miRNA Promoters Research Articles

Abstract B42: An integrated view on genetic and epigenetic mechanisms revealed aberrant DNA methylation as an important source for miRNA deregulation in prostate cancer

Abstract Prostate cancer (PCa) the most common malignant tumor in males and the third leading cause of cancer-related deaths in Western developed countries. The clinical spectrum of PCa ranges from indolent tumors requiring no therapy to highly aggressive and often metastatic diseases. MicroRNAs (miRNAs) have emerged as important regulators in human tumorigenesis and tumor progression during the last decade. miRNA expression is strongly deregulated in many human cancers including PCa. We investigated the contribution of genetic and epigenetic events to the deregulation of miRNAs in PCa by utilizing the profiling data of 19 Early-Onset Pca (EOPC) from International Cancer Genome Consortium (ICGC) (http://www.icgc.org). For 31% (154/491) of the downregulated miRNAs and for 23% (133/578) of the upregulated miRNAs, DNA hyper- and hypomethylation respectively of a promoter region was observed in at least one of the 13 PCa. Deletions were detected for 26% (126/491) of the downregulated miRNAs in at least one sample. We observed a single translocation event leading to disruption of miR-1244-2 from its putative regulatory region that might contribute to expression decrease. We did not detect any SNV within miRNA precursors. Only one miRNA precursor (miR-4307) was amplified in one patient, but the corresponding mature miRNA was not expressed at all. We demonstrated that, among deletions, methylation of miRNA promoters is one of the major mechanisms leading to miRNA silencing or activation, respectively in PCa. 37 miRNA were downregulated by hypermethylation of regulatory regions and 10 miRNAs were upregulated by hypomethylation in both the ICGC EOPC cohort and the validation dataset on 50 prostate cancer and 48 normal prostate tissues. 82% (9/11) of the miRNAs/miRNA clusters analyzed regulate genes that are involved in PI3K/AKT/PTEN signaling, which represents one of the PCa key pathways being deregulated in more than one third of primary PCa. Note: This abstract was not presented at the conference. Citation Format: Olga Bogatyrova, Daniela Wuttig, Lei Gu, Yassen Assenov, Ruprecht Kuner, Constance Baer, Lars Feuerbach, Clarissa Gerhäuser, Dieter Weichenhan, Thorsten Schlomm, Ronald Simon, Guido Sauter, Holger Sültmann. An integrated view on genetic and epigenetic mechanisms revealed aberrant DNA methylation as an important source for miRNA deregulation in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B42.

Aberrant DNA methylation of miR‐219 promoter in long‐term night shiftworkers

The idea that shiftwork may be carcinogenic in humans has gained widespread attention since the pioneering work linking shiftwork to breast cancer over two decades ago. However, the biomolecular consequences of long-term shiftwork exposure have not been fully explored. In this study, we performed a genome-wide CpG island methylation assay of microRNA (miRNA) promoters in long-term night shiftworkers and day workers. This analysis indicated that 50 CpG loci corresponding to 31 miRNAs were differentially methylated in night shiftworkers compared to day workers, including the circadian-relevant miR-219, the expression of which has been implicated in several cancers. A genome-wide expression microarray assay was carried out in a miR-219-overexpressed MCF-7 breast cancer cell line, which identified 319 differentially expressed transcripts. The identified transcriptional targets were analyzed for network and functional interrelatedness using the Ingenuity Pathway Analysis (IPA) software. Overexpression of miR-219 in MCF-7 breast cancer cells resulted in accentuated expression of apoptosis- and proliferation-related anti-viral immunodulators of the Jak-STAT and NF-κβ pathways. These findings suggest that long-term night shiftwork exposure may lead to the methylation-dependent downregulation of miR-219, which may in turn lead to the downregulation of immunomediated antitumor activity and increased breast cancer risk.

Global survey on sequence characteristics of plant microRNA genes: Cis-regulatory SNPs in promoters and microRNA precursors

MicroRNAs (miRNAs) are tiny molecules with multiple essential roles in plant growth and development. So far, numerous miRNA families, both highly conserved and species-specific, have been identified and functionally characterized. However, we are still far from a thorough understanding of these molecules, including their sequence characteristics. In this bioinformatics study, cis-acting regulatory elements located within plant miRNA promoters were predicted, and a comprehensive list of single nucleotide polymorphisms (SNPs) residing within these cis-elements, designated as cis-regulatory SNPs, was identified. These SNPs potentially contribute to the distinct expression patterns of miRNA genes in various Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) subspecies. Additionally, sequence conservation analysis was carried out for all the currently miRBase-registered pre-miRNAs (precursor miRNAs) belonging to numerous plant species. Together, these analyses provide a basis to gain deeper insights into the biological role of miRNA sequence characteristics.

