Transcription factor and microRNA interactions in lung cells: an inhibitory link between NK2 homeobox 1, miR-200c and the developmental and oncogenic factors Nfib and Myb
© Tagne et al.; licensee BioMed Central. 2015
Received: 2 December 2014
Accepted: 30 January 2015
Published: 13 February 2015
The transcription factor NK2 homeobox 1 (Nkx2-1) plays essential roles in epithelial cell proliferation and differentiation in mouse and human lung development and tumorigenesis. A better understanding of genes and pathways downstream of Nkx2-1 will clarify the multiple roles of this critical lung factor. Nkx2-1 regulates directly or indirectly numerous protein-coding genes; however, there is a paucity of information about Nkx2-1-regulated microRNAs (miRNAs).
Methods and results
By miRNA array analyses of mouse epithelial cell lines in which endogenous Nkx2-1 was knocked-down, we revealed that 29 miRNAs were negatively regulated including miR-200c, and 39 miRNAs were positively regulated by Nkx2-1 including miR-1195. Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs. Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs. Promoter reporter assays indicated that 1kb of the miR-200c 5′ flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3′UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c.
These studies suggest that Nkx2-1 controls the expression of specific miRNAs in lung epithelial cells. In particular, we identified a regulatory link between Nkx2-1, the known tumor suppressor miR-200c, and the developmental and oncogenic transcription factors Nfib and Myb, adding new players to the regulatory mechanisms driven by Nkx2-1 in lung epithelial cells that may have implications in lung development and tumorigenesis.
KeywordsmicroRNA Transcription factors Gene expression Lung epithelial cells Targets
The NK2 homeobox 1 (Nkx2-1, Ttf1, T/ebp) gene controls lung, thyroid and brain gene expression in development and tumors [1-3]. In lung development, Nkx2-1 is essential for epithelial branching morphogenesis and bronchiolar and alveolar epithelial cell differentiation [2,3]. Mutations of NKX2-1 lead to lung epithelial hyperplasia, interstitial disease, and postnatal respiratory distress . In tumors, NKX2-1 has oncogenic and tumor suppressor functions, depending on the cell context, suggesting a dual role as a lineage specific factor contributing to lung cancer progression [5-8]. The downstream genes controlled by Nkx2-1 mediate its multiple functions in different cell contexts. In previous genome-wide studies we and others identified Nkx2-1 regulated protein-coding genes (mRNA) and Nkx2-1 direct binding targets [9-13] in mice and humans. However, non-coding microRNAs (miRNAs) regulated by Nkx2-1 have not been identified. Regulation of gene expression by miRNAs is a major mechanism of gene silencing , that controls translation and stability of target mRNAs in a cell and tissue specific manner. In the lung, the expression patterns and functions of specific miRNAs have been described during cell differentiation, development and in diseases such as lung fibrosis and cancer [14-16]. In development, specific miRNAs are differentially regulated over time and between sexes ; the miR-17-92 cluster plays important roles in cell differentiation and growth [18,19], whereas the Gata6-regulated cluster miR-302-367  controls multiple aspects of lung endoderm progenitor cell behavior. Several microRNAs including miR-29, miR-365, and miR-17-92 [14,19] control tumor cell proliferation, invasion and survival. However, the link between the key lung transcription factor Nkx2-1, downstream miRNAs and their predicted targets has not been addressed. In this study we have characterized miRNAs regulated by Nkx2-1 in a mouse lung cell line system by genome-wide analysis of mRNA and miRNA profiles and confirm the expression patterns of highly regulated miRNAs in normal mouse lung and in lungs expressing phosphorylation mutant Nkx2-1. In particular, we found a regulatory link between Nkx2-1, miR-200c and the nuclear factor I/B (Nfib) and myeloblastosis oncogene (Myb). These findings add new components to the gene regulatory network controlled by Nkx2-1 in lung epithelial cells that may have implications in the various roles of Nkx2-1 in development and disease.
