Abstract
Purpose: :
Our previous studies demonstrated that overexpression of pigment epithelium-derived factor (PEDF) inhibited ocular melanoma growth and metastasis. The purpose of this study is to determine if down-regulation of endogenous PEDF affects the growth of ocular melanoma, and to know the functional consequences of PEDF down-regulation during malignant progression of ocular melanoma.
Methods: :
To establish a melanoma cell line which expresses the down-regulated PEDF gene, PEDF shRNA or control shRNA lentiviral particles were transduced into mouse melanoma cell line B16LS9. After puromycin selection for 2 weeks, PEDF expression was detected by RT-PCR and western blot. CyQUANT® cell proliferation assay was performed in B16LS9 with PEDF down-regulation (B16LS9-PEDFshRNA) and B16LS9 with control shRNA transduction (B16LS9-shRNA). 2.5x105/2µl of B16LS9-PEDFshRNA and B16LS9-shRNA cells were inoculated into posterior chamber in C57/BL6 mice. At the 7th day after inoculation, the eye was checked with histological methods, and the largest area of ocular tumor was measured under microscope with Image J.
Results: :
PEDF expression was found in B16LS9-shRNA. The loss of PEDF expression in B16LS9-PEDFshRNA cells was confirmed using RT-PCR and western blot. Tumor cell proliferation in vitro was increased by PEDF knockout. In vivo experiments showed the area of ocular tumor with B16LS9-PEDFshRNA was significantly larger than one with B16LS9-shRNA (P<0.01).
Conclusions: :
Endogenous PEDF plays an important role in the proliferation of ocular melanoma. Loss of PEDF promotes the growth in ocular melanoma.
Keywords: melanoma • proliferation • gene modifiers