRT-PCR)测定人NSCLC细胞系与正常肺上皮细胞系内EZH2 mRNA表达。将A549细胞分为对照组、GSK126组、ROS抑制剂(NAC)+GSK126组、SB203580+GSK126组,进行相应处理后,MTT法、EdU染色及细胞克隆形成实验检测各组细胞增殖情况,酶联免疫吸附法(ELISA)测定各组细胞培养液上清血管内皮生长因子(VEGF)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)含量,DCFH-DA荧光探针法检测各组细胞内活性氧(ROS)水平,蛋白质免疫印迹(Western blot)实验测定各组细胞内p38丝裂原活化蛋白激酶(p38 MAPK)磷酸化水平。结果:NSCLC组织内EZH2阳性表达率显著高于肿瘤边缘正常组织(P<0.05),人NSCLC细胞系A549、HCC827、H1975、H1299、SPC-A-1中EZH2 mRNA相对表达量显著高于正常肺上皮细胞系BEAS-2B(P<0.05)。与对照组比较,GSK126组细胞存活率、EdU阳性率显著降低(P<0.05),细胞克隆形成数目显著减少(P<0.05),细胞培养液上清中免疫因子VEGF、IFN-γ、TNF-α含量均显著减少(P<0.05),而细胞内ROS水平及p38 MAPK磷酸化水平均显著升高(P<0.05)。与GSK126组比较,NAC+GSK126组和SB203580+GSK126组细胞存活率、EdU阳性率显著升高(P<0.05),细胞克隆形成数目显著增加(P<0.05),细胞培养液上清中VEGF、IFN-γ、TNF-α含量均显著增加(P<0.05),同时,细胞内ROS水平、p38 MAPK磷酸化水平均显著下降(P<0.05)。结论:EZH2在NSCLC中高表达,并可促进肿瘤细胞生长与VEGF、IFN-γ、TNF-α分泌,该作用机制可能与调控ROS/p38 MAPK磷酸化途径有关。
Objective:To explore the effect and mechanism of enhancer of Zeste homolog 2(EZH2)on cell growth and cytokine expression in non-small cell lung cancer(NSCLC).Methods:EZH2 protein expression in NSCLC tissues and normal tissues at tumor margins was observed by immunohistochemical staining.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to determine EZH2 mRNA expression in human NSCLC cell lines and normal lung epithelial cell line.A549 cells were divided into control group,GSK126 group,ROS inhibitor(NAC)+GSK126 group,SB203580+GSK126 group.After the corresponding treatment,MTT assay,EdU staining and cell clonal formation assay were used to detect the cell proliferation in each group.The contents of vascular endothelial growth factor(VEGF),interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)in the supernatant of cell culture medium in each group were determined by enzyme-linked immunosorbent assay(ELISA).DCFH-DA fluorescence probe was used to detect the levels of intracellular reactive oxygen species(ROS),and the phosphorylation levels of p38 mitogen activated protein kinase(p38 MAPK)were determined by Western blot.Results:The positive expression rate of EZH2 in NSCLC tissues was significantly higher than that in tumor margin normal tissues(P<0.05).The relative expression level of EZH2 mRNA in human NSCLC cell lines A549,HCC827,H1975,H1299 and SPC-A-1 was significantly higher than that in normal lung epithelial cell line BEAS-2B(P<0.05).Compared with the control group,the cell survival rate and the positive rate of EdU in GSK126 group were significantly decreased(P<0.05),the number of cell clone formation was significantly decreased(P<0.05),the contents of immune factors VEGF,IFN-γand TNF-αin the supernatant of cell culture medium were significantly decreased(P<0.05),but the intracellular ROS levels and phosphorylation levels of p38 MAPK were significantly increased(P<0.05).Compared with GSK126 group,the cell survival rate and the positive rate of EdU in NAC+GSK126 group and SB203580+GSK126 group were significantly increased(P<0.05),and the number of cell clones formed was significantly increased(P<0.05),the contents of immune factors VEGF,IFN-γand TNF-αin the supernatant of cell culture medium were significantly increased(P<0.05),while the levels of intracellular ROS and phosphorylation of p38 MAPK were significantly decreased(P<0.05).Conclusion:EZH2 is highly expressed in NSCLC and promotes the growth of tumor cells and the secretion of VEGF,IFN-γ,TNF-α,which may be related to the regulation of ROS/p38 MAPK phosphorylation pathway.
作者
李瑶
呼群
LI Yao;HU Qun(Department of Oncology,Affiliated Hospital of Inner Mongolia Medical University,Inner Mongolia Hohhot 010050,China)
出处
《现代肿瘤医学》
CAS
北大核心
2023年第20期3715-3721,共7页
Journal of Modern Oncology
基金
内蒙古自治区自然科学基金项目(编号:2021LHMS08046)。
