| Background and objectmicroRNA (miRNA), a class of 20-23 nucleotides in length, non-coding endogeno- us RNAs, can negatively regulate the expression of protein-coding genes through mRNA degradation or translational inhibition by binding with the 3′untranslated regions (3′UTR) of their mRNA targets. They can affect fundamental cellular processes such as differentiation, proliferation, and cell cycle, apoptosis. Growing evidence indicated alteration of miRNAs expression in tumors play an important role in tumor development and progress. Lung cancer is by far the leading cause of cancer death worldwide. As the most important environment risk-factor of lung cancer, cigarette smoking accounts for the 90% of lung cancer incidence.During cigarette smoking, more than 60 kinds of carcinogens are produced, among them benzo[a]pyrene (B[a]P) is one of the most important strong carcinogen in smoking as well as a ubiquitous environmental pollutant, which is a representative polycyclic aromatic hydrocarbon (PAH). Anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide(anti-BPDE) is the most important metabolite of B[a]P and an important chemical carcinogens cause cancer. However, the molecular mechanisms of anti-BPDE involved in chemical carcinogenesis are complex and less well understood. Our primary studies have found that in anti-BPDE-induced malignant transformed cells many tumor related genes and miRNA have abnormal expression. N-Ras expression at both mRNA and protein levels in anti-BPDE-induced cells were the highest among three Ras members (H-Ras, K-ras, N-Ras) and play an important role in chemically induced oncogenesis were observed in our earlier study. Our primary studies have focused on the over-expression of miRNAs during the anti-BPDE-induced malignant transformed cells, whereas little study has investigated on the molecular functions of low expression'miRNAs. Many studies have shown that miRNA can influence the development and progress of tumor through target on oncogenes or tumor suppressor genes, and the different expression levels of miRNAs contribute to divergent biological effects. In this study, in order to explore the potent function of the lower expressed miRNAs, according to our former study results, we selected and investigated the possible role of miR-506 and miR-542-3p which were predicted to target on N-Ras gene and might regulate the N-Ras expression by transient transfecting specificial miRNA mimic in the transformed cells, and further revealed the functions of those miRNAs on the anti-BPDE-induced carcinogenic progress.Methods1. Bioinformatic analyses: Bioinformatic analyses websites or softwares such as TargetScan, miRanda, miRnet and RNA22 etc. were used to predict the potent miRNAs which could target in the 3'untranslated region of N-Ras gene.2. Transient transfection of miRNA mimics: Cells were transfected using Lipofectamine-2000 according to the manufacturer's instruction. After 5.5h post-transfection, the transfection efficiency was examined with FAM-conjugated negative control by ?uorescence microscopy and the medium was replaced.3. Detection of miRNA and N-Ras expression: TaqMan MicroRNA assays kits and SYBR Green methods were used to identify the mRNA expression levels of miR-506 and miR-542-3p, as well as N-Ras in 16HBE-N and 16HBE-T cells, and N-Ras protein levels were detected by western blot.4. Cell viability assay: 16HBE-T cells were plated in the 96-well plates (3×103 cells per well) and cultured for 24 h, then transfected with miRNA mimics. The cell viability was evaluated by a cell counting kit CCK-8 at 24h, 48h post-transfection.5. Cell cycle At 24h of serum-starvation and 48h after transfection cells were stained with propidium iodide and RNase A, and then analyzed by flow cytometry.6. Apoptosis assay: The cells were harvested by 0.25% trypsin without EDTA after 48h post-transfection and evaluated by Annexin V-FITC/PI apoptosis kit and flow cytometry.7. Scrath assay: 24h post-transfection,cells were seed on 6-well plate and allowed to grow to confluence.Confluent monolayers were scratched with a pipette tip and maintained under serum-starvation for 24h then photographed.8. Soft agar assay: 1000 cells were resuspended in 2ml of 0.35% low melting point agarose, and