MiRNAs Regulating Growth Of Anti-BPDE-transformed Human Bronchial Epithelial Cells And Lung Cancer Cells

Background and ObjectmicroRNA, about 22-nucleotide non-coding RNA, is cleaved from long transcripts pri-microRNA and pre-microRNA, could bind to complementary sequences in the 3′un-translated regions (UTR) of their target mRNAs and induce mRNA degradation or translational repression. The human genome may encode over 1000 miRNAs which may target approximately 60% of mammalian genes, and are abundant in many human cell types. Evidences demonstrate miRNA regulate various biological processes including cellular differentiation, proliferation and death. Recently increasingly studies were focused on roles of miRNA in biomarkers, diagnosis, prevention, therapy, and prognosis of various cancers. Recently, we compared the miRNA profiles between malignant transformed 16HBE-T cells and the control 16HBE cells, and a set of miRNA was found to be expressed differentially in 16HBE-T cells. Our recent studies have found that miR-494, miR-22 and miR-106a function as oncogenes in 16HBE transformation induced by anti-BPDE. However, there is little research on miRNA relative to chemocarcinogenesis or anticarcinogenic miRNA and its mechanisms. The ability of natural agents including resveratrol to suppress carcinogenesis has attracted the widespread attention of cancer prevention and treatment researchers. Extensive study over the past decade has shown the chemopreventive and chemotherapeutic potential of resveratrol. Recently, evidence indicated that the anti-tumor activities of resveratrol also could be relative to miRNA. To discuss application of miRNA in clinical prevention and therapy in lung cancer, we investigated anticarcinogenic miRNA function in chemical cancerogenesior anticarcinogenesis in lung cancer.MethodsPart 1. miR-622 impedes growth of anti-BPDE-transformed human bronchial epithelial cells via targeting K-RasThe MiRNA Expression Profiling Assay was adopted for compare miRNA expression between the 16HBE-T cells that were treated with and without resveratrol. Total RNA was abstracted by using Trizol reagent for 16HBE-T cells that were treated with and without resveratrol. The qRT-PCR was used for validated results from miRNA profiles and qunantify miR-622 expression levels in 16HBE-T, H460 and 16HBE cells. To elevate or depress expression levels of miR-622, cells were transfected transiently by Lipofectamine 2000 with miR-622 mimic or miR-622 inhibitor. Transfected with miR-622 mimic or miR-622 inhibitor for 48 h, 16HBE-T and H460 cells were collected for cell proliferation measured by CCK-8 assay and cell cycle content distribution anylized by flow cytometer. After 24 h transfection, 16HBE-T cells was collected for soft-agar colony formation assay and tumor xenograft model study. Therefore, we searched the targets for miR-622 by bioinformatic softwares, and we found the K-Ras target for miR-622. Transfected with miR-622 mimic for 48 h, 16HBE-T cells ware collected for extracting toatal RNA and protein to dectect the expression levels of K-Ras mRNA and protein by qRT-PCR and western blotting. Then, we constructed a reporter expression vector to verify the putative target K-Ras for miR-622 by luciferase activity assay. At last, pre-treatment with FTS (K-Ras inhibitor) for 4 days, after validating K-Ras mRNA and protein expression by qRT-PCR and western blotting, 16HBE-T cells then transfected with miR-622 for another 48 h for cell preoliferation by CCK-8 assay and cell cycle analysis by flow cytometer. Part 2. miR-497 and miR-34a regulate growth of lung cancer cell via cyclin E1In Part 1, we found that upregulated expression levels of miR-34a and miR-497 by resveratrol, which were selected for validating their function in lung cancer. First, we detected the expression of miR-34a and miR-497 in normal human bronchial epithelial cell line 16HBE and human lung cancer cell lines A549, H460, H1299, H446 and QG-56 by qRT-PCR. To elevate or depress expression levels of miR-34a or miR-497, cells were transfected transiently by using Lipofectamine 2000 with miR-34a mimic, miR-497 mimic, miR-34a inhibitor or miR-497 inhibitor. Human lung cancer cell lines A549, H460 and H1299 were transfected with miR-34a or miR-497 mimic for 48 h. Cell proliferation was detected by CCK-8 assay, and cell cycle content distribution was anylized by flow cytometer. A549 cells transfected with miR-34a or miR-497 mimic for 24 h were collected for soft-agar colony formation assay and tumor