miR Promoters Research Articles

Short Chain Fatty Acid Sodium Butyrate Increases miR-21, miR-143 and miR-145 Expression in Human Colorectal Cancer HCT-116 Cell Line.

Sodium butyrate (NaBu) is a short-chain fatty acid; it is one of the histone deacetylase inhibitors, which can alter both genetic and epigenetic expressions. The present study aimed to elucidate the effect of Na-Bu on the expression of miR-21, miR-143, and miR-145 in human colorectal cancer HCT-116 cell lines. This study was done in Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. HCT-116 cell line was treated with diverse concentrations of NaBu (6.25 mM to 200 mM) at 24, 48, and 72 h. MTT assay was used for assessing the cytotoxicity. Quantitative Real-Time-PCR was performed to investigate the gene expression of miR-21, miR-143, and miR-145. IC50 values were evaluated by MTT assay. IC50 for HCT-116 was 50 mM, 12.5 mM, and 6.25 mM for 24, 48, and 72 h of incubation, respectively. According to the Real-Time-PCR results, 50 mM NaBu after 24 h caused a significant up-regulation in the expression of the miR-21, miR-143, and miR-145 (P<0.05). In 48 h, incubation, 12.5 mM NaBu caused a significant up-regulation in the expression of the miR-21, miR-143, and miR-145 (P<0.05). In treated cells with 6.25 mM NaBu after 72 h of incubation caused a significant up-regulation in the expression of the miR-21, miR-143, and miR-145 compared with untreated cells (P<0.05). The upregulation of miR-21, miR-143, and miR-145 expression are mediated by transcriptional regulation and the activation of this miR promoter is modulated by histone acetylation. The employment of NaBu may represent a promising approach for improving HDACi drug-based therapies for colon cancers.

A mechanism by which gut microbiota elevates permeability and inflammation in obese/diabetic mice and human gut

ObjectiveAmple evidence exists for the role of abnormal gut microbiota composition and increased gut permeability (‘leaky gut’) in chronic inflammation that commonly co-occurs in the gut in both obesity and...

Spliceosome disassembly factors ILP1 and NTR1 promote miRNA biogenesis in Arabidopsis thaliana.

The intron-lariat spliceosome (ILS) complex is highly conserved among eukaryotes, and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, Increased Level of Polyploidy1-1D (ILP1) and NTC-Related protein 1 (NTR1), positively regulate microRNA (miRNA) biogenesis by facilitating transcriptional elongation of MIRNA (MIR) genes in Arabidopsis thaliana. ILP1 and NTR1 formed a stable complex and co-regulated alternative splicing of more than a hundred genes across the Arabidopsis genome, including some primary transcripts of miRNAs (pri-miRNAs). Intriguingly, pri-miRNAs, regardless of having introns or not, were globally down-regulated when the ILP1 or NTR1 function was compromised. ILP1 and NTR1 interacted with core miRNA processing proteins Dicer-like 1 and Serrate, and were required for proper RNA polymerase II occupancy at elongated regions of MIR chromatin, without affecting either MIR promoter activity or pri-miRNA decay. Our results provide further insights into the regulatory role of spliceosomal machineries in the biogenesis of miRNAs.

Epigenetic Modification of MicroRNA-200b Contributes to Diabetic Vasculopathy

Hyperglycemia (HG) induces genome-wide cytosine demethylation. Our previous work recognized miR-200b as a critical angiomiR, which must be transiently downregulated to initiate wound angiogenesis. Under HG, miR-200b downregulation is not responsive to injury. Here, we demonstrate that HG may drive vasculopathy by epigenetic modification of a miR promoter. In human microvascular endothelial cells (HMECs), HG also lowered DNA methyltransferases (DNMT-1 and DNMT-3A) and compromised endothelial function as manifested by diminished endothelial nitric oxide (eNOS), lowered LDL uptake, impaired Matrigel tube formation, lower NO production, and compromised VE-cadherin expression. Bisulfite-sequencing documented HG-induced miR-200b promoter hypomethylation in HMECs and diabetic wound-site endothelial cells. In HMECs, HG compromised endothelial function. Methyl donor S-adenosyl-L-methionine (SAM) corrected miR-200b promoter hypomethylaton and rescued endothelial function. Invivo, wound-site administration of SAM to diabetic mice improved wound perfusion by limiting the pathogenic rise of miR-200b. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics and ingenuity pathway analysis identified HG-induced proteins and principal clusters in HMECs sensitive to the genetic inhibition of miR-200b. This work presents the first evidence of the miR-200b promoter methylation as a critical determinant of diabetic wound angiogenesis.

