GPX4对肺腺癌干细胞特性及侵袭转移能力影响

Effects of GPX4 on the stem cells characteristics,invasion and metastasis of lung adenocarcinoma

目的肿瘤干细胞(cancer stem cells,CSCs)是影响肿瘤发生发展的关键因素,谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)作为机体内重要膜脂过氧化调控酶类,被发现与多种肿瘤恶性表型关系密切。本研究对GPX4在肺腺癌组织中的表达情况及其与肺腺癌干细胞特性及体外侵袭转移能力的关系进行了探讨。方法收集河南省胸科医院胸外科2017-06-01-2018-03-31肺腺癌及癌旁组织各46例,实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR)技术检测组织中GPX4mRNA及干细胞标志物CD133和Sox-2mRNA的表达情况,分析各基因mRNA在不同组织中的表达水平及相关性。蛋白质印迹法检测正常支气管上皮细胞及各肺腺癌细胞系中GPX4蛋白的表达,通过慢病毒转染空白对照载体及表达GPX4siRNA的载体于H1299细胞中,分别作为H1299Control对照细胞及siGPX4干扰细胞,Transwell检测两组细胞侵袭及迁移能力,qPCR技术和蛋白质印迹法检测两组细胞GPX4、CD133和Sox-2mRNA蛋白表达水平。结果肺腺癌组织中GPX4、CD133和Sox-2的表达水平高于癌旁组织,均P<0.01;GPX4、CD133(r=0.561,P=0.003)和Sox-2(r=0.688,P<0.001)表达水平均呈正相关;Ⅰ、Ⅱ、Ⅲ和Ⅳ期患者肺腺癌组织中GPX4mRNA表达水平分别为0.033±0.006、0.046±0.010、0.094±0.011和0.138±0.029,Ⅲ、Ⅳ期患者肺腺癌组织中GPX4mRNA表达水平高于Ⅰ、Ⅱ期患者(P<0.05),各肺腺癌细胞系GPX4蛋白表达水平均明显高于正常支气管上皮细胞HBE,且H1299细胞高于其他肺腺癌细胞系;H1299Control和siGPX4细胞侵袭细胞数分别为(174.3±21.6)和(75.9±10.4)个,siGPX4低于Control细胞,t=8.305,P<0.01。H1299Control和siGPX4细胞迁移细胞数分别为(225.8±24.2)和(97.4±8.6)个,siGPX4低于Control细胞,t=9.346,P<0.01。H1299siGPX4细胞GPX4(t=13.006,P<0.001)、CD133(t=10.124,P<0.001)和Sox-2mRNA(t=3.521,P=0.026)表达水平均低于H1299Control细胞;H1299siGPX4细胞GPX4、CD133和Sox-2蛋白表达水平低于H1299Control细胞。结论 GPX4在肺腺癌组织中高表达,且可促进干细胞特性及细胞侵袭转移能力。

OBJECTIVE Cancer stem cells (CSCs) is a key factor affecting the occurrence and development of tumors.Glutathione peroxidase 4 (GPX4), as an important membrane lipid peroxidation regulator, has been found to be closely related to many malignant phenotypes of tumors.In this study,the expression of GPX4 in lung adenocarcinoma tissues and its relationship with the characteristics of stem cells of lung adenocarcinoma cells and its invasion and metastasis in vitro were explored.METHODS Totally 46 cases of lung adenocarcinoma and para cancerous tissue in the Thoracic Surgery Department of Henan Thoracic Hospital were collected from 2017.6 to 2018.3.Real-time fluorescence quantitative PCR(qPCR) technology was used to detect the expression of GPX4 mRNA and stem cell markers CD133 and Sox-2 mRNA in tissues,and the expression levels and correlations of mRNA in different tissues were analyzed.Western Blot was used to detect the expression of GPX4 protein in normal bronchial epithelial cells and each lung adenocarcinoma cell lines respectively.Lentivirus transfect the blank and GPX4 siRNA vector to H1299 cell as H1299 Control cell and siGPX4 interfere cell.Trans well invasion and migration assay were used to detect the ability of invasion and migration in both groups of cell.qPCR technology and Western Blot were used to detect the expression of GPX4 , CD133 and Sox-2 mRNA and protein.RESULTS The expression levels of GPX4 ,CD133 and Sox-2 in lung adenocarcinoma tissues were significantly higher than those in adjacent tissues (P<0.01 ).The expression level of GPX4 was significantly positively correlated with the expression level of CD133 and Sox-2 (r=0.561 and 0.688,P=0.003 and<0.001).The expression levels of GPX4 mRNA in lung adenocarcinoma tissues of stage Ⅰ,Ⅱ,Ⅲ and Ⅳ patients were 0.033±0.006,0.046±0.010±0.094±0.011 and 0.138±0.029,and the expression levels of GPX4 mRNA in lung adenocarcinoma tissues of stage Ⅲ and Ⅳ patients were significantly higher than those of stage I and D patients (P<0.05).The expression level of GPX4 prot&n in lung ado nocarcinoma cell line was significantly normal bronchial epithelial cell HBE, and the expression of H1299 was higher than others.The number of invasive cells in H1299 Control and siGPX4 cells were 174.3±21.6 and 75.9± 10.4,and siGPX4 was significantly lower than that in Control cells(i=8.305,P<0.01).The number of migrated cells in H1299 Control and siGPX4 cells were 225.8±24.2 and 97.4±8.6,and siGPX4 was significantly lower than that in Control cells(t=9.346,P<0.01).The expression levels of GPX4,CD133 and Sox-2 mRNA in H1299 siGPX4 cells were significantly lower than those in H1299 Control cells (t=13.006, P<0.001;t=10.124 , P<0.001;t=3.521, P=0.026), and the expression levels of GPX4,CD133 and Sox-2 protein in H1299 siGPX4 cells were significantly lower than those in H1299 Control cells.CONCLUSION GPX4 is highly expressed in lung adenocarcinoma tissues and cell lines, and can promote stem cell characteristics?cell invasion and metastasis.