Silencing of N-Ras gene expression using shRNA decreases transformation efficiency and tumor growth in transformed cells induced by anti-BPDE.
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 G(0)/G(1) 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.
Institute for Chemical Carcinogenesis, State Key Laboratory of Respiratory Diseases, Guangzhou Medical College, Guangzhou 510182, China.
Lanlan
L
Zhou
Yiguo
Y
Jiang
Aijun
A
Tan
Anne R
AR
Greenlee
Yuelan
Y
Shen
Linhua
L
Liu
Qiaoyuan
Q
Yang
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D000818:Animals; D015153:Blotting, Western; D002273:Carcinogens / Q000633:toxicity*; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D002461:Cell Line, Transformed; D049109:Cell Proliferation / Q000187:drug effects*; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000235:genetics* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D011905:Genes, ras; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000235:genetics* / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014410:Tumor Stem Cell Assay; D015854:Up-Regulation
D002273:Carcinogens; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide