State Key Laboratory of Molecular Oncology, Cancer Institute and Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Acta Pharmacol Sin. 2012 Oct;33(10):1301-10. doi: 10.1038/aps.2012.123. Epub 2012 Sep 17.
Filamin binding LIM protein 1, also known as migfilin, is a skeleton organization protein that binds to mitogen-inducible gene 2 at cell-extracellular matrix adhesions. The aim of this study was to investigate the role of migfilin in cisplatin-induced apoptosis in human glioma cells, to determine the functional domains of migfilin, and to elucidate the molecular mechanisms underlying the regulation of cisplatin-related chemosensitivity.
The human glioma cell lines Hs683, H4, and U-87 MG were transfected with pEGFP-C2-migfilin to elevate the expression level of migfilin. RNA interference was used to reduce the expression of migfilin. To determine the functional domains of migfilin, U-87 MG cells were transfected with plasmids of migfilin deletion mutants. After treatment with cisplatin (40 μmol/L) for 24 h, the cell viability was assessed using the MTS assay, and the cell apoptotic was examined using the DAPI staining assay and TUNEL analysis. Expression levels of apoptosis-related proteins were detected by Western blot analysis.
Overexpression of migfilin significantly enhanced cisplatin-induced apoptosis in Hs683, H4, and U-87 MG cells, whereas downregulation of migfilin expression inhibited the chemosensitivity of these cell lines. The N-terminal region of migfilin alone was able to enhance the cisplatin-induced apoptosis. However, despite the existence of the N-terminal region, mutants of migfilin with any one of three LIM domains deleted led to a function loss. Furthermore, apoptotic proteins (PARP and caspase-3) and the anti-apoptotic protein Bcl-xL were modulated by the expression level of migfilin in combination with cisplatin.
The LIM1-3 domains of migfilin play a key role in sensitizing glioma cells to cisplatin-induced apoptosis through regulation of apoptosis-related proteins.
细丝结合 LIM 蛋白 1，也称为迁移蛋白，是一种骨架组织蛋白，它与细胞-细胞外基质黏附中的有丝分裂原诱导基因 2 结合。本研究旨在探讨迁移蛋白在顺铂诱导人神经胶质瘤细胞凋亡中的作用，确定迁移蛋白的功能域，并阐明调节顺铂相关化疗敏感性的分子机制。
用 pEGFP-C2-migfilin 转染人神经胶质瘤细胞系 Hs683、H4 和 U-87 MG，以提高 migfilin 的表达水平。用 RNA 干扰降低 migfilin 的表达。为了确定 migfilin 的功能域，用 migfilin 缺失突变体的质粒转染 U-87 MG 细胞。用顺铂（40 μmol/L）处理 24 h 后，用 MTS 测定法评估细胞活力，用 DAPI 染色法和 TUNEL 分析检测细胞凋亡。用 Western blot 分析检测凋亡相关蛋白的表达水平。
过表达 migfilin 显著增强了 Hs683、H4 和 U-87 MG 细胞中顺铂诱导的凋亡，而下调 migfilin 表达则抑制了这些细胞系的化疗敏感性。单独的 migfilin N 端区域能够增强顺铂诱导的凋亡。然而，尽管存在 N 端区域，缺失三个 LIM 结构域之一的 migfilin 突变体导致功能丧失。此外，凋亡蛋白（PARP 和 caspase-3）和抗凋亡蛋白 Bcl-xL 的表达水平与顺铂共同调节 migfilin。
迁移蛋白的 LIM1-3 结构域通过调节凋亡相关蛋白在使神经胶质瘤细胞对顺铂诱导的凋亡敏感方面发挥关键作用。
