Silencing of n-ras gene expression using shRNA decreases transformation efficiency and tumor growth in transformed cells induced by Anti-BPDE

Abstract

Anti-benzo[a]pyrene-trans-7,8-diol-9,10-epoxide (anti-BPDE) is the most important metabolite of benzo[a]pyrene which is a ubiquitous environmental pollutant, and may cause human cancer, especially of the lung. Ras genes (H, K, and N) are activated in 40% of human tumors and may contribute to carcinogenesis. Here, we used malignant human bronchial epithelial cells transformed by anti-BPDE (16HBE-T) to help characterize possible molecular mechanisms of carcinogenesis. We compared H-, K-, and N-Ras mRNA and protein expression levels in 16HBE-T cells and untransformed control 16HBE cells (16HBE-N), using reverse transcription-PCR (RT-PCR) and Western blotting. We further used short hairpin RNA to silence N-Ras gene expression in 16HBE-T cells to determine the effects of silencing on the cell cycle, transformation efficiency and tumor growth. We observed overexpression of H-, K-, and N-Ras genes at both mRNA and protein levels in 16HBE-T cells, compared with 16HBE-N cells. Silencing of N-Ras in 16HBE-T cells using stable RNA interference increased the proportion of cells in G0/ G1 phase, decreased the proportion in S-phase, decreased transformation efficiency, and inhibited tumor growth. Our findings suggest that overexpression of N-Ras gene plays an important role in malignant transformation of 16HBE cells by anti-BPDE. N-Ras gene may be a useful target for gene therapy. © The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.