then seeded onto the bottom layer of 0.6% low melting point agarose in 6-well plates. Colonies with at least 50 cells were counted at 2 weeks post-plated.9. Tumorigenicity assay: 24h after transfection, transfected or untransfected cells were harvested and resuspended with PBS to a unity density. Four mice per group were each given 4×106 cells in 150μl of PBS and were injected subcutaneously into the fold inguen of each mouse. The mice were monitored every 5 days for tumor formation, then sacrificed after 20 days post-injected and the tumor xenografts were excised and weighed.10. Statistial analyses: All statistical analysis was done with SPSS11.5 software. Values are expressed as mean±S.D. All experiments were done at least three times. Differences between groups were analyzed by double-sided Student's t-test for the compare of two groups and multiple comparisons were executeed by one-way ANOVA. The differences were considered to be significant when p<0.05.Results1. Bioiformatic analyses: Two putative bindingsites for miR-506 and miR-542-3p in N-Ras 3'UTR were obtained from miRNA target prediction softwares. The sequen- ce of miR-506, miR-542-3p and the targeting sites in N-Ras 3′UTR are all conser- ved among kinds of mammals.2. Original expression level of miRNA and N-Ras: In comparison with the 16HBE-N group, the expression level of miR-506 and miR-542-3p was decreased to (0.35±0.08) fold(,0.39±0.07)fold in 16HBE-T cells respectively,meanwhile, the expression level of N-Ras mRNA and protein was increased to(3.56±0.50), (1.62±0.13) fold in 16HBE-T cells respectively (p < 0.05) .3. Changes of miRNAs and N-Ras after transfection: At 48h post-transfection,the expression level of miR-506 and miR-542-3p in miR-506/542-3p-mimic group was respectively upregulated to(4.70±0.63)fold and (5.23±0.55) fold compared with miR-nc groups (P<0.05). The expression levels of N-Ras mRNA and protein in miR-506-mimic group decreased to (0.45±0.08) fold and (0.57±0.10) fold, were both significantly below the levels seen in the miR-nc groups(P<0.05).There's no statistically significant differences with the expression level of mRNA (0.94±0.18)fold or protein (0.85±0.11) fold of N-ras in miR-542-3p-mimic group(P>0.05).4. Change of Cell cycle: miR-506/miR-542-3p-mimic-transfected cells differed significantly in their cell cycle distribution with a higher proportion of cells within the G0/G1 phase and a reduction in the S and G2/M phase when compared to the miR-nc-transfected cells (P<0.05).5. Alteration of cell proliferation: The cell viability decresed to (73.42±6.35)% and (62.06±5.61)% ,respectively,in miR-506-mimic group and miR-542-3p-mimic group when compared to the miR-nc-transfected cells after 48h transfection (P<0.05).6. Alteration of cell apoptosis: There were no statistically significant differences with the apoptosis rates in miR-506-mimic/miR-542-3p-mimic -transfected cells when compared to the miR-nc-transfected cells after 24h or 48h transfection (P>0.05).7. Wound-scratch healing assay: In comparison with the miR-nc group, coverage areas ratio of wound-scratch in miR-506-mimic group and miR-542-3p-mimic group reduce to (0.48±0.10), (0.31±0.08) respectively (P<0.05).8. Soft agar assay: Rates of anchorage-independent colony formation in miR-506-mimic group (4.46±0.81)% and miR-542-3p-mimic group (5.87±0.67)% were both decreased, comparing with miR-nc group (9.87±0.63)% (P<0.05).9. Tumorigenicity in vivo: Whether volume or weight, the group of mice inoculated with miR-506-mimic-transfected cells grew smaller tumors (592.36±55.29 mm3) and less weight (694.00±124.39 mg) than miR-nc transfected cells (969.82±62.31 mm3, 1152.50±131.24 mg) (p < 0.05). Conclusion1. lower-expression of muture miR-506 and mature miR-542-3p in malignantly transformed human bronchial epithelial cells induced by anti-BPDE.2. Changes in muture miR-506 and muture miR-542-3p expression can affect the cell biological properties of 16HBE-T.3. Up-regulation of miR-506 and miR-542-3p by transfection of miRNA mimics in 16HBE-T cells induced growth suppression and decrease malignancy degree,and act as anti-oncogenic miRNA in malignant transformed cells.4. N-Ras is a putative target of miR-506. |
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