STV1, a ribosomal protein, binds primary microRNA transcripts to promote their interaction with the processing complex in Arabidopsis

MicroRNAs (miRNAs) are key regulators of gene expression. They are processed from primary miRNA transcripts (pri-miRNAs), most of which are transcribed by DNA-dependent polymerase II (Pol II). miRNA levels are precisely controlled to maintain various biological processes. Here, we report that SHORT VALVE 1 (STV1), a conserved ribosomal protein, acts in miRNA biogenesis in Arabidopsis A portion of STV1 localizes in the nucleus and binds pri-miRNAs. Using pri-miR172b as a reporter, we show that STV1 binds the stem-loop flanked by a short 5' arm within pri-miRNAs. Lack of STV1 reduces the association of pri-miRNAs with their processing complex. These data suggest that STV1 promotes miRNA biogenesis through facilitating the recruitment of pri-miRNAs to their processing complex. Furthermore, we show that STV1 indirectly involves in the occupancy of Pol II at the promoters of miRNA coding genes (MIR) and influences MIR promoter activities. Based on these results, we propose that STV1 refines the accumulation of miRNAs through its combined effects on pri-miRNA processing and transcription. This study uncovers an extraribosomal function of STV1.

Targeting BTK through microRNA in chronic lymphocytic leukemia

AbstractBruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

MicroRNA-Based Therapy of GATA2-Deficient Vascular Disease.

The transcription factor GATA2 orchestrates the expression of many endothelial-specific genes, illustrating its crucial importance for endothelial cell function. The capacity of this transcription factor in orchestrating endothelial-important microRNAs (miRNAs/miR) is unknown. Endothelial GATA2 was functionally analyzed in human endothelial cells in vitro. Endogenous short interfering RNA-mediated knockdown and lentiviral-based overexpression were applied to decipher the capacity of GATA2 in regulating cell viability and capillary formation. Next, the GATA2-dependent miR transcriptome was identified by using a profiling approach on the basis of quantitative real-time polymerase chain reaction. Transcriptional control of miR promoters was assessed via chromatin immunoprecipitation, luciferase promoter assays, and bisulfite sequencing analysis of sites in proximity. Selected miRs were modulated in combination with GATA2 to identify signaling pathways at the angiogenic cytokine level via proteome profiler and enzyme-linked immunosorbent assays. Downstream miR targets were identified via bioinformatic target prediction and luciferase reporter gene assays. In vitro findings were translated to a mouse model of carotid injury in an endothelial GATA2 knockout background. Nanoparticle-mediated delivery of proangiogenic miR-126 was tested in the reendothelialization model. GATA2 gain- and loss-of-function experiments in human umbilical vein endothelial cells identified a key role of GATA2 as master regulator of multiple endothelial functions via miRNA-dependent mechanisms. Global miRNAnome-screening identified several GATA2-regulated miRNAs including miR-126 and miR-221. Specifically, proangiogenic miR-126 was regulated by GATA2 transcriptionally and targeted antiangiogenic SPRED1 and FOXO3a contributing to GATA2-mediated formation of normal vascular structures, whereas GATA2 deficiency led to vascular abnormalities. In contrast to GATA2 deficiency, supplementation with miR-126 normalized vascular function and expression profiles of cytokines contributing to proangiogenic paracrine effects. GATA2 silencing resulted in endothelial DNA hypomethylation leading to induced expression of antiangiogenic miR-221 by GATA2-dependent demethylation of a putative CpG island in the miR-221 promoter. Mechanistically, a reverted GATA2 phenotype by endogenous suppression of miR-221 was mediated through direct proangiogenic miR-221 target genes ICAM1 and ETS1. In a mouse model of carotid injury, GATA2 was reduced, and systemic supplementation of miR-126-coupled nanoparticles enhanced miR-126 availability in the carotid artery and improved reendothelialization of injured carotid arteries in vivo. GATA2-mediated regulation of miR-126 and miR-221 has an important impact on endothelial biology. Hence, modulation of GATA2 and its targets miR-126 and miR-221 is a promising therapeutic strategy for treatment of many vascular diseases.

