The effect of ROCK-1 activity change on the adhesive and invasive ability of Y79 retinoblastoma cells
Department of Ophthalmology, Ruijin Hospital Affiliated Medical School, Shanghai Jiaotong University, 197 Ruijin No. 2 Road, Shanghai 200025, PR China
BMC Cancer 2014, 14:89 doi:10.1186/1471-2407-14-89Published: 14 February 2014
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.