Cell lines and tissues
microRNA and mRNA microarray experiments
Total RNA, isolated from three independently transduced Nkx2-1-shRNA and control MLE15 cell lines , was enriched in low molecular weight RNA using miRNeasy kit (Qiagen, Valencia, CA). RNA was labeled with FlashTag kit (Genisphere Inc., Hatfield, PA), and was hybridized to miRNA Galaxy arrays (Affymetrix, Santa Clara, CA). Arrays were scanned using Affymetrix GeneArray Scanner 3000 7G Plus. miRNA QC Tool software version 126.96.36.199 was used for background subtraction, detection p-value calculation and normalization. A two-sample t-test was performed to identify differentially regulated miRNA expression in Nkx2-1 knockdown cells; the Benjamini-Hochberg False Discovery Rate (FDR) was used to correct for multiple hypothesis comparisons . Genes with a p < 0.05 and an FDR adjusted p value < 0.2 were considered to be differentially expressed.
For mRNA expression analysis we followed the GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix, Santa Clara, CA) as described previously . The labeled fragmented DNA was hybridized to the Gene Arrays 1.0ST. After scanning, data were summarized using Affymetrix Expression Console (version 1.1). Robust Multi-Array Analysis algorithm  was used to generate gene-level data. A two-sample t-test was performed to identify differentially regulated mRNA expression in Nkx2-1 knockdown cells adjusted for multiple hypothesis comparisons . Genes with an adjusted p < 0.0005 were considered significant. Both mRNA and microRNA expression data are deposited in the Gene Expression Omnibus GSE47055. Predicted targets of miR-200c were downloaded from TargetScanMouse 6.2 (aggregate PCT > 0.1) . Gene Ontology database analysis was performed using GATHER (Gene Annotation Tool to Help Explain Relationships) . Detailed microRNA and mRNA array methods are described in Additional file 1.
Real time RT-PCR
mRNA expression was analyzed by RT-qPCR in total RNA samples using methods described previously . miRNA expression was analyzed in 1μg of total RNA, reverse transcribed using MicroRNA Reverse Transcription Kit (Applied Biosystems, Grand Island, NY). miRNA RT-qPCR analyses were performed with MicroRNA Assays (Applied Biosystems) in a StepOne Real-Time PCR System (Applied Biosystems). We used rnuRNA-6B in cell lines and snoRNA-202 in mouse tissues since the expression of these endogenous genes was more stable in the conditions tested in each system. Quantitative analysis was performed by the 2-ΔΔCt method.
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation assays (ChIPs) were performed as described previously , using 1 × 106 cells and 10 μl of NKX2-1 antibody (07-601-Upstate, Millipore, Billarica, MA) or IgG control (Santa Cruz Biotechnology, Dallas, TX). Equal volumes of immunoprecipitated DNA solution and 10% of the input DNA fragments were amplified by qPCR using a custom designed TaqMan assay (Applied Biosystems) within -1kb relative to the first nucleotide of the pre-miRNA sequence indicated in the UCSC Genome Browser mm10  (miR-1195, chr17:70860551–70861600; miR-200c, chr6:124718366–124719390) and quantified using TaqMan Master Mix (Applied Biosystems). Data were normalized to IgG and expressed as percentage of the input. Detailed ChIP methods are described in Additional file 1.
miRNA promoter cloning, transfections and luciferase assays
Genomic regions (1.1 kb) 5′ to the first nucleotide of miR-200c and of miR-221 pre-miRNA sequences were retrieved from the UCSC Mouse Genome Browser . We used oligonucleotides with restriction enzyme adaptors to amplify ~ 0.9-1kb of each region by PCR (miR-200c F 5′-SacI CAGGCAGACACTGCCATCT-3′, R 5′-HindIII CTACCCAACCAGTCCACCTCC-3′; miR-221 F 5′-SacI AGGAGAGGCCCTTGGTATAG-3′, R 5′-HindIII GTTCAGCCTGCAAATTATCC). Amplicons were ligated into the pGL3-basic luciferase vector (Promega, Madison, WI) and confirmed by sequencing. After several attempts we were unable to clone the 5′flanking region of miR-1195. Thus, miR-200c-Luc and miR-221-Luc plasmids were transiently transfected using Lipofectamine 2000 (Life Technologies, Grans Island, NY) in the cell lines described above. A renilla luciferase expressing vector was co-transfected for transfection efficiency control. Cells were harvested 48 h after transfection and luciferase activity measured using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase signal was normalized to Renilla luciferase and data expressed relative to the pGL3-basic vector.