Identification of new p53 target microRNAs by bioinformatics and functional analysis

BackgroundThe tumor suppressor p53 is a sequence-specific transcription factor that regulates an extensive network of coding genes, long non-coding RNAs and microRNAs, that establish intricate gene regulatory circuits influencing many cellular responses beyond the prototypical control of cell cycle, apoptosis and DNA repair.MethodsUsing bioinformatic approaches, we identified an additional group of candidate microRNAs (miRs) under direct p53 transcriptional control. To validate p53 family-mediated responsiveness of the newly predicted target miRs we first evaluated the potential for wild type p53, p63β and p73β to transactivate from p53 response elements (REs) mapped in the miR promoters, using an established yeast-based assay.ResultsThe REs found in miR-10b, -23b, -106a, -151a, -191, -198, -202, -221, -320, -1204, -1206 promoters were responsive to p53 and 8 of them were also responsive to p63β or p73β. The potential for germline p53 mutations to drive transactivation at selected miR-associated REs was also examined. Chromatin Immuno-Precipitation (ChIP) assays conducted in doxorubicin-treated MCF7 cells and HCT116 p53+/+ revealed moderate induction of p53 occupancy at the miR-202, -1204, -1206, -10b RE-containing sites, while weak occupancy was observed for the miR-23b-associated RE only in MCF7 cells. RT-qPCR analyses cells showed modest doxorubicin- and/or Nutlin-dependent induction of the levels of mature miR-10b, -23b, -151a in HCT116 p53+/+ and MCF7 cells. The long noncoding RNA PVT1 comprising miR-1204 and −1206 was weakly induced only in HCT116 p53+/+ cells, but the mature miRs were not detected. miR-202 expression was not influenced by p53-activating stimuli in our cell systems.ConclusionsOur study reveals additional miRs, particularly miR-10b and miR-151a, that could be directly regulated by the p53-family of transcription factors and contribute to the tuning of p53-induced responses.

Signal Transducer and Activator Of Transcription 3 Activates and Deregulates Micro RNA Expression In Chronic Lymphocytic Leukemia Cells

MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL. Disclosures:No relevant conflicts of interest to declare.

Identification and analysis of the proximal promoters of microRNA genes in Arabidopsis

Endogenous microRNAs (miRNAs) modulate gene expression at the post-transcription level. In plants, a vast majority of MIR genes are thought to be transcribed by RNA Polymerase II (Pol II). However, promoter organization is currently unknown for most plant MIR genes. This deficiency prevents a comprehensive understanding of miRNA-mediated gene networks. In this study, we performed Pol II chromatin immunoprecipitation (ChIP) analysis in Arabidopsis using a genome tiling microarray. Distinct Pol II binding was found at most MIR loci, which allowed prediction of the transcription start sites (TSSs) for 167 MIR genes in Arabidopsis that was validated by average free energy profiling. By employing 99 position weight matrices (PWM), we systematically scanned the regulatory regions upstream of the TSSs. We discovered eleven and ten cis-elements that are statistically over- and under-represented in MIR promoters, respectively. Thus, analysis of Pol II binding provides a new perspective for studying miRNAs in plants.

Abstract 2286: p53-miR-dependent post-transcriptional circuits: mechanisms, targets and inter-individual variation

Abstract Using bioinformatics approaches, we identified a group of candidate microRNAs (miRs) for direct p53 transcriptional control. To validate p53 family-mediated control of the newly predicted target miRs we evaluated the potential for wild type p53, p63α and p73α to transactivate from p53 response elements (REs) mapped at the miR promoters. Results obtained with an established yeast-based assay indicated that the REs found at miR-10b, -23b, -106a, -151, -191, -198, -202, -221, -320, -1204, -1026 genes were responsive to p53 of which 8 were also responsive to p63α or p73α. ChIP assays in doxorubicin-treated HCT116 cells -p53+/+ vs p53-/– revealed moderate p53 occupancy at the miR-202, -1206 sites, and weak occupancy at miR-10b, -191. RT-qPCR analyses in HCT116 and MCF7 cells showed modest doxorubicin- and/or p53-dependent regulation for miR-23b, -151, -191. We then focused on miR-191 and its potential impact on MDM4, an important modulator of p53. The A&amp;gt;C SNP (rs4245739) in the proximal portion of the MDM4 3′UTR occurs in a predicted binding site for miR-191, with the A allele resulting in a mismatch at the predicted miR-191 seed. This SNP is in strong association with the tag-SNP (rs2369244) within the MDM4 haplotype that was reported to impact on cancer risk. Using MCF7 and HCT116 cells that are heterozygous for rs4245739, we examined at the endogenous MDM4 gene level the impact of varying the expression of miR-191. A slight allelic imbalance was observed by allele-specific qPCR when miR-191 was over-expressed, and the co-transfection of miR-34a, for which seed sequences are predicted in the distal portion of the MDM4-3′UTR, had an additive effect, leading to ∼20% reduction of the C-allele. Western blot analyses indicated that steady-state MDM4 protein levels were reduced by miR-191 and/or miR-34a overexpression. Our study reveals additional miRs that could be directly regulated by the p53-family of transcription factors and could contribute to the tuning of p53-induced responses with the possibility of inter-individual variations due to functional SNPs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2286. doi:1538-7445.AM2012-2286