Methylation of promoters of microRNAs and their host genes in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of hematopoietic malignancies characterized by ineffective hematopoiesis. Recently, we identified MDS-associated microRNAs (miRNAs) that are down-regulated in MDS. This study examines possible explanations for that observed down-regulation of miRNA expression in MDS. Since genomic losses are insufficient to explain the down-regulation of all our MDS-associated miRNAs, we explored other avenues. We demonstrate that these miRNAs are predominantly intragenic, and that, in many cases, they and their host genes are expressed in a similar pattern during myeloid maturation, suggesting their co-regulation. This co-regulation is further supported by the down-regulation of several of the host genes in MDS and increased methylation of the shared promoters of several miRNAs and their respective host genes. These studies identify a role of hypermethylation of miRNA promoters in the down-regulation of MDS-associated miRNAs, unifying research on miRNAs in MDS and epigenetic regulation in MDS into a common pathway.

Abstract 4156: Aberrant DNA methylation of miR-219 is associated with long-term shift work and cancer-relevant biological pathways.

Abstract Background: Shift work that involves circadian disruption has been deemed by IARC to be "probably carcinogenic" to humans. Although several theories have been put forth to explain the connection between shift work and cancer, including suppression of melatonin production and deregulation of circadian genes involved in cancer-related pathways, other potential cancer-relevant pathways remain unexplored. In this study, we test the hypothesis that aberrant methylation events in the CpG islands of specific miRNA promoters may be induced by prolonged exposure to shift work in an all-female population, which in turn can lead to cancer development. Material and Methods: We performed a genome-wide methylation assay of miRNA promoters using DNA extracted from 10 pairs of long-term shift workers and day workers selected from participants of the Danish “Diet, Cancer and Health” prospective cohort. A genome-wide expression microarray analysis was then carried out using a miR-219-overexpressed MCF-7 breast cancer cell line. Differentially expressed transcripts were then analyzed for network and functional interrelatedness using the Ingenuity Pathway Analysis (IPA) software. Result: The genome-wide methylation analysis indicated that 50 CpG loci of 30 miRNAs, including miR-10a, miR-219, miR-7-3, miR34b/c, and 25 other newly discovered miRNAs, were differentially methylated in shift workers compared to day workers. Of these, miR-219 has previously been implicated in circadian-related pathways. Genome-wide expression microarray analysis identified 319 differentially expressed transcripts in miR-219-overexpressed MCF-7 cells, which were subsequently analyzed by IPA. The most statistically significant network formed from these transcripts suggests that overexpression of miR-219 induced apoptotic and tumor-suppressive activities in MCF-7. Additional analysis indicated that a number of signaling pathways were affected following miR-219 overexpression, including interferon-α/β (IFN-α/β) singnaling, interferon-gamma (IFN-γ) singnaling, and JAK/STAT signaling. In addition, miR-219 appears to also be involved in the initiation of apoptosis signaling via the increase of TRAIL expression and induction of the caspase cascade. Conclusion: Our results suggest that long-term shift work can lead to aberrant methylation patterns in miRNAs, including miR-219. Hypermethylation of the miR-219 promoter, as observed in our long-term shift workers, may suppress the expression of miR-219, potentially leading to downregulation of miR-219-mediated apoptosis and increased risk for cancer. This study may help us develop a more complete understanding of a potential mechanism for the increased risk of breast cancer observed in female long-term shift workers, which could be useful for providing additional insight in the development of preventative and therapeutic strategies. Citation Format: Fengqin Shi, Yong Zhu, Alan Fu, Johnni Hansen, Richard Stevens, Ronghua Wang. Aberrant DNA methylation of miR-219 is associated with long-term shift work and cancer-relevant biological pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4156. doi:10.1158/1538-7445.AM2013-4156

Abstract 3087: Epigenetic regulation of microRNAs in early stage lung cancer.