3′-UTR luciferase assays
All plasmids and reagents were obtained from Genecopeia (Rockville, MD). Plasmids harboring a Firefly luciferase reporter and the 3′UTR flanking region of the mir-200c predicted targets Myb (MmiT029722-MT01), Nfib (MmiT027729a2-MT01), and Six1 (MmiT028207-MT01), or a control vector (CmiT000001-MT01) were co-transfected in MLE15 cells either with a pre-miR-200c plasmid (MmiR3304-MR01) or with an scrambled control clone (CmiR0001-MR01) using EndoFectin Plus transfection kit. Different ratios of 3′UTR plasmids and miR-200c plasmid were evaluated (data not shown). A 1:5 ratio rendered the most consistent effect with all plasmids. Renilla luciferase expressing vector was co-transfected for transfection efficiency control. Cells were harvested 48 h after transfection and luciferase activity was measured using the Luc-Pair miR Luciferase Assay. Firefly luciferase signal was normalized to Renilla luciferase and data expressed relative to the Control Luciferase vector.
miRNA-1195 antagomir assay
MLE15 cells were transduced with miRVana miR-1195 inhibitor (4464084 ID MH13628 (Life Technologies) using Lipofectamine RNAiMAX (Life Technologies) protocol.
Data were obtained from at least three independent experiments (N = 3) and presented as mean ± SEM. The significance of differences was calculated using t-test for two-group unpaired comparisons. P < 0.05 was considered statistically significant.
miRNAs downstream of Nkx2-1 in lung epithelial cells
miRNAs differentially expressed in Nkx2-1shRNA vs non-silencing control in MLE15 cells
miRNA expression is altered in Nkx2-1 phosphorylation-mutant lungs
Nkx2-1 null mice have an extreme lung phenotype due to failing in branching morphogenesis resulting in a large sac lined by a simple epithelium [2,3]. Therefore, we tested the effect of Nkx2-1 on specific miRNAs in vivo in wild type and in Nkx2-1 phosphorylation-mutant lungs at E19.5 by RT-qPCR. These mice have normal branching morphogenesis but show alterations in distal epithelial differentiation and expression of lung function genes. By RT-qPCR analysis we showed that absence of phosphorylated NKX2-1 results in a significant increment in the levels of miR-200c and miR-221; miR-1195 is down-regulated although no significantly, showing a similar trend than in Nkx2-1 knockdown experiments in MLE15 cells (Figure 2C).
NKX2-1 protein binds to miRNA regulatory regions
Nkx2-1 controls transcriptional activity of the miR-200c 5′ flanking region
To evaluate the transcriptional activity of the 5′flanking regions of selected microRNAs we cloned ~1kb 5′ to the miR-200c and to the miR-221 transcripts in a luciferase reporter vector. Both fragments contain several NKX2-1 consensus binding core CAAG/CTTG sites (Figure 3A). The miR-200c 5′flanking fragment is highly conserved in humans where it is transcriptionally active . We transiently transfected the mouse constructs in MLE15 cell lines previously transduced with lentivirus expressing Nkx2-1-shRNA or non-silencing control (Figure 3D). The 5′ flanking region of miR-200c exhibits intense transcriptional activity (15-fold ± 0.66; p = 0.0005) with normal levels of Nkx2-1. Unexpectedly, we found that knock-down of Nkx2-1 resulted in lower transcriptional activity of this 1kb fragment (8-fold ± 0.24; p = 1.6 × 10−6). The 1kb fragment 5′ to miR-221 was transcriptionally inactive in the same conditions (Figure 3D). So, Nkx2-1 has a strong effect in controlling the levels of miR-200c expression but this control might be indirect. Nkx2-1 is recognized to act mainly as a transcriptional activator , binding to the promoters/enhancers of 58% of activated downstream genes but only to 23% of genes repressed by Nkx2-1. Therefore, most genes repressed by Nkx2-1 do so by mechanisms other than direct promoter binding as may be the case for miR-200c. Alternatively, the elements mediating Nkx2-1 repression of miR-200c might be located in regulatory regions beyond the -1kb 5′ flanking region. Analysis of histone marks in distinct microRNA loci to identify putative transcription start sites (TSS) indicates that a putative TSS of miR-200c in mouse cells might be within 5 kb of the pre-miRNA sequence .