Abstract 977: Elevated MDM2 protein contributes to activity of MDM2 antagonists by enabling p53 induction of antitumor microRNAs

Abstract Small-molecule inhibitors of p53-MDM2 binding, the nutlins, can free p53 from MDM2 control and effectively activate its growth suppressive and pro-apoptotic activity in cancer cells (Vassilev et al., Science, 2004, 303, 844). Optimized members of this family of molecules are currently under clinical investigation as novel therapeutic modality for cancers expressing wild-type p53. We have shown previously that MDM2 antagonists are most effective in cancer cells that overproduce MDM2 as a result of mdm2 gene amplification (Tovar et al., PNAS, 2006, 103, 1888). Here, we show that reduction of MDM2 protein by RNA interference decreases the effectiveness of nutlin-3a in osteosarcoma cells with mdm2 gene amplification, suggesting that MDM2 protein contributes to antitumor activity of MDM2 antagonists. In search of the mechanism behind this phenomenon, we found that nutlin-induced p53 activates the transcription of a specific subset of microRNAs in cancer cells with mdm2 amplification but not in cells expressing normal levels of MDM2 protein. Five specific members of the class (miR1, miR133, miR141, miR184, miR375) were studied and showed that their p53-dependent transcription is also dependent on the presence of high levels of MDM2 protein, generated by continuous p53 activation of MDM2 in the presence of nutlin. Weak p53 response elements were found in the promoter regions of all five miRs and p53 binding was conformed by ChIP analysis of cell lysates and p53 reporters in experiments with miR375. Transcriptional cofactor p300 was found elevated in cancer cells with mdm2 gene amplification and dependent on the presence of high MDM2 protein levels. Our experimental data suggests that in the presence of high p300, and high MDM2, generated by nutlin treatment, p53 acquires the ability to activate weak p53-responsive miR promoters in MDM2-amplified cells. Interestingly, miR mimics of two of the five p53-induced miRs showed growth suppressive effects in cancer cells and enhanced the activity of low dose nutlin-3a. These findings reveal a novel mechanism for modulation of p53 transcriptional activity and indicate that the high levels of MDM2, generated in mdm2-amplified cancer cells in the presence of nutlin, contribute to its antitumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 977. doi:1538-7445.AM2012-977

Inhibitory effect of miR-29a on the chondrogenic differentiation of mesenchymal stem cells

Backgroundand objectivesMesenchymal stem or stromal cells (MSC) are multipotent cells that can differentiate into different lineages, particularly osteoblasts and chondrocytes. The differentiation process of MSC is regulated by various molecules...

Abstract 1191: Role of histone deacetylases in silencing functionally relevant microRNAs in CLL

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the selective down-regulation of a class of gene regulators, the microRNA (miRs) in comparison to healthy B- lymphocytes. Low levels of miRs are associated with multiple aspects of CLL pathology such as the genesis of disease, apoptotic resistance, progression, and overall survival, suggesting that miRs may function as tumor suppressors in this disease. However, despite the central role of miRs in regulating CLL pathobiology, the mechanisms by which they become under-expressed in CLL are not well known. We hypothesized that the gene repressive action of the histone deacetylases (HDACs) may lead to the silencing of functionally relevant miRs in CLL. HDACs lead to the deacetylation of histones at gene promoters, an action that triggers a loss in the levels of the transcriptionally required histone modification, H3K4me3, which leads to gene silencing. Therefore, we used chromatin immunoprecipitation directed at the H3K4me3 mark to conduct a genome wide analysis of the levels of this transcriptionally activating modification at all miR promoters in normal CD19+ B-cells and in CLL cells before and following HDAC inhibition. Our data demonstrates that normal B-cells displayed the H3K4me3 mark at miR promoters. In contrast, primary CLL samples lacked the H3K4me3 mark at miR promoters but accumulated this activating modification at multiple promoters upon HDAC inhibition. Correspondingly, accumulation of H3K4me3 was associated with an increase in the expression levels of the cognate miRNAs in CLL samples. To determine whether the epigenetically re-expressed miR genes had functional relevance in CLL we first performed a pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. This analysis indicated that the targets of the epigenetically repressed miRs had roles in regulating apoptosis, proliferation, or as transcription factors indicating that the miRs that underwent HDAC-mediated silencing in CLL had important functional roles in regulating multiple, critical cellular processes in CLL. In addition, a detailed functional evaluation of a panel of representative miRs that underwent HDAC-mediated silencing identified both Mcl-1 and E3 ligases that negatively regulate p73-dependent apoptotic signaling among their cellular targets. Finally, our data also identified the presence of HDAC1 at epigenetically silenced miR promoters in CLL suggesting that HDAC1 is an integral component of the repressor complex that silences miR promoters in CLL. Thus our data delineates the complement of miR that is silenced by HDACs in CLL, identifies that such silencing may have functional consequences in facilitating apoptotic resistance or progression in CLL and may identify a new target for therapeutic manipulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1191. doi:10.1158/1538-7445.AM2011-1191

