Department of Pathology, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, Japan.
Oncol Rep. 2011 Jul;26(1):91-9. doi: 10.3892/or.2011.1267. Epub 2011 Apr 15.
Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell marker during central nervous system development. Nestin is expressed in gliomas, and its expression levels are higher in gliomas with high WHO histopathological classification grades than in those with low grades. In the present study, we examined whether nestin regulates the biological activities of human glioma cells. Immunohistochemically, the nestin expression patterns in 10 human glioblastoma patients were examined. The expression levels of nestin in A172, a human high-grade glioma cell line, and KG-1-C, a human low-grade glioma cell line, were examined using real-time PCR, Western blot and immunofluorescence analyses. An expression vector carrying a short hairpin RNA targeting nestin was stably transfected into A172 (Sh) cells. The effects of decreased expression levels of nestin in Sh cells on cell growth, migration, invasion, adhesion to extracellular matrices and fibrillar actin expression on three-dimensional culture plates were examined. The nestin expression vector was transiently transfected into KG-1-C (Nes) cells, and the effects of the nestin overexpression on cell growth and migration were examined. Nestin was expressed in the cytoplasm of the glioblastoma cells in all cases examined. Sh cells showed marked decreases in the expression levels of nestin mRNA and protein, and the growth rate of Sh cells was lower than that of sham (Sc) cells. In contrast, the adhesion activity of Sh cells to types I and IV collagens, fibronectin and laminin was higher than that of Sc cells. Fibrillar actin was clearly detected at the periphery of colonies of Sh cells at the attachment sites on three-dimensional culture plates. The migration and invasion of Sh cells were markedly inhibited compared with those of Sc cells. In contrast, the levels of nestin expression markedly increased in the Nes cells, which were transiently transfected with the nestin expression vector. The growth rate and motility of Nes cells were higher than those of the mock cells. In conclusion, nestin plays important roles in cell growth, migration, invasion and adhesion to extra-cellular matrices in glioma cells. Nestin may serve as a novel candidate for molecular-targeted therapy for gliomas, including glioblastomas.
巢蛋白是一种 VI 类中间丝蛋白，最初在中枢神经系统发育过程中被描述为神经元干细胞标志物。巢蛋白在神经胶质瘤中表达，其在 WHO 组织病理学分级较高的神经胶质瘤中的表达水平高于分级较低的神经胶质瘤。在本研究中，我们研究了巢蛋白是否调节人神经胶质瘤细胞的生物学活性。用免疫组化方法检测了 10 例人脑胶质母细胞瘤患者的巢蛋白表达模式。用实时 PCR、Western blot 和免疫荧光分析检测了人高级别神经胶质瘤细胞系 A172 和人低级别神经胶质瘤细胞系 KG-1-C 中的巢蛋白表达水平。将携带巢蛋白短发夹 RNA 的表达载体稳定转染入 A172(Sh)细胞。在三维培养板上，通过检测 Sh 细胞中巢蛋白表达水平降低对细胞生长、迁移、侵袭、细胞外基质黏附和纤维状肌动蛋白表达的影响，研究了下调 Sh 细胞中巢蛋白表达水平对细胞生长、迁移、侵袭、细胞外基质黏附和纤维状肌动蛋白表达的影响。将巢蛋白表达载体瞬时转染入 KG-1-C(Nes)细胞，研究了巢蛋白过表达对细胞生长和迁移的影响。在所有检测的胶质母细胞瘤细胞中，巢蛋白均表达于细胞质中。Sh 细胞中巢蛋白 mRNA 和蛋白的表达水平明显降低，Sh 细胞的生长速度低于 sham(Sc)细胞。相反，Sh 细胞对 I 型和 IV 型胶原、纤连蛋白和层粘连蛋白的黏附活性高于 Sc 细胞。在三维培养板的附着部位，Sh 细胞集落的边缘清晰地检测到纤维状肌动蛋白。与 Sc 细胞相比，Sh 细胞的迁移和侵袭明显受到抑制。相反，瞬时转染巢蛋白表达载体的 Nes 细胞中巢蛋白的表达水平明显增加。Nes 细胞的生长速度和运动能力均高于对照细胞。综上所述，巢蛋白在神经胶质瘤细胞的生长、迁移、侵袭和细胞外基质黏附中发挥重要作用。巢蛋白可能成为神经胶质瘤，包括胶质母细胞瘤的分子靶向治疗的新候选药物。