Expression patterns of predicted targets of Nkx2-1-regulated miRNAs
miR-200c controls expression of its predicted targets Nfib and Myb
A significant number of miR-200c targets whose expression was negatively correlated to miR-200c in the microarray analysis were overrepresented in the transcriptional regulation GO category (Additional file 4: Table S3). The transcription factors Gata4 , Ntf3  and Sox2  are experimentally validated targets of miR-200c in human cells. We selected Nfib, Six1, and Myb to perform 3′UTR-luciferase reporter assays in MLE 15 cells expressing pre-miR-200c or scrambled control. Significant reduction of luciferase activity was observed in Nfib 3′UTR-Luciferase (0.63 ± 0.06; p = 0.016) and in Myb 3′UTR-Luciferase (0.62 ± 0.04; p = 0.002) transfected cells in the presence of pre-miR-200c compared to the scrambled control (Figure 4D). No difference in luciferase activity was observed in cells co-transfected with Six-1-Luciferase and pre-miR-200c or scrambled plasmids.
miR-1195 controls expression of its predicted targets
To further evaluate the effect of miR-1195 on predicted downstream genes, we transfected MLE15 cells with a miR-1195 inhibitor to reduce its expression levels. After 48 h of transduction, expression levels of miR-1195 were measured by RT-qPCR confirming that miR-1195 was reduced by 50% (Figure 5D). Expression of Map3k2 and Cyp2s1 was significantly up-regulated by the miR-1195 inhibitor (Figure 5D). Because miR-1195 is a mouse specific miRNA no further analyses were performed to determine direct regulation of the identified targets. hsa-miR-584 shares partial homology to mmu-miR-1195  and will be analyzed in future studies.
In this study we have identified microRNAs whose expression is influenced by knock-down of Nkx2-1. Using genome- wide miRNA expression profiling in a lung adenocarcinoma-derived mouse epithelial cell line (MLE15) , we observed that reduction of Nkx2-1 levels to approximately half of that in control cells promoted significant and reproducible changes in miRNA expression patterns, including a high up-regulation of miR-200c. The top up- and down- regulated miRNAs are expressed in normal mouse fetal lung and their level of expression is also altered in mice lacking functional phosphorylated-Nkx2-1. Furthermore, we present evidence of a regulatory link between Nkx2-1, mirR-200c and the downstream transcription factors Nfib and Myb. The studies indicate that down-regulation of Nkx2-1 de-represses miR-200c, either by a direct or an indirect mechanism. Downstream, miR-200c reduces the expression of its predicted targets Nfib and Myb.
miR-200c was initially identified as a lung-specific miRNA in rats . Expression of miR-200c is higher in adult rat lung alveolar cells than fibroblasts, and its expression is lower during development than in adult lung. Most studies about miR-200c have been performed in the mesenchymal-epithelial transition (MET) context. One well studied function of the miR-200 family is the induction of an epithelial phenotype by inhibiting the transcriptional repressor Zeb2 and thereby enhancing E-cadherin expression [31,35]. Supporting its role in maintaining an epithelial phenotype in the lung, miR-200c expression is significantly reduced in the lung of mice with experimental fibrosis and in lungs of IPF patients where the epithelium undergoes profound alterations by acquiring some mesenchymal characteristics. In lung adenocarcinoma a high-level of NKX2-1 expression is significantly associated with longer overall survival , whereas a high-level of miR-200c expression is associated with shorter overall survival  supporting the inverse correlation. But in epithelial cell contexts, as the ones described in this work, increased miR-200c may regulate cellular processes other than MET, such as proliferation, or survival of lung cells.
