ToxSci Advance Access originally published online on June 20, 2008
Toxicological Sciences 2008 105(2):286-294; doi:10.1093/toxsci/kfn122
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Silencing of N-Ras Gene Expression Using shRNA Decreases Transformation Efficiency and Tumor Growth in Transformed Cells Induced by Anti-BPDE
* Institute for Chemical Carcinogenesis, State Key Laboratory of Respiratory Diseases, Guangzhou Medical College, Guangzhou 510182, China
Disease Control and Prevention Center of Zhuhai, Zhuhai 519000, China
Eastern Oregon University and Center for Research on Occupational and Environmental Toxicology, La Grande, Oregon 97850
1 To whom correspondence should be addressed at Institute for Chemical Carcinogenesis, Guangzhou Medical College, 195 Dongfengxi Road, Guangzhou 510182, China. Fax: +86-20-8134-0724. E-mail: jiangyiguo{at}yahoo.com.
Received May 22, 2008; accepted June 11, 2008
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Abstract |
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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.
Key Words: anti-BPDE; short hairpin RNA; RNA interference; H-Ras, K-Ras, N-Ras; human bronchial epithelial cells; malignant transformation.