ABSTRACT
<p><b>OBJECTIVE</b>To screen miRNA profiles of malignantly transformed human bronchial epithelial cells, 16HBE-T, induced by anti-benzo[a]pyrene-trans-7,8-diol-9,10-epoxide (anti-BPDE), and to analyze putative miR-10a targets in 16HBE-T.</p><p><b>METHODS</b>A novel microarray platform was employed to screen miRNA profiles of 16HBE-T cells transformed by anti-BPDE. Microarray data for miR-10a and miR-320 were validated using quantitative real time polymerase chain reaction (QRT-PCR). The expression of a putative target for miR-10a, HOXA1, was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and QRT-PCR.</p><p><b>RESULTS</b>In comparison with the vehicle-treated cells (16HBE-N), 16HBE-T exhibited differential expression of 54 miRNAs, in which, 45 were over-expressed and 9 were down-regulated. The five most highly expressed miRNAs were miR-494, miR-320, miR-498, miR-129, and miR-106a. The lowest expressed miRNAs were miR-10a, miR-493-5p, and miR-363*. Three members of miR-17-92 cluster, miR-17-5p, miR-20a, and miR-92, showed significantly higher abundance in 16BHE-T as miR-21, miR-141, miR-27a, miR-27b, miR-16 and miRNAs of the let-7 family. The putative target for miR-10a, HOXA1 mRNA was up-regulated 3-9-fold in 16HBE-T, as compared with 16HBE-N.</p><p><b>CONCLUSION</b>The findings of the study provide information on differentially expressed miRNA in malignant 16HBE-T, and also suggest a potential role of these miRNAs in cell transformation induced by anti-BPDE. HOXA1 is similarly up-regulated, suggesting that miR-10a is associated with the process of HOXA 1-mediated transformation.</p>
Subject(s)
Humans , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide , Toxicity , Carcinogens , Toxicity , Cell Transformation, Neoplastic , Genetics , Metabolism , Cells, Cultured , Gene Expression Profiling , Homeodomain Proteins , Genetics , Metabolism , MicroRNAs , Metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To establish the stable inhibition of HER2/neu expression by vector-mediated small hairpin RNA in malignant transformed human bronchial epithelial cell line induced by anti-benzo(a)pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide (anti-BPDE).</p><p><b>METHODS</b>The pSIREN-RetroQ-neu recombinant vector targeting HER2/neu was constructed and confirmed by restriction and sequencing analysis, then it was transfected into anti-BPDE malignant transformed 16HBE cells (16HBE-T) through lipofectamine 2000. The control groups included the 16HBE-T cells transfected with negative control vector (negative control) and 16HBE-T. The cells transfected with vectors were screened by puromycin. The HER2/neu mRNA and protein expressions in the vector-transfected 16HBE-T cells were detected by RT-PCR and Western blot method respectively.</p><p><b>RESULTS</b>The pSIREN-RetroQ-neu recombinant vector which inhibited HER2/neu mRNA and protein expressions in the 16HBE-T was constructed. The level of HER2/neu mRNA in the 16HBE-T cells transfected with pSIREN-RetroQ-neu was significantly reduced as compared to the negative control and blank control cells (0.114 +/- 0.003 vs.blank control 0.186 +/- 0.001, t = 39.154, P < 0.05; and negative control 0.182 +/- 0.015, t = 7.564, P < 0.05), while its level did not differ significantly between negative control cells and blank control of 16HBE-T (t = -0.409, P > 0.05). HER2/neu protein level in pSIREN-RetroQ-neu transfected cells was inhibited by 40% and 39% respectively.</p><p><b>CONCLUSION</b>Plasmid-based shRNA expression systems targeted against HER2/neu gene were generated successfully, which resulted in down-regulation of HER2/neu gene expression in the 16HBE-T efficiently.</p>
Subject(s)
Humans , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide , Toxicity , Base Sequence , Cell Line, Transformed , Epithelial Cells , Metabolism , Gene Expression , Genes, erbB-2 , Molecular Sequence Data , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To screen microRNA (miRNA) profiles of malignantly transformed cells induced by anti-benzo-a-pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide (BPDE) and to look for miRNAs which is expressed differently between malignantly transformed cells and normal human bronchial epithelial cells 16HBE.</p><p><b>METHODS</b>Experimental group was the malignantly transformed 16HBE which was induced by cultured with final concentration 2.0 micromol/L of BPDE which was dissolved in dimethyl sulphoxide. The control group was 16HBE that was cultured with minimal essential medium including dimethyl sulphoxide. 327 miR-NAs were tested be-tween those two groups with miRNA microarray analysis. MiR-10a that was down expressed and miR-320 that was overexpressed were selected to be validated by miRNA specific quantitative real-time reverse transcriptase chain reaction (miR qRT-PCR).</p><p><b>RESULTS</b>327 human miRNAs were tested with miRNA microarray analysis. 55 miRNAs were found expressing differently between those two groups and of which 46 were overexpressed and 9 were down expressed. Some data were validated by quantitative RT-PCR.</p><p><b>CONCLUSION</b>miRNAs expressed significantly between malignantly transformed 16HBE and normal cells and this helps us look for unique miRNAs of malignantly transformed cells induced by BPDE, but there should have more sufficient evidences to prove their functions in malignant cells.</p>