Down-regulation of PEDF expression by ribozyme transgene in endothelial and lung cancer cells and its impact on angiogenesis in vitro

Abstract

Pigmental epithelial-derived factor (PEDF) is an important anti-angiogenesis factor. It has many different functions in ocular cells. The inhibition on angiogenesis by PEDF is associated with endothelial cell apoptosis. Until now, there have been few reports on the PEDF's role in cancer cell progression. This study examined the effect of a ribozyme transgene, designed to inhibit human PEDF expression, and its impact on in vitro lung cancer, endothelial cell growth and the angiogenesis forming process, in vitro. A transgene encoding ribozymes to specially target human PEDF was constructed using pEF6/v5-his vector. The human lung cancer cell line, A549, and endothelial cells, HECV, were transfected with PEDF ribozyme. MTT assay was used to analyse cell growth alternation after the PEDF gene was knocked out. An in vitro endothelial tubule formation assay was employed to analyse the microtubule forming change after the PEDF gene was knocked out in HECV cells. The PEDF message and protein were successfully removed with the PEDF ribozyme as shown by reverse transcription-PCR and Western blotting, respectively. The growth of both A549 and HECV cells were significantly accelerated when the PEDF gene was knocked out. The HECV cell ability to form microtubules increased following PEDF knockout. Furthermore, HECV significantly enhanced the ability to form microtubules when co-cultured with A549 cells whose PEDF expression was lost by way of the ribozyme transgene. Ribozyme transgenes targeting PEDF in lung cancer and endothelial cells can reduce the growth of these cells. Expression of PEDF in both cancer cells and endothelial cells are correlated with the angiogenic process' paracrine and autocrine pattern.