J Mol Neurosci. 2013 Nov;51(3):1046-51. doi: 10.1007/s12031-013-0096-3.
Crk-like (CrkL) is an adapter protein that has crucial roles in cell proliferation, adhesion, and migration. However, the expression pattern and potential mechanism of CrkL protein in glioblastoma multiforme (GBM) have not been fully elucidated. To determine roles of CrkL in cell signaling, proliferation, and migration, small interfering RNAs and plasmids transfection were used to suppress or overexpress CrkL in U87 and U251; soft-agar assay and wound-healing assay were used to observe cell invasiveness, migration, and proliferation. Erk1/2, Smad2, and matrix metalloproteinase 9 (MMP9) were also analyzed by western blot. CrkL was expressed in U87 and U251 cell lines and can be activated by transforming growth factor-beta 1 (TGF-β1) in vitro; CrkL knockdown significantly suppressed the expression of phosph-ERK1/2 and MMP9 but enhanced phosph-Smad2 expression compared with control (p <0.001). Overexpression of CrkL against control upregulated phosph-ERK1/2 and MMP9 and, at the same time, downregulated phosph-Smad2 (p <0.01). On the other hand, CrkL knockdown could significantly affect U87 and U251 invasiveness (p <0.01) and wound closure (p <0.01) using soft-agar assay and wound-healing assay. These studies suggest that CrkL efficiently mediates cell proliferation, migration, and invasion induced by TGF-β pathway in glioblastoma. Furthermore, CrkL can be used as a potential and efficient therapeutic target of GBM and may also mediate other signaling pathway.
卷曲相关蛋白（CrkL）是一种衔接蛋白，在细胞增殖、黏附和迁移中发挥着关键作用。然而，CrkL 蛋白在多形性胶质母细胞瘤（GBM）中的表达模式和潜在机制尚未完全阐明。为了确定 CrkL 在细胞信号转导、增殖和迁移中的作用，使用小干扰 RNA 和质粒转染来抑制或过表达 U87 和 U251 中的 CrkL；使用软琼脂测定和划痕愈合测定来观察细胞侵袭、迁移和增殖。还通过 Western blot 分析 Erk1/2、Smad2 和基质金属蛋白酶 9（MMP9）。CrkL 在 U87 和 U251 细胞系中表达，并且可以在体外被转化生长因子-β1（TGF-β1）激活；与对照相比，CrkL 敲低显著抑制了磷酸化-Erk1/2 和 MMP9 的表达，但增强了磷酸化-Smad2 的表达（p<0.001）。与对照相比，CrkL 的过表达上调了磷酸化-Erk1/2 和 MMP9，同时下调了磷酸化-Smad2（p<0.01）。另一方面，CrkL 敲低可以显著影响 U87 和 U251 的侵袭性（p<0.01）和划痕愈合（p<0.01），使用软琼脂测定和划痕愈合测定。这些研究表明，CrkL 有效地介导了 TGF-β 通路在胶质母细胞瘤中诱导的细胞增殖、迁移和侵袭。此外，CrkL 可以作为 GBM 的潜在和有效治疗靶点，也可能介导其他信号通路。
