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目的分析Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响,以探讨脑胶质瘤浸润性生长的机理。方法从U87细胞中扩增Ezrin基因CDS区片段,克隆至表达载体pEGFP-C1中,构建Ezrin基因表达质粒pEGFP-C1/Ezrin。将pEGFP-C1/Ezrin、Ezrin基因shRNA质粒shRNA-Ezrin-2和pEGFP-C1以脂质体LipofectimineTM2000介导分别转染U87细胞,Western blot分析转染细胞中Ezrin蛋白的表达;划痕试验检测转染细胞的迁移情况。结果克隆的Ezrin基因与GenBank中登录的Homo sapiens ezrin(EZR),transcript variant 1,mRNA序列同源性达99%;该基因编码的氨基酸序列与ezrin[Homo sapiens](Sequence ID:ref|NP_003370.2|,Length:586)的一致性为99%,在第S66P、K258R、P265L和K577R位发生变异,读码框正确。pEGFP-C1/Ezrin转染的U87细胞中Ezrin蛋白的相对表达量(1.17)高于shRNA-Ezrin-2转染组(0.47)和pEGFP-C1转染组(0.82)。划痕试验显示,shRNA-Ezrin-2转染组有少量细胞迁移至划痕处,pEGFP-C1/Ezrin转染组细胞几乎占满划痕处。结论 Ezrin基因敲低可阻止U87细胞迁移,其过表达可促进细胞迁移,表明U87细胞浸润性生长与Ezrin基因的表达有关。
Objective To analyze the effect of Ezrin gene knockdown and overexpression on the migration of glioma U87 cells in order to explore the mechanism of glioma invasive growth. Methods The CDS region of Ezrin gene was amplified from U87 cells and cloned into expression vector pEGFP-C1 to construct Ezrin gene expression plasmid pEGFP-C1 / Ezrin. Eukaryotic expression plasmids pEGFP-C1 / Ezrin and Ezrin were transfected into U87 cells with shRNA-Ezrin-2 and pEGFP-C1 respectively. The expression of Ezrin protein in the transfected cells was detected by Western blot. Dye migration. Results The cloned Ezrin gene was 99% homologous to the Homo sapiens ezrin (EZR), transcript variant 1, and mRNA sequences registered in GenBank. The amino acid sequence of this gene was identical to that of ezrin [Homo sapiens] (Sequence ID: ref | NP_003370). 2 |, Length: 586) was 99% identical with the S66P, K258R, P265L and K577R mutants, and the reading frame was correct. The relative expression level of Ezrin protein (1.17) in pEGFP-C1 / Ezrin transfected U87 cells was higher than that in shRNA-Ezrin-2 transfection group (0.47) and pEGFP-C1 transfection group (0.82). Scratch tests showed that a small amount of cells in the shRNA-Ezrin-2 transfection group migrated to the scratches, and cells in the pEGFP-C1 / Ezrin transfection group almost occupied the scratches. Conclusion Ezrin knockdown can prevent the migration of U87 cells. The overexpression of Ezrin gene can promote the migration of U87 cells, indicating that the invasive growth of U87 cells is related to the expression of Ezrin gene.