The Functions Of MiR-106a And MiR-17-5p In Malignant Transformation Of Cells Induced By Anti-bpde

Background and objectBenzo[a]pyrene (B[a]P) is the first environmentally chemical carcinogen to be found widely distributed in the environment. The ultimate carcinogens of B[a]P is anti-BPDE produced by the metabolic activation. MiRNA are an abundant class of small noncoding RNAs that primarily function as oncogenes and tumor suppressors by mediating translational repression or mRNA degradation via binding target genes. In this study, malignant human bronchial epithelial cells transformed by anti-BPDE were used to help characterize possible mechanisms of miRNA function in chemical carcinogenesis. The expression levels of mature miR-106a and mature miR-17-5p were measured by the qRT-PCR method, and inhibitors or mimics were further used to down-regulate or up-regulate their activity in malignantly transformed cells to determine the effects of lower-expression and over-expression of miR-106a and miR-17-5p on the biological properties of the cell. Compared with control cells, we observed over-expression of miR-106a and miR-17-5p in transformed cells. Silencing miR-106a and miR-17-5p by transfection with inhibitors suppressed cell proliferation, induced cell-cycle arrest and apoptosis, inhibited anchorage-independent growth. In contrast, increasing miR-106a or miR-17-5p in malignantly transformed cells by transfection with mimics gave the opposite results. We further conducted nude mice assay with miR-106a inhibitor and mimic and found tumors in anti-miR-106a-injected group growed extremely slowly while that in miR-106a mimic-injected group growed significantly fast. Our findings suggest that miR-106a and miR-17-5p may function as oncogenes in transformation induced by a chemical carcinogen. Thus, knockdown of miR-106a and miR-17-5p in malignant cells is a potentially therapeutic strategy.Methods1. Detection of miRNA expression: Using TaqMan MicroRNA assays kits to identify the expression levels of miRNAs in 16HBE-N and 16HBE-T cells.2. Transient transfection of miRNA inhibitors and mimics: Transient transfection was performed according to instructions for Lipofectamine 2000 reagent. MiRNA mimic treatment has three groups, specific miRNA mimics (miR-106a mimic and miR-17-5p mimic) and negative control (mimic NC). MiRNA inhibitor treatment has three groups, specific miRNA inhibitors (anti-miR-106a and anti-miR-17-5p) and negative control (inhibitor NC). The control groups also include Lipofectamine 2000 reagent negative control group (Lipo-2000 NC). 5h post-transfection, transfection efficiency was confirmed and miRNAs were detected at 72h.3. Cell cycle assay: 72 h post-transfection, the cells were stained with propidium iodide and RNase A, then analyzed by flow cytometry.4. Cell proliferation assay: Equal numbers of 16HBE-T cells were seeded in 96-well plates and cultured for 24 h, followed by infection. Cell culture was continued for 5 days and assessed using the CCK-8 and microplate reader.5. Apoptosis assay: Apoptotic rates of transfected or untransfected cells were evaluated by Annexin V/PI apoptosis kit and flow cytometry 72 h post-transfection.6. Soft agar assay: 500 cells were resuspended in 1ml of 0.3% low melting point agarose, then were plated on top of 1 ml of semi-solid 0.6% low melting point agarose in the same medium in 12-well plates. Low melting point agarose was prepared with MEM cotaining 10% serum. Colonies were counted at 3 weeks.7. Tumorigenicity assay: 24h after transfection, transfected or untransfected cells were collected and resuspended with PBS to a density of 10×106/ml. 0.2 ml of PBS were inoculated subcutaneously bilateral behind the posterior limb, four five-week-old BALB/c nude mice per group. Growth curves for xenografts were determined by externally measuring tumour volumes every five days after tumor formation. After 42 days, the mice were killed, the xenografts were excised, weighed and fixed in 4% buffered formalin for pathological