xenograft model study. We searched the targets for miR-34a and miR-497 by bioinformatic softwares, and cyclin E1 mRNA CCNE1 was predicted to be a co-target for miR-34a and miR-497. A549, H460 and H1299 cells transfected with miR-34a or miR-497 mimic for 48 h were collected for dectected the expression levels of cyclin E1 mRNA CCNE1 by qRT-PCR and the expression levels of cyclin E1 by western blotting. Then, we constructed 3 reporter expression vectors to verify the putative target CCNE1 for miR-34a and miR-497 by luciferase activity assay. We knock-downed expression of cyclin E1 by using the synthesized CCNE1 siRNA. Co-transfected the CCNE1 siRNA with miR-34a or miR-497 mimic for 48 h, A549 cell proliferation was detected by CCK-8 assay, and cell cycle content distribution was analized by flow cytometer.ResultsPart 1. miR-622 impedes growth of anti-BPDE-transformed human bronchial epithelial cells via targeting K-Ras1. Expression of miR-622 is up-regulated in resveratrol-treated 16HBE-T cellsResveratrol induced growth inhibition of 16HBE-T cells in a time- and dose-dependent manner and mostly inhibited growth at 50μM for a 48 h treatment. We further compared the miRNA expression profiles of 16HBE-T cells with or without treatment of 50μM resveratrol for 48 h. Among 735 human miRNAs analyzed, 105 of them exhibited significantly differential expression in resveratrol-treated 16HBE-T cells. The top up-regulated miRNA miR-622 was selected for further study. We first examined the expression levels of miR-622 by the Taqman miRNA assay and validated that miR-622 up-regulated upon treatment of resveratrol.2. Expression levels of miR-622we assessed the expression level of miR-622 by Taqman miRNA assay in 16HBE-T cells compared with the control 16HBE cells. Expression levels of miR-622 in 16HBE-T cells was about 3-fold change less than that in control 16HBE cells. Interestingly, we observed the same phenomenon that resveratrol induced growth inhibition and miR-622 was up-regulated upon resveratrol in lung cancer cell line H460. The levels of miR-622 were significantly increased or reduced by transfection with miR-622 mimic or miR-622 inhibitor in 16HBE-T cells and NCI H460, suggesting that oligonucleotides are introduced efficiently into 16HBE-T and H460 cells to enhance or regress miR-622 expression.3. miR-622 mimic affect cell proliferation, cell cycle content distribution and cell clony formationUp-regulating expression level of miR-622 in 16HBE-T cells induced cell growth inhibition, which reduced the cell viability to 72.18%±2.01%, p<0.01. The same effect of miR-622 was observed in H460 cells. The effect of miR-622 on 16HBE-T cell cycle progression showed that miR-622 induced cell cycle arrest at G0 phase. The miR-622 arrested cells at the G0 phase of the cell cycle. 68.96%±1.75% of 16HBE-T cells with miR-622 mimic-transfected were found in the G0 phase after 48 h transfection in comparison to 56.86%±2.01% of untransfected control cells, while S phase decreased from 28.76%±1.15% to 18.05%±1.22% upon transfection with miR-622 mimic. The similar effect that miR-622 induced cell cycle arrest at G0 was observed in H460 cells.We assessed the ability of colony formation of 16HBE-T cells with miR-622 mimic. Cells with miR-622 mimic transfected (27.7±6.4, p<0.05) showed fewer and smaller colony than NC mimic group (55±3.3).4. miR-622 mimic affect tumorigenicity of 16HBE-T cells16HBE-T cells with NC mimic- and miR-622 mimic-transfected were inoculated s.c. into the bilateral inguino-abdominal flanks of the same nude mice, respectively. Among 7 mice, average gross (0.18±0.03 g, p<0.05) and volume (0.58±0.14 cm3, p<0.05) of mice with miR-622 mimic transfection were lighter and smaller than NC mimic control group (0.35±0.10 g or 1.09±0.24 cm3).5. K-Ras is a target for miR-622K-Ras is a target for miR-622 predicted by bioinformatic softwares Targetscan, RNA22 and RNAhybrid. miR-622 mimic didn't affect abundance of E2f1, c-myc and Cdk6 proteins, while significantly reduced level of K-Ras protein was observed in 16HBE-T cells with miR-622 mimic transfected. The reporter plasmid co-transfected with wt-miR-622 mimic depressed luciferase activity compared to that in the empty vector control or untransfected group. Whereas, the plasmid co-transfected with mt-miR-622 mimic actually diminished down-regulation of luciferase activity.6. K-Ras is involved in growth inhibition induced by miR-622Treatment with 50μM FTS for 4 days remarkably decreased the K-Ras mRNA and protein