Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, 197 Ruijin No, 2 Road, Shanghai 200025, PR China.
BMC Cancer. 2014 Feb 14;14:89. doi: 10.1186/1471-2407-14-89.
Retinoblastoma (Rb) is the most common intraocular tumor in childhood worldwide. It is a deadly pediatric eye cancer. The main cause of death in Rb patients is intracranial and systemic metastasis. ROCK is the main downstream effector of Ras-homologous (Rho) family of GTPases which are involved in many cellular functions, such as cell proliferation, invasion and metastasis. Overexpression of ROCK promotes invasion and metastasis of many solid tumors. However, the effect of ROCK in Rb is largely unknown.
ROCK-1 and ROCK-2 mRNA expression in Y79 cell lines were examined by RT-PCR. Protein expression in the Y79 cell line were examined by western blot analyses. ROCK-1 and ROCK-2 siRNA were transfected into Y79 cells with Lipofectamine 2000. Cell proliferation was evaluated by CCK-8 assay after exposure to ROCK inhibitor (Y-27632). We examined the effect of ROCK inhibitors (Y-27632, ROCK-1 and ROCK-2 siRNA) on Y79 cell adhesive capacity by cell adhesion assay. Cell invasion assay through matrigel was used to study the effect of ROCK inhibitors on Y79 cell invasive capacity.
The expression of mRNA of ROCK-1 was more than that of ROCK-2 in the Y79 cell line. The protein expression levels of ROCK-1 and ROCK-2 were downregulated in the cells transfected with siRNA. Y-27632 treatment didn't lead to any changes of Y79 cells proliferation. Adhesive ability of Y79 cells was enhanced following Y-27632 or ROCK-1 siRNA treatment. The invasive capacity of Y79 cells showed an inverse relationship with increasing Y-27632 concentration. Invasiveness of Y79 cells also decreased in Y79 cells transfected with ROCK-1 siRNA. However, there was no change in adhesive ability or invasive capacity in Y79 cells transfected with siRNA against ROCK-2.
The findings of this study demonstrate that ROCK-1 protein plays a key role in regulating metastasis and invasion of Y79 cells, suggesting that the ROCK-1 dependent pathway may be a potential target for therapy of Rb.
视网膜母细胞瘤（Rb）是全球儿童中最常见的眼内肿瘤。它是一种致命的小儿眼癌。Rb 患者死亡的主要原因是颅内和全身转移。ROCK 是 Ras-homologous（Rho）家族 GTPases 的主要下游效应物，参与许多细胞功能，如细胞增殖、侵袭和转移。ROCK 的过表达促进了许多实体瘤的侵袭和转移。然而，ROCK 在 Rb 中的作用在很大程度上是未知的。
通过 RT-PCR 检测 Y79 细胞系中 ROCK-1 和 ROCK-2 mRNA 的表达。通过 Western blot 分析检测 Y79 细胞系中的蛋白表达。用 Lipofectamine 2000 将 ROCK-1 和 ROCK-2 siRNA 转染到 Y79 细胞中。用 CCK-8 法检测 ROCK 抑制剂（Y-27632）处理后细胞增殖情况。通过细胞黏附试验研究 ROCK 抑制剂（Y-27632、ROCK-1 和 ROCK-2 siRNA）对 Y79 细胞黏附能力的影响。通过基质胶侵袭试验研究 ROCK 抑制剂对 Y79 细胞侵袭能力的影响。
在 Y79 细胞系中，ROCK-1 mRNA 的表达量多于 ROCK-2。用 siRNA 转染后，细胞中 ROCK-1 和 ROCK-2 的蛋白表达水平下调。Y-27632 处理不会导致 Y79 细胞增殖发生任何变化。Y-27632 或 ROCK-1 siRNA 处理后，Y79 细胞的黏附能力增强。Y79 细胞的侵袭能力与 Y-27632 浓度的增加呈反比关系。用 ROCK-1 siRNA 转染的 Y79 细胞的侵袭能力也降低。然而，用针对 ROCK-2 的 siRNA 转染 Y79 细胞不会改变其黏附能力或侵袭能力。
本研究结果表明，ROCK-1 蛋白在调节 Y79 细胞的转移和侵袭中起着关键作用，提示 ROCK-1 依赖性途径可能成为治疗 Rb 的潜在靶点。
