MiR-494 And MiR-22 Regulate PTEN Expression In Transformed Cells Induced By Anti-BPDE

Background and objectBenzo[a]pyrene (B[a]P) is a ubiquitous environmental pollutant which is a indirect carcinogen requires metabolic activation, generated from cigarette smoke and coal combustion. Accumulating evidence indicated human lung cancer is associated with B[a]P expose. Inactivation of PTEN tumor suppressor gene involves the initiation and progression of human tumors. MicroRNA is a class of evolutionally conserved, regulatory non-coding small RNAs. Recent researches reveal that the presence of miRNAs alternations paralleling with aberrant gene expression. Transformed 16HBE cells passaged 30 times (16HBE-T30) and 15 times (16HBE-T15), induced by anti-BPDE, were used to detect the miRNA expression. Bioiformatic tools were used to predict the miRNA target and a putative target gene PTEN was gained. Hypothesis was validated, overexpression and inhibition of miRNA expression in the transformed cell model followed by PTEN detection. Target sequences were indentified by luciferase assay, and the miRNA functionality in carcinogenesis were further elucidated. This research demonstraed the role of miRNA engaging in the cell transformation and regulatory role on PTEN regulation, provided perspective for lung cancer therapy and prenvention.Methods1. Bioinformatic analyses: TargetScan, Pictar and RNAhybrid were used in the PTEN 3'untranslated region (3'UTR) target analyses.2. Detection of miRNA and PTEN expression: Using real-time RT-PCR to identify the mRNA expression of miRNAs and PTEN in 16HBE-T30 and 16HBE-T15 cells, and PTEN protein were detected by western blot.3. Transient transfection of miRNA precursors and inhibitors: Transfection were performed according to the manufacturers'protocol. MiRNA precursor treatment has three groups, specific miRNA precursors (pre-miR-494 and pre-miR-22) and negative control (pre-miR-nc). MiRNA inhibitor treatment has three groups, specific miRNA inhibitors (anti-miR-494 and anti-miR-22) and negative control (pre-miR-nc). 24h post-transfection, transfection efficiency was confirmed and miRNA and PTEN were deteced at 72h.4. Plasmid constrction and luciferase assay: Two sequences containing the putative wild-type binding sites and mutated binding sites for miR-494 and miR-22, respectively, were inserted into the XbaI enzyme site of pGL3 vector and named WT and MT vector. Reconstructed plasmids were checked by EcoR I digestion and sequencing. Luciferase assays were carried out using dual-luciferase detection kit. Co-transfected with RNA and plasmid DNA using Lipofectamin2000, and pRL-TK renal luciferase was used as a normalization.5. Apoptosis assay: Cell transfection were performed in 96 well plate, 48h post transfection, caspase-3/7 activities were detected using ApoONE Homogeneous Caspase-3/7 according to the manufacturer'recommendations. DNA fragment staining were using Hoechst 33258, transfected cell were fixed at room temperature with 4% formaldehyden in PBS, followed by twice washes apoptotic cells were counted under a flourenscence microscope.6. Soft agar assay: 1000 cells resuspended in 2ml top low melting point agarose were seed into 6-well plates coated with bottom low melting point agarose in MEM cotaining 10% serum. Incubated at 37℃in 5% CO2 for 14d, colonies were counted7. Scrath assay: Transfected cell were seed on 24-well plate and allowed to grow to confluence. Confluent monolayers were scratched with a pipette tip and maintained under standard conditions for 24 h. Plates were washed once with fresh medium to remove nonadherent cells and then photographed.