Our data shows that miR-200c represses Nfib and Myb genes. These transcription factors [38,39] and other known miR-200c targets such as E2F3  and Kras  have been linked to lung epithelial proliferation in development and in tumorigenesis acting as oncogenes. Nfib is highly expressed in the embryonic lung epithelium and in the mesenchyme, but its expression gets restricted to the epithelium at late gestation. Absence of Nfib in null mutant mice results in early postnatal lethality with severe lung hypoplasia . In small cell lung carcinomas NFIB regulates cell viability and proliferation  and it is considered an oncogene. The other predicted target, Myb has been recently linked to the differentiation of airway epithelial cells , and was shown to be regulated by miR-200c in glioblastomas  and breast cancer cells . In the latter, the human MYB 3′UTR was shown to contain miR-200c binding sites. Therefore, a reduction of Nkx2-1may modulate the proliferative activity of lung epithelial cells not only by direct inhibition of cyclin B, as it was previously described [12,13], but also through direct or indirect activation of miR-200c to inhibit downstream oncogenes.
We identified other miRNAs regulated by Nkx2-1 expression. miR-1195, for instance, is positively regulated by Nkx2-1 in the lung epithelial cell system and in normal vs. phosphorylation mutant lungs. miR-1195 is a mouse specific miRNA , enriched in epithelial structures in the embryo and moderately homologous to the human miRNA hsa-miR-584-3p (miRBase ). Predicted targets of miR-1195, including the mitogen-activated protein kinase Map3k2  and the extra-hepatic cytochrome P450 enzyme Cyp2s1 [47,48], responded to changes in miR-1195 levels. Because miR-1195 is not found in humans, its close homolog hsa-miR-584-3p will be the focus of further studies.
Overall, our findings suggest that modulation of the level of expression of Nkx2-1 has a high impact on downstream regulatory events mediated by miRNAs in mouse lung epithelial cell lines and in lung tissue. Particularly, miR-200c, negatively regulated by Nkx2-1, reduces the expression of downstream targets Nfib and Myb. Because of the high conservation between mouse and human homologs of Nkx2-1, Nfib, Myb, and miR-200c it is likely that these links apply to human cells. The individual functions of NKX2-1 [5-7,49], miR-200c [30,50,51], MYB [39,52], and NFIB  in lung development and/or lung cancer have been widely documented. Thus, our future analyses are aimed at characterizing these novel regulatory links between NKX2-1:miR-200c: NFIB, or MYB in propagating fluctuations in the levels of NKX2-1 in human lung tumors.
We thank Drs. Yuxia Cao, Avi Spira and Catalina Perdomo for numerous suggestions and discussions about the project. We thank the Boston University Microarray Resource Facility and Qinying Gao for technical support. Supported by NIH P01 HL47049 (MIR), K01 HL121028 (JBT) and UL1-TR000157 (CTSI Bioinformatics Support).