Human papillomavirus type 16 E6 induces cervical cancer cell migration through the p53/microRNA-23b/urokinase-type plasminogen activator pathway

Deregulation of microRNA (miRNA or miR) expression in human cervical cancer is associated frequently with human papillomavirus (HPV) integration. miR-23b is often downregulated in HPV-associated cervical cancer. Interestingly, urokinase-type plasminogen activator (uPA), the miR-23b target, is detected in cervical cancer, but not in normal cervical tissues. Thus, the importance of miR-23b and uPA in HPV-associated cervical cancer development is investigated. In this study, the high-risk subtype HPV-16 E6 oncoprotein was found to decrease the expression of miR-23b, increase the expression of uPA, and thus induce the migration of human cervical carcinoma SiHa and CaSki cells. uPA is the target gene for miR-23b as the miR repressed uPA expression and interacted with the 3'-untranslated region of uPA mRNA. The tumor suppressor p53 is known to be inactivated by HPV-16 E6. A consensus p53 binding site is detected in the promoter region of miR-23b, whereas p53 trans-activated and also interacted with the miR's promoter. Therefore, p53 is believed to mediate the HPV-16 E6 downregulation of miR-23b. From the above, miR-23b/uPA are confirmed to be involved in HPV-16 E6-associated cervical cancer development.

Abstract LB-358: Carcinogen-induced epigenetic silencing of miR-205 and miR-200 family promotes EMT and transformation of lung epithelial cells

Abstract An in vitro carcinogen-induced transformation model was developed using immortalized bronchial epithelial cells (HBECs) to identify key molecular changes in preneoplastic lung epithelial cells that occur during the development of lung cancer in smokers. HBECs were treated for twelve weeks (one exposure per week) with low-doses of tobacco carcinogens (MNU and BPDE) that resulted in transformation from genotoxic stress (DNA damage). The HBECs displayed a morphological change consistent with an epithelial-to-mesenchymal transition (EMT) after 4 weeks of carcinogen treatment that persisted in colonies that were recovered and expanded from soft agar (transformed cell line). Expression of mesenchymal markers vimentin, fibronectin, and N-cadherin increased significantly, while the epithelial marker E-cadherin was significantly decreased. The most notable changes associated with EMT included 4 - 55 fold reduced expression of miR-200b, -200c, and -205 that correlated with 2-1500 fold increased expression of E-box binding transcription factors (SNAI1, ZEB1, ZEB2) and basic helix-loop-helix transcription factor (TWIST1). Restoring the expression of miR-200b, -200c or -205 in carcinogen transformed cells prevented growth in soft agar; meanwhile the cells maintained a fibroblast-like mesenchymal appearance. Levels of DNA methyltransferase 1 (DNMT1) protein also increased significantly during carcinogen treatment and stable knockdown of DNMT1, but not DNMT3A and DNMT3B prior to carcinogen prevented EMT and transformation. ChIP for H3K4me2 (indicative of active/open chromatin) revealed a progressive decrease of this mark at the promoters of miR-200b, -200c, and -205 over 12 weeks of carcinogen treatment and during transformation. A modest enrichment for H3K9me2 (inactive/closed chromatin mark) was observed at all time points. In contrast, H3K27me3 (indicative of inactive/closed chromatin) was enriched at the miR promoters after 4 weeks of carcinogen treatment and enrichment declined over time. Bisulfite sequencing revealed CpG DNA methylation increased from 3% to 42% in the miR-200 family and miR-205 promoters during transformation. To examine the clinical relevance of miR-200b, -200c, and -205 their expression was analyzed in lung tumors relative to distant normal lung tissue from 24 patients. Reduced expression of at least one miR was observed in 50% of lung adenocarcinomas. These findings implicate a major role for epigenetic regulation of EMT that extends beyond that of cancer metastasis to causality for development of premalignant lung cancer. Supported by ES008801. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-358.