Abstract Lung cancer is the most common diagnosed cancer, and a leading cause of cancer-associated deaths worldwide. It is imperative to understand the molecular mechanisms underlying early lung cancer development, to identify better diagnostic and treatment options. We have conducted a multi-platform genomic screen of sixty paired early stage tumor and non-tumor adjacent tissues excised from a cohort of lung cancer patients recruited in our longstanding NCI-MD Case-Control Study. Tissues were characterized through miRNA and mRNA expression, and methylation profiling. Our aim is to provide a framework that can be interrogated to illuminate biological differences associated with racial disparities, to find diagnostic/prognostic biomarkers of lung cancer, and to define miRNA transcriptional networks operational in lung cancer. MicroRNAs (miRNAs) are 20-22 nucleotide-long non-coding RNAs that regulate mRNA transcription and translation through sequence-specific base pairing. MiRNAs are transcribed from large pri-miRNAs, as independent transcripts or within introns of protein-coding genes. It is unclear whether they possess independent promoter regions. However, several miRNAs are adjacent to CpG islands (regions rich in CG dinucleotides). CpG island methylation is a well-described mechanism by which genes with tumor-suppressive function are silenced in cancer. It has been reported that some tumor-suppressive miRNAs are epigenetically controlled through CpG island methylation. Such regulation could have important implications for progression of early stage lung cancer. Here, tumor and adjacent non-tumor tissue DNA were analyzed using the Illumina Infinium HumanMethylation27 BeadChip assays, featuring genome-wide coverage of CpG sites. This platform covers 110 miRNA promoters, which are annotated by being close to gene-associated CpGs. We found seven miRNA loci hypermethylated in tumors. Of these, 4 miRNAs are located within homeobox gene clusters, miR-196b (HOXA9), miR-615 (HOXC5), miR-10a (HOXB4), and miR-10b (HOXD4). Two other miRNAs are located adjacent to genes with significant changes in methylation, miR-638 (DNM2) and miR-639 (GPSN2). Lastly, methylation of miR-34b/c (BTG4) was consistent with prior studies. A comparative analysis of methylation at these loci showed a high concordance between our study and methylation profiling in lung adenocarcinoma conducted by TCGA (The Cancer Genome Atlas), with r2: 0.8. Therefore, methylation at these sites is a generalized finding. Additionally, we confirmed the methylation of miR-615 in A549 lung cancer cell line and its demethylation and reactivation by exposure to 5-aza/TSA. Downstream targets of miR-615 are being explored using available transcriptomic data to gain insight into the functional role of miRNAs in lung cancer progression. We are also expanding the analysis to other lung cancer cohorts to evaluate the association of miRNA methylation with patient survival. Citation Format: Saikartik A. Kumar, Ana I. Robles, Hirokazu Okayama, Curtis C. Harris. Epigenetic regulation of microRNAs in early stage lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3087. doi:10.1158/1538-7445.AM2013-3087

The role of Arabidopsis MYB2 in miR399f-mediated phosphate-starvation response

In plants, microRNA399 (miR399) is a major regulator of phosphate (Pi) homeostasis by way of post-transcriptional mechanisms including transcript cleavage and transcriptional repression. Although miRNA genomic organization, biogenesis, and mode of action in plants are known, the regulatory mechanisms affecting miRNAs are poorly understood. We have shown that AtMYB2 functions as a transcriptional activator for miR399f expression in the context of phosphate homeostasis. AtMYB2 directly binds to a MYB-binding site in the promoter of the miR399f precursor and regulates miR399f expression. In addition, AtMYB2 transcripts are induced under Pi deficiency. The overexpression of AtMYB2 affects root system architecture (RSA), indicated by suppression of primary root growth and enhanced development of root hairs. AtMYB2 and miR399f are expressed and localized in the same tissues under Pi limitation. This study establishes that AtMYB2 regulates Pi-starvation responses (PSR) by activating of miR399f transcript, suggesting that an analysis of this miRNA promoter could reveal the existence and extent of crosstalk with other signaling mechanisms.

MiRNA Gene Promoters Are Frequent Targets of Aberrant DNA Methylation in Human Breast Cancer

miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

Genome-Wide Epigenetic Regulation of miRNAs in Cancer

Aberrant microRNA (miRNA) expression contributes to tumorigenesis and cancer progression. Although the number of reported deregulated miRNAs in various cancer types is growing fast, the underlying mechanisms of aberrant miRNA regulation are still poorly studied. Epigenetic alterations including aberrant DNA methylation deregulate miRNA expression, which was first shown by reexpression of miRNAs upon pharmacologic DNA demethylation. However, studying the influence of DNA methylation on miRNA transcription on a genome-wide level was hampered by poor miRNA promoter annotation. Putative miRNA promoters were identified on a genome-wide level by using common promoter surrogate markers (e.g., histone modifications) and were later validated as such in different tumor entities. Integrating promoter datasets and global DNA methylation analysis revealed an extensive influence of DNA hyper- as well as hypomethylation on miRNA regulation. In this review, we summarize the current knowledge of the field and discuss recent efforts to map miRNA promoter sequences and to determine the contribution of epigenetic mechanisms to the regulation of miRNA expression. We discuss examples of tumor suppressive and oncogenic miRNAs such as the miR-34 and miR-21 family, respectively, which highlight the complexity and consequences of epigenetic miRNA deregulation.

