MiR-320 and miR-494 affect cell cycles of primary murine bronchial epithelial cells exposed to benzo[a]pyrene.

MicroRNAs (miRNAs) are a class of small noncoding RNA molecules with profound impact on various biological processes. Some miRNAs are involved in tumorigenesis by regulation of cell cycle progression. Here, we cultured primary murine bronchial epithelial cells and then examined the expression of miR-320 and miR-494 in cells exposed to benzo[a]pyrene (B[a]P). To better characterize roles of miR-320 and miR-494 in cell cycle progression, we used miRNA inhibitors to downregulate expression of miRNAs and determined cell cycle distribution and expression of cyclin-dependent kinases 6 (CDK6) by flow cytometric analysis. Treating cells with 1 microM B[a]P for 24h resulted in time-dependent increases in miR-320 and miR-494 expression. Moreover, G1 arrest and downregulated expression of CDK6 were shown in the treated cells. Flow cytometric analysis indicated a relief of G1 arrest and an elevated expression of CDK6 after inhibition of the expressions of miR-320 and miR-494 in cells exposed to B[a]P. These results suggest that expression levels of miRNA-320 and miR-494, which regulate B[a]P-exposed cell cycle progression, may impact G1/S transition through CDK6, and provide further insights into functions of miRNAs in cell cycle of primary murine bronchial epithelial cells exposed to B[a]P.

Effects of silencing of HER2/neu gene in anti-BPDE-transformed cells.

Anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) is a metabolite of benzo[a]pyrene (B[a]P) and acts as a potent mutagen in mammalian systems. However, the molecular mechanisms related to anti-BPDE-induced carcinogenesis are poorly understood. We have used malignant human bronchial epithelial cells (16HBE-T) transformed by exposure to anti-BPDE to help characterize these possible molecular mechanisms. We have previously observed overexpression of HER2/neu in 16HBE-T. To further investigate the effects of HER2/neu on 16HBE-T cell biologic phenotype, we inhibited HER2/neu expression using RNA interference. Silencing of HER2/neu in 16HBE-T cells was performed in vitro using retrovirus-delivered short hairpin RNA (shRNA). Silencing of HER2/neu in 16HBE-T cells resulted in significant increases and decreases in the proportions of cells in G0/G1 phase (67.1+/-2.1%) and in S phase (17.3+/-4.1%), respectively, and significantly reduced cell viability and colony formation rate. These results may help to explain epithelial cell transformation following exposure to anti-BPDE, and suggest an oncogenic role for HER2/neu in anti-BPDE-induced carcinogenesis.