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目的:研究新型表皮生长因子受体(epidermal growth factor receptor,EGFR)异构体EGFRv A对人恶性胶质瘤U87MG细胞迁移的影响,并深入探讨其可能的机制。方法 :首先将p WPT-GFP[重组有绿色荧光蛋白(green l uorescent protein,GFP)基因,作为空白对照]、p WPT-EGFRwt(重组有野生型EGFR基因,作为阴性对照)和p WPT-EGFRv A慢病毒表达载体分别与慢病毒包装质粒ps PAX2和p MD2.G一起共转染293T细胞,获得慢病毒颗粒,然后感染胶质瘤U87MG细胞,建立U87MG GFP、U87MG EGFRwt和U87MG EGFRv A细胞系。采用蛋白质印迹法和FCM法验证EGFRwt和EGFRv A分别在感染后的U87MG EGFRwt和U87MG EGFRv A细胞中成功过表达。采用Transwell小室实验检测各组细胞迁移能力的变化。实时荧光定量PCR法验证前期基因芯片技术筛选出的各组细胞之间表达水平有明显变化的52个基因的m RNA水平,并用蛋白质印迹法检测其中4个有显著差异的肿瘤转移相关基因的蛋白表达水平。结果 :稳定感染U87MG细胞后,U87MG EGFRwt和U87MG EGFRv A细胞中分别过表达EGFRwt和EGFRv A。相对于EGFRwt过表达组,过表达的EGFRv A更能促进U87MG细胞发生迁移(P<0.001)。U87MG EGFRv A细胞中癌基因FBLN 2和ROBO 1的m RNA水平高于U87MG EGFRwt细胞,而抑癌基因CDH 13和SOX 2的m RNA水平则明显下调(P值均<0.05)。在蛋白质水平上,U87MG EGFRv A细胞中与肿瘤转移相关的CDH13和SOX2表达水平也明显下调(P值均<0.05)。结论 :EGFRv A过表达可以促进人胶质瘤U87MG细胞的迁移,它可能是通过影响肿瘤细胞中FBLN2、ROBO1、CDH13和SOX2基因的表达来发挥作用。
OBJECTIVE: To study the effect of epidermal growth factor receptor (EGFR) isoform EGFRv A on the migration of human malignant glioma U87MG cells and to explore its possible mechanism. Methods: p WPT-GFP [green fluorescent protein (GFP) gene as blank control], p WPT-EGFRwt (recombinant wild type EGFR gene as negative control) and p WPT-EGFRv A lentivirus expression vector were co-transfected with the lentiviral packaging plasmid ps PAX2 and p MD2.G 293T cells to obtain lentivirus particles, and then infected glioma U87MG cells to establish U87MG GFP, U87MG EGFRwt and U87MG EGFRv A cell line . Western Blotting and FCM were used to verify that EGFRwt and EGFRv A were overexpressed in infected U87MG EGFRwt and U87MG EGFRv A cells, respectively. Transwell chamber experiments were used to detect the changes of cell migration ability in each group. The real-time quantitative PCR method was used to verify the m RNA levels of 52 genes with significant changes among the groups of cells screened by pre-gene chip technique. Four proteins with significantly different metastasis-related genes were detected by Western blotting The expression level. Results: After stable infection of U87MG cells, EGFRwt and EGFRv were overexpressed in U87MG EGFRwt and U87MG EGFRv A cells, respectively. Overexpression of EGFRv A promoted U87MG cell migration more than EGFRwt overexpression group (P <0.001). The mRNA levels of oncogenes FBLN 2 and ROBO 1 in U87MG EGFRv A cells were significantly higher than those in U87MG EGFRwt cells, while the m RNA levels of tumor suppressor genes CDH 13 and SOX 2 were significantly down-regulated (all P <0.05). At the protein level, the expressions of CDH13 and SOX2 in U87MG EGFRv A cells were also significantly down-regulated (P <0.05). CONCLUSION: Overexpression of EGFRv A can promote the migration of human glioma U87MG cells. It may play an important role by affecting the expression of FBLN2, ROBO1, CDH13 and SOX2 in tumor cells.