- Boggaram V. Thyroid transcription factor-1 (TTF-1/Nkx2.1/TITF1) gene regulation in the lung. Clin Sci (Lond). 2009;116(1):27–35.View ArticleGoogle Scholar
- Kimura S, Hara Y, Pineau T, Fernandez-Salguero P, Fox CH, Ward JM, et al. The T/ebp null mouse: thyroid-specific enhancer-binding protein is essential for the organogenesis of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev. 1996;10(1):60–9.PubMedView ArticleGoogle Scholar
- Kimura S, Ward JM, Minoo P. Thyroid-specific enhancer-binding protein/thyroid transcription factor 1 is not required for the initial specification of the thyroid and lung primordia. Biochimie. 1999;81(4):321–7.PubMedView ArticleGoogle Scholar
- Galambos C, Levy H, Cannon CL, Vargas SO, Reid LM, Cleveland R, et al. Pulmonary pathology in thyroid transcription factor-1 deficiency syndrome. Am J Respir Crit Care Med. 2010;182(4):549–54.PubMedPubMed CentralView ArticleGoogle Scholar
- Kwei KA, Kim YH, Girard L, Kao J, Pacyna-Gengelbach M, Salari K, et al. Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer. Oncogene. 2008;27(25):3635–40.PubMedPubMed CentralView ArticleGoogle Scholar
- Mu D. The complexity of thyroid transcription factor 1 with both pro- and anti-oncogenic activities. J Biol Chem. 2013;288(35):24992–5000.PubMedPubMed CentralView ArticleGoogle Scholar
- Perner S, Wagner PL, Soltermann A, LaFargue C, Tischler V, Weir BA, et al. TTF1 expression in non-small cell lung carcinoma: association with TTF1 gene amplification and improved survival. J Pathol. 2009;217(1):65–72.PubMedView ArticleGoogle Scholar
- Winslow MM, Dayton TL, Verhaak RG, Kim-Kiselak C, Snyder EL, Feldser DM, et al. Suppression of lung adenocarcinoma progression by Nkx2-1. Nature. 2011;473(7345):101–4.PubMedPubMed CentralView ArticleGoogle Scholar
- Kolla V, Gonzales LW, Gonzales J, Wang P, Angampalli S, Feinstein SI, et al. Thyroid transcription factor in differentiating type II cells: regulation, isoforms, and target genes. Am J Respir Cell Mol Biol. 2007;36(2):213–25.PubMedView ArticleGoogle Scholar
- Maeda Y, Tsuchiya T, Hao H, Tompkins DH, Xu Y, Mucenski ML, et al. Kras(G12D) and Nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung. J Clin Invest. 2012;122(12):4388–400.PubMedPubMed CentralView ArticleGoogle Scholar
- Snyder EL, Watanabe H, Magendantz M, Hoersch S, Chen TA, Wang DG, et al. Nkx2-1 represses a latent gastric differentiation program in lung adenocarcinoma. Mol Cell. 2013;50(2):185–99.PubMedPubMed CentralView ArticleGoogle Scholar
- Tagne JB, Gupta S, Gower AC, Shen SS, Varma S, Lakshminarayanan M, et al. Genome-wide analyses of Nkx2-1 binding to transcriptional target genes uncover novel regulatory patterns conserved in lung development and tumors. PLoS One. 2012;7(1):e29907.PubMedPubMed CentralView ArticleGoogle Scholar
- Watanabe H, Francis JM, Woo MS, Etemad B, Lin W, Fries DF, et al. Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target. Genes Dev. 2013;27(2):197–210.PubMedPubMed CentralView ArticleGoogle Scholar
- Sessa R, Hata A. Role of microRNAs in lung development and pulmonary diseases. Pulm Circ. 2013;3(2):315–28.PubMedPubMed CentralView ArticleGoogle Scholar
- Dong J, Jiang G, Asmann YW, Tomaszek S, Jen J, Kislinger T, et al. MicroRNA networks in mouse lung organogenesis. PLoS One. 2010;5(5):e10854.PubMedPubMed CentralView ArticleGoogle Scholar
- Pandit KV, Milosevic J, Kaminski N. MicroRNAs in idiopathic pulmonary fibrosis. Transl Res. 2011;157(4):191–9.PubMedView ArticleGoogle Scholar
- Mujahid S, Logvinenko T, Volpe MV. Nielsen HC: miRNA regulated pathways in late stage murine lung development. BMC Dev Biol. 2013;13(1):13.PubMedPubMed CentralView ArticleGoogle Scholar
