Department of Pathology, The University of Hong Kong, Hong Kong, China.
Liver Int. 2010 Oct;30(9):1315-23. doi: 10.1111/j.1478-3231.2010.02307.x.
Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene, encodes a Rho GTPase-activating protein (RhoGAP) with GAP activity specific for RhoA. It exhibits tumour suppressor functions and inhibits tumour cell proliferation, migration as well as transformation.
In this study, we aimed to investigate the underlying mechanisms of the DLC2 gene in suppressing cell migration and cell growth. HepG2 hepatoma cells were stably transfected with the DLC2γ isoform, which contains the RhoGAP domain.
On performing immunofluorescence staining and Western blot analysis, the expression of the focal adhesion protein paxillin was found to be much reduced in DLC2γ-stable clones. Upon flow cytometric analysis of the cell cycle profiles, the DLC2γ-stable clones were shown to have a higher population of cells arrested at the G1 phase than the EGFP vector-stable clone, suggesting that downregulation of RhoA activity in DLC2γ-stable clones inhibited cell cycle progression. In the DLC2γ-stable clone, the levels of Raf-1 and extracellular signal-regulated kinase (ERK) 1/2 were decreased as compared with those of the parental HepG2, EGFP vector and DLC2γ-GAP defective mutant-stable clones. Furthermore, the ribosomal kinase p70S6K, a downstream target of ERK1/2, was suppressed in the DLC2-stable clones. On the contrary, when DLC2 was knocked down by siRNA in HepG2 cells, the expression levels of phospho-p70S6K and phospho-ERK1/2 were upregulated.
Our data show that DLC2 inhibits the activity of Raf-1-ERK1/2-p70S6K via its RhoGAP function, resulting in the suppression of cell growth. Further studies on the molecular signalling between DLC2 and p70S6K may provide an insight into its growth suppressor function.
肝癌缺失基因 2（DLC2）是一种假定的肿瘤抑制基因，编码一种 Rho GTP 酶激活蛋白（RhoGAP），具有针对 RhoA 的 GAP 活性。它具有肿瘤抑制功能，可抑制肿瘤细胞增殖、迁移和转化。
本研究旨在探讨 DLC2 基因抑制细胞迁移和细胞生长的潜在机制。用包含 RhoGAP 结构域的 DLC2γ 同工型稳定转染 HepG2 肝癌细胞。
通过免疫荧光染色和 Western blot 分析，发现焦点黏附蛋白 paxillin 的表达在 DLC2γ 稳定克隆中明显减少。通过细胞周期谱的流式细胞术分析，发现 DLC2γ 稳定克隆中处于 G1 期的细胞比例高于 EGFP 载体稳定克隆，表明 DLC2γ 稳定克隆中 RhoA 活性的下调抑制了细胞周期的进展。在 DLC2γ 稳定克隆中，与亲本 HepG2、EGFP 载体和 DLC2γ-GAP 缺陷突变体稳定克隆相比，Raf-1 和细胞外信号调节激酶（ERK）1/2 的水平降低。此外，ERK1/2 的下游靶标核糖体激酶 p70S6K 在 DLC2 稳定克隆中受到抑制。相反，当 HepG2 细胞中的 DLC2 被 siRNA 敲低时，磷酸化 p70S6K 和磷酸化 ERK1/2 的表达水平上调。
我们的数据表明，DLC2 通过其 RhoGAP 功能抑制 Raf-1-ERK1/2-p70S6K 的活性，从而抑制细胞生长。进一步研究 DLC2 与 p70S6K 之间的分子信号可能为其生长抑制功能提供深入了解。