examination.8. Statistial analyses: Data were expressed as mean±S.D.. Differences between groups were assessed with one-way ANOVA or student's t-test with SPSS 17.0 for windows, according to data feature. Differences were determined to be significant when P < 0.05.Results1. Over-expression of miR-106a and miR-17-5p in 16HBE-T: The levels of miR-106a and miR-17-5p were 2.9 (±0.2)-fold and 2.3±(0.1)-fold higher in 16HBE-T than in 16HBE-N cells (P<0.05).2. Change of miRNAs after transfections: Compared with 16HBE-T, the level of miR-106a was reduced to 0.3 (±0.1)-fold or increased to 1.9 (±0.3)-fold following transfection with miR-106a inhibitor or miR-106a mimic (P<0.05), and the level of miR-17-5p was reduced to 0.4 (±0.1)-fold or increased to 1.7 (±0.2)-fold following transfection with miR-17-5p inhibitor or miR-17-5p mimic (P<0.05).3. Change of Cell cycle: Anti-miR-106a- and anti-miR-17-5p-transfected cells differed significantly in their cell-cycle distribution when compared to the inhibitor NC-transfected cells with a higher proportion of cells within the G0/G1 phase and a reduction in the S phase (P<0.05). In contrast, miR-106a mimic-transfected cells showed a lower proportion of cells in the G0/G1 phase and accumulation in the S phase when compared to mimic NC-transfected cells (P <0.05).4. Alteration of cellular proliferation: An 0.37 (±0.01)-fold decrease and an 0.45 (±0.07)-fold increase in cell growth were observed in anti-miR-106a-transfected cells and miR-106a mimic-transfected cells, and an 0.29 (±0.03)-fold decrease and an 0.34 (±0.02)-fold increase in cell growth were observed in anti-miR-17-5p-transfected cells and miR-17-5p mimic-transfected cells, when compared to control groups at 5 days (P <0.05).5. Alteration of cell apoptosis: Significantly higher apoptotic rates were found in the anti-miR-106a- and anti-miR-17-5p-transfected cells when compared to the inhibitor NC group (P <0.05), whereas negative control-transfected groups revealed a frequency of apoptosis similar to that of non-transfected 16HBE-T cells. Meanwhile, the apoptotic rates of miR-106a mimic- and miR-17-5p mimic-transfected cells were significantly less than that in the NC mimic group (P <0.05).6. Alteration of clonal growth: Cells transfected with anti-miR-106a or anti-miR-17-5p displayed fewer colonies compared with the NC inhibitor group and non-transfected cells (P <0.05). However, transfection with miR-106a mimic or miR-17-5p mimic increased the number of colonies of 16HBE-T cells compared with NC mimic transfectant (P <0.05).7. tumorigenicity in vivo: Compared to negative control groups, tumours derived from anti-miR-106a-transfected cells grew slowest (P <0.05) and tumours derived from miR-106a mimic-transfected cells grew fastest throughout the period of tumour growth (P <0.05). There was no significant difference of tumour growth among mice receiving 16HBE-T, Lipo-2000 NC, inhibitor NC or mimic NC-transfected cells. Tumours were harvested on day 42 post-injection (P >0.05). The average weight of tumours derived from anti-miR-106a-transfected cells (35.7±9.6 mg) was significantly lighter than those derived from NC inhibitor -transfected cells (110.8±15.3 mg) (P <0.05). In contrast, the average weight of tumours derived from miR-106a mimic-transfected cells (340.4±23.1 mg) was significantly heavier than those derived from NC mimic-transfected cells (127.6±21.9 mg) (P <0.05). Pathological examination indicated that mice injected with six kinds of cells showed growth of squamous cell carcinoma.Conclusion1. Over-expression of muture miR-106a and mature miR-17-5p in malignantly transformed human bronchial epithelial cells induced by anti-BPDE.2. Changes in muture miR-106a and muture miR-17-5p expression can affect the cell biological properties of 16HBE-T.3. MiR-106a and miR-17-5p play an important role in malignantly transformed cells induced by environmentally chemical carcinogens and are two micro-oncogenes, which can inhibit cell apoptosis and increase cell malignancy.