- Lu Y, Thomson JM, Wong HY, Hammond SM, Hogan BL. Transgenic over-expression of the microRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Dev Biol. 2007;310(2):442–53.PubMedPubMed CentralView ArticleGoogle Scholar
- Ventura A, Young AG, Winslow MM, Lintault L, Meissner A, Erkeland SJ, et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell. 2008;132(5):875–86.PubMedPubMed CentralView ArticleGoogle Scholar
- Tian Y, Zhang Y, Hurd L, Hannenhalli S, Liu F, Lu MM, et al. Regulation of lung endoderm progenitor cell behavior by miR302/367. Development. 2011;138(7):1235–45.PubMedPubMed CentralView ArticleGoogle Scholar
- Wikenheiser KA, Vorbroker DK, Rice WR, Clark JC, Bachurski CJ, Oie HK, et al. Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc Natl Acad Sci U S A. 1993;90(23):11029–33.PubMedPubMed CentralView ArticleGoogle Scholar
- Cao Y, Vo T, Millien G, Tagne JB, Kotton D, Mason RJ, et al. Epigenetic mechanisms modulate thyroid transcription factor 1-mediated transcription of the surfactant protein B gene. J Biol Chem. 2010;285(3):2152–64.PubMedView ArticleGoogle Scholar
- DeFelice M, Silberschmidt D, DiLauro R, Xu Y, Wert SE, Weaver TE, et al. TTF-1 phosphorylation is required for peripheral lung morphogenesis, perinatal survival, and tissue-specific gene expression. J Biol Chem. 2003;278(37):35574–83.PubMedView ArticleGoogle Scholar
- Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B Methodol. 1995;57(1):289–300.Google Scholar
- Varma S, Cao Y, Tagne JB, Lakshminarayanan M, Li J, Friedman TB, et al. The transcription factors grainyhead-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differentiation. J Biol Chem. 2012;287(44):37282–95.PubMedPubMed CentralView ArticleGoogle Scholar
- Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics. 2003;4(2):249–64.PubMedView ArticleGoogle Scholar
- Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009;19(1):92–105.PubMedPubMed CentralView ArticleGoogle Scholar
- Chang JT, Nevins JR. GATHER: a systems approach to interpreting genomic signatures. Bioinformatics. 2006;22(23):2926–33.PubMedView ArticleGoogle Scholar
- Karolchik D, Hinrichs AS, Kent WJ. The UCSC genome browser. Curr Protoc Bioinformatics. 2012;Chapter 1:Unit1 4.PubMedGoogle Scholar
- Tejero R, Navarro A, Campayo M, Vinolas N, Marrades RM, Cordeiro A, et al. miR-141 and miR-200c as markers of overall survival in early stage non-small cell lung cancer adenocarcinoma. PLoS One. 2014;9(7):e101899.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang B, Zhang Z, Xia S, Xing C, Ci X, Li X, et al. KLF5 activates microRNA 200 transcription to maintain epithelial characteristics and prevent induced epithelial-mesenchymal transition in epithelial cells. Mol Cell Biol. 2013;33(24):4919–35.PubMedPubMed CentralView ArticleGoogle Scholar
- Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, Johnstone S, et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell. 2008;134(3):521–33.PubMedPubMed CentralView ArticleGoogle Scholar
- Huang HN, Chen SY, Hwang SM, Yu CC, Su MW, Mai W, et al. miR-200c and GATA binding protein 4 regulate human embryonic stem cell renewal and differentiation. Stem Cell Res. 2014;12(2):338–53.PubMedView ArticleGoogle Scholar
- Howe EN, Cochrane DR, Cittelly DM, Richer JK. miR-200c targets a NF-kappaB up-regulated TrkB/NTF3 autocrine signaling loop to enhance anoikis sensitivity in triple negative breast cancer. PLoS One. 2012;7(11):e49987.PubMedPubMed CentralView ArticleGoogle Scholar
- Wellner U, Schubert J, Burk UC, Schmalhofer O, Zhu F, Sonntag A, et al. The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs. Nat Cell Biol. 2009;11(12):1487–95.PubMedView ArticleGoogle Scholar