expression in 16HBE-T cells. FTS attenuated the growth retardation effect of miR-622 mimic as detected by CCK-8 assay. Cells with miR-622 ectopic expression upon FTS treatment have no effect on cell-cycle progression, which means that K-Ras may be a necessary for miR-622 to exert its role in growth inhibition.Part 2. miR-497 and miR-34a regulate growth of lung cancer cell via cyclin E11. Expression levels of miR-497 and miR-34aIn Part 1, we found that upregulated expression levels of miR-497 and miR-34a by resveratrol, which were selected for validating their function in lung cancer. The levels of miR-497 and miR-34a were decreased in lung cancer cells compared to that in 16HBE normal bronchial epithelial cells. The levels of miR-497 or miR-34a were significantly increased or reduced by transfection with mimic for miR-497 or miR-34a or inhibitor for miR-497 or miR-34a in A549, H460 or H1299 cells.2. miR-497 or miR-34a mimic affect cell proliferation, cell cycle content distributionUp-regulating expression level of miR-497 in A549, H460, and H1299 cells transfected with miR-497 mimic induced cell growth inhibition, which reduced the cell viability to 65.07%±1.46%, 82.94%±1.35% and 79.08%±0.20%, p<0.05. Up-regulating expression level of miR-34a in A549, H460, and H1299 cells transfected with miR-34a mimic induced cell growth inhibition, which reduced the cell viability to 65.07%±1.46%, 82.94%±1.35% and 79.08%±0.20%, p<0.05. The effect of miR-497 or miR-34a on A549 cell cycle progression showed that miR-497 or miR-34a induced cell cycle arrest at G0/G1 phase. The miR-497 or miR-34a arrested cells at the G0/G1 phase of the cell cycle. 82.38%±1.75% or 78.27%±1.27% of A549 cells with miR-497 or miR-34a mimic-transfected were found in the G0 phase after 48 h transfection in comparison to 61.06%±2.33%of untransfected control cells, while S phase decreased from 27.50%±2.44% to 13.67%±3.75% or 15.72%±1.99% upon transfection with miR-497 or miR-34a mimic.3. miR-497 or miR-34a mimic affect cell clony formation and tumorigenicity in vivoWe assessed the ability of colony formation on A549 cells with NC mimic, miR-497 mimic, miR-34a mimic or without any transfection. Cells with miR-497 or miR-34a transfected (31.3±2.4, p<0.05 or 21.0±4.0, p<0.01) showed much fewer and smaller colony than that in NC mimic control group (71.0±9.3). We further examined effect of miR-497 or miR-34a on tumorigenicity in vivo. A549 cells with NC mimic, miR-497 mimic, or miR-34a mimic transfected were inoculated sc into the bilateral inguino-abdominal flanks of the same nude mice, respectively. Among 7 mice, average gross (0.29±0.01 g) and volume (0.59±0.11 cm3) of mice with transfected with miR-497 mimic were much lighter and smaller than controls (0.43±0.07 g or 1.29±0.23 cm3), while average gross (0.07±0.01 g) and volume (0.26±0.13 cm3) of mice with transfected with miR-34a mimic were much lighter and smaller than controls (0.27±0.13 g or 0.92±0.64 cm3), p<0.05.4. CCNE1 is a putative co-target of miR-497 and miR-34aCCNE1 is a co-target for miR-622 predicted by bioinformatic softwares RNA22 and RNAhybrid. CCNE1 protein levels in A549, H460 and H1299 cells transfected with miR-34a or miR-497 were decreased compared with that in transfected with NC mimic. The reporter plasmid co-transfected with miR-497 mimic markedly depressed luciferase activity to 65.44%±1.13%, p<0.01, while plasmid co-transfected with miR-497 inhibitor restored luciferase activity. The reporter plasmid co-transfected with miR-34a mimic markedly depressed luciferase activity to 46.58%±3.16%, p<0.01, while plasmid co-transfected with miR-34a inhibitor actually diminished down-regulation of reporters.5. CCNE1 is involved in growth retardation by miR-497 or miR-34aCyclin E1 knockdown induces cell growth inhibition, cell cycle arrest at G0/G1, and inhibited anchorage-independent growth by 60%-80% in A549 cells. Co-transfection CCNE1 siRNA with miR-497 mimic or miR-34a mimc in A549 cells, the cell viability and cell cycle content distribution were not affected.Conclusion1. miR-622 induced the growth inhibition, clony formation and tumorigenicity of 16HBE-T cells, which is via negatively regulating oncogene K-Ras.2. miR-497 or miR-34a induced the growth inhibition, clony formation and tumorigenicity of lung cancer cells, which is through CCNE1.3. miR-622, miR-497 or miR-34a up-regulated in resvertrol-treatment in lung cancer, which are Anticarcinogenic miRNAs functioning in chemical cancerogenesis or anticarcinogenesis in lung cancer.