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 15.0 for windows, according to data feature. Differences were determined to be significant when P < 0.05.Results1. Bioiformatic analyses: Three putative targets for miR-494 and miR-22 in PTEN 3'UTR were obtained from bioinformatic tools, and having favorable minimum free energy of the miRNA︰mRNA complex, species-cross conserved sequence were also observed.2. MiRNA and PTEN expression in 16HBE-T15 and 16HBE-T30 cells: MiR-494 and miR-22 were significantly up-regulated by 12.6±1.7- and 2.3±0.1-fold, respectively (P<0.05), while both miRNA expression levels in 16HBE-T15 cells were 0.34±0.1-fold and 0.62±0.1-fold, respectively (P<0.05), comparing with 16HBE-N15 cells, but with no PTEN mRNA changes. The PTEN protein level in 16HBE-T15 was slightly increased, while in 16HBE-T30 cells PTEN levels were significantly reduced by 40.0±12.5% (P < 0.05) The protein level of PTEN and the expression level of miRNA were inversely correlated.3. MiRNA expression validation: Cells transfected with precursor or inhibitor 72h post-transfection showed increased and decreaded expression of miRNA, respectively. Inversely, PTEN protein upregulated in inhibitor treatment groups (P<0.05). PTEN protein downregulated in precursor treatment groups (P<0.05) .4. Plasmid construct and luciferase assay: Inserted sequences were checked by enzyme digestion and by sequencing. 16HBE-N30 cells transfected with the WT-vector showed higher luciferase activity than 16HBE-T30 cells. The luciferase activity of the MT-vector was higher than the WT-vector in 16HBE-T30 cells (P<0.05). Anti-miR-494 and anti-miR-22 treatment increased WT-vector luciferase activity relative to anti-miR-nc. Luciferase activities of MT groups had no significant differences, but were higher than the WT-vector with anti-miR-nc. Enhanced miR-494 and miR-22 expression levels decreased the WT-vector luciferase activity, however, no changes were observed among MT-vectors with precursors. These results indicated that miR-494 and miR-22 regulate PTEN expression through binding with the binding sites in PTEN 3'UTR, and mutations in the binding site abrogate the regulation of PTEN.5. MiR-494 and miR-22 engaged in cell apoptosis: Increase in caspase-3 and caspase-7 activities were found for anti-miR-494 (1.6±0.2 fold) and anti-miR-22 (1.5±0.1 fold) in 16HBE-T30 cells when compared to control (P<0.05). Decreased caspase-3/7 activity levels occurred in the overexpression studies, decreased to 0.82±0.03- and 0.84±0.05- fold, respectively. The overexpression studies showed high expression of miR-494 and miR-22 reduced the activities of caspase-3/7 (0.73±0.10- and 0.87±0.02- fold, respectively) in 16HBE-N30 cells (P<0.05). Hoechst 33258 staining for DNA fragmentation showed inhibition of miR-494 or miR-22 in 16HBE-T30 cells increased the number of apoptotic cells, in comparison with the control group (P<0.05).6. Soft agar assay: Rates of anchorage-independent colony growth in anti-miR-494 group (5.9%±0.9%) and anti-miR-22 group (6.2%±1.1%) were both decreased, comparing with anti-miR-nc group (11.6%±0.9%) (P<0.05).7. Wound-scratch healing assay: Wound-scratch healing assays showed that 16HBE-T30 cells transfected with anti-miR-494 (50.6%±1.5%) or anti-miR-22 (74.8%±2.5%) had lower motility than cells transfected with anti-miR-nc (93.9±4.4)(P<0.05).Conclusion1. MiR-494 and miR-22 were deregulated in early malignant transformed cell induced by anti-BPDE, while upregulated in typical malignant cells. PTEN protein showed inversely trend to the miRNA expression.2. MiR-494 and miR-22 target on PTEN through binding with their target sites in PTEN 3'UTR. Mutated target sites and silence of miR-494 and miR-22 can abrogate their regulatory role on PTEN. These may provide strategy for lung cancer prevention and therapy.3. MiR-494 and miR-22 are two micro-oncogenes, which can inhibit cell apotosis. In 16HBE-T30 cells, silence of miR-494 and miR-22 decreased cell colony formation and cell motility.