- Griffiths-Jones S, Saini HK, van Dongen S. miRBase: tools for microRNA genomics. Nucleic Acids Res. 2008;36(Database issue):D154–8.PubMedGoogle Scholar
- Wang Y, Weng T, Gou D, Chen Z, Chintagari NR, Liu L. Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research. BMC Genomics. 2007;8:29.PubMedPubMed CentralView ArticleGoogle Scholar
- Grunder A, Ebel TT, Mallo M, Schwarzkopf G, Shimizu T, Sippel AE, et al. Nuclear factor I-B (Nfib) deficient mice have severe lung hypoplasia. Mech Dev. 2002;112(1–2):69–77.PubMedView ArticleGoogle Scholar
- Griffin CA, Baylin SB. Expression of the c-myb oncogene in human small cell lung carcinoma. Cancer Res. 1985;45(1):272–5.PubMedGoogle Scholar
- Tao T, Liu D, Liu C, Xu B, Chen S, Yin Y, et al. Autoregulatory feedback loop of EZH2/miR-200c/E2F3 as a driving force for prostate cancer development. Biochim Biophys Acta. 2014;1839(9):858–65.PubMedView ArticleGoogle Scholar
- Kopp F, Wagner E, Roidl A. The proto-oncogene KRAS is targeted by miR-200c. Oncotarget. 2014;5(1):185–95.PubMedGoogle Scholar
- Dooley AL, Winslow MM, Chiang DY, Banerji S, Stransky N, Dayton TL, et al. Nuclear factor I/B is an oncogene in small cell lung cancer. Genes Dev. 2011;25(14):1470–5.PubMedPubMed CentralView ArticleGoogle Scholar
- Cesi V, Casciati A, Sesti F, Tanno B, Calabretta B, Raschella G. TGFbeta-induced c-Myb affects the expression of EMT-associated genes and promotes invasion of ER+ breast cancer cells. Cell Cycle. 2011;10(23):4149–61.PubMedView ArticleGoogle Scholar
- Siebzehnrubl FA, Silver DJ, Tugertimur B, Deleyrolle LP, Siebzehnrubl D, Sarkisian MR, et al. The ZEB1 pathway links glioblastoma initiation, invasion and chemoresistance. EMBO Mol Med. 2013;5(8):1196–212.PubMedPubMed CentralView ArticleGoogle Scholar
- Yuan Z, Sun X, Liu H, Xie J. MicroRNA genes derived from repetitive elements and expanded by segmental duplication events in mammalian genomes. PLoS One. 2011;6(3):e17666.PubMedPubMed CentralView ArticleGoogle Scholar
- Kesavan K, Lobel-Rice K, Sun W, Lapadat R, Webb S, Johnson GL, et al. MEKK2 regulates the coordinate activation of ERK5 and JNK in response to FGF-2 in fibroblasts. J Cell Physiol. 2004;199(1):140–8.PubMedView ArticleGoogle Scholar
- Thum T, Erpenbeck VJ, Moeller J, Hohlfeld JM, Krug N, Borlak J. Expression of xenobiotic metabolizing enzymes in different lung compartments of smokers and nonsmokers. Environ Health Perspect. 2006;114(11):1655–61.PubMedPubMed CentralGoogle Scholar
- Wang SL, He XY, Hong JY. Human cytochrome p450 2s1: lack of activity in the metabolic activation of several cigarette smoke carcinogens and in the metabolism of nicotine. Drug Metab Dispos. 2005;33(3):336–40.PubMedView ArticleGoogle Scholar
- Yamaguchi T, Hosono Y, Yanagisawa K, Takahashi T. NKX2-1/TTF-1: an enigmatic oncogene that functions as a double-edged sword for cancer cell survival and progression. Cancer Cell. 2013;23(6):718–23.PubMedView ArticleGoogle Scholar
- Ceppi P, Mudduluru G, Kumarswamy R, Rapa I, Scagliotti GV, Papotti M, et al. Loss of miR-200c expression induces an aggressive, invasive, and chemoresistant phenotype in non-small cell lung cancer. Mol Cancer Res. 2010;8(9):1207–16.PubMedView ArticleGoogle Scholar
- Feng X, Wang Z, Fillmore R, Xi Y. MiR-200, a new star miRNA in human cancer. Cancer Lett. 2014;344(2):166–73.PubMedView ArticleGoogle Scholar
- Yang ZH, Zheng R, Gao Y, Zhang Q, Zhang H. Abnormal gene expression and gene fusion in lung adenocarcinoma with high-throughput RNA sequencing. Cancer Gene Ther. 2014;21(2):74–82.PubMedView ArticleGoogle Scholar
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