Comparative Analysis of MicroRNA Promoters in Arabidopsis and Rice

Endogenously-encoded microRNAs (miRNAs) are a class of small regulatory RNAs that modulate gene expression at the post-transcriptional level. In plants, miRNAs have increasingly been identified by experiments based on next-generation sequencing (NGS). However, promoter organization is currently unknown for most plant miRNAs, which are transcribed by RNA polymerase II. This deficiency prevents a comprehensive understanding of miRNA-mediated gene networks. In this study, by analyzing full-length cDNA sequences related to miRNAs, we mapped transcription start sites (TSSs) for 62 and 55 miRNAs in Arabidopsis and rice, respectively. The average free energy (AFE) profiles in the vicinity of TSSs were studied for both species. By employing position weight matrices (PWM) for 99 plant cis-elements, we discovered that three cis-elements were over-represented in the miRNA promoters of both species, while four and ten cis-elements were over-represented in Arabidopsis only and in rice only. Thus, comparison of miRNA promoters between Arabidopsis and rice provides a new perspective for studying miRNA regulation in plants.

Tamoxifen Represses miR-200 MicroRNAs and Promotes Epithelial-to-Mesenchymal Transition by Up-Regulating c-Myc in Endometrial Carcinoma Cell Lines

Although tamoxifen (TAM), a selective estrogen receptor modulator, has been widely used in the treatment of hormone-responsive breast cancer, its estrogen-like effect increases the risk of endometrial cancer. However, the molecular mechanisms of TAM-induced endometrial carcinoma still remain unclear. In this report, we explored the role of microRNAs (miRNAs) in TAM-induced epithelial-mesenchymal transition (EMT) in ECC-1 and Ishikawa endometrial cancer cell lines and found miR-200 is involved in this process via the regulation of c-Myc. When treated with TAM, ECC-1 and Ishikawa cells were characterized by higher invasiveness and motility and underwent EMT. miR-200, a miRNA family with tumor suppressive functions in a wide range of cancers, was found reduced in response to TAM treatment. Consistent with zinc finger E-box binding homeobox 2, which was confirmed as a direct target of miR-200b in endometrial cancer cell lines, some other key factors of EMT such as Snail and N-cadherin increased, whereas E-cadherin decreased in the TAM-treated cells, contributing to TAM-induced EMT in these endometrial cancer cells. In addition, we showed that c-Myc directly binds to and represses the promoter of miR-200 miRNAs, and its up-regulation in TAM-treated endometrial cancer cells leads to the down-regulation of miR-200 and eventually to EMT. Collectively, our data suggest that TAM can repress the miR-200 family and induce EMT via the up-regulation of c-Myc in endometrial cancer cells. These findings describe a possible mechanism of TAM-induced EMT in endometrial cancer and provide a potential new therapeutic strategy for it.

PROmiRNA: a new miRNA promoter recognition method uncovers the complex regulation of intronic miRNAs

The regulation of intragenic miRNAs by their own intronic promoters is one of the open problems of miRNA biogenesis. Here, we describe PROmiRNA, a new approach for miRNA promoter annotation based on a semi-supervised statistical model trained on deepCAGE data and sequence features. We validate our results with existing annotation, PolII occupancy data and read coverage from RNA-seq data. Compared to previous methods PROmiRNA increases the detection rate of intronic promoters by 30%, allowing us to perform a large-scale analysis of their genomic features, as well as elucidate their contribution to tissue-specific regulation. PROmiRNA can be downloaded from http://promirna.molgen.mpg.de.

Novel miRNA Genes Methylated in Lung Tumors

MicroRNAs play an important role in the regulation of expression of many genes and are involved in carcinogenesis. The regulation of miRNA gene expression can involve the methylation of promoter CpG islands. In this work, the methylation of six miRNA genes (mir-107, mir-125b-1, mir-130b, mir-137, mir-375, and mir-1258) in non-small-cell lung cancer (NSCLC) was studied for the first time by methylation-specific PCR using a representative set of specimens (39 cases). Four new genes (mir-125b-1, mir-137, mir-375, and mir-1258) methylated in primary NSCLC tumors were identified with frequencies of 56, 31, 56, and 36%, respectively. The frequencies of miRNA promoter methylation in DNA of tumors and histologically normal tissues differed significantly (P < or = 0.05 by Fisher's test). In lung tissues of 20 donors without a history of cancer, these genes were only methylated in a few cases. It was also shown that the previously unstudied promoter CpG islands of mir-107 and mir-130b were not methylated in NSCLC. The frequencies of mir-125b-1 and mir-137 methylation were shown for the first time to correlate with NSCLC progression (clinical stage and metastasis).

Spatiotemporal clustering of the epigenome reveals rules of dynamic gene regulation

Spatial organization of different epigenomic marks was used to infer functions of the epigenome. It remains unclear what can be learned from the temporal changes of the epigenome. Here, we developed a probabilistic model to cluster genomic sequences based on the similarity of temporal changes of multiple epigenomic marks during a cellular differentiation process. We differentiated mouse embryonic stem (ES) cells into mesendoderm cells. At three time points during this differentiation process, we used high-throughput sequencing to measure seven histone modifications and variants—H3K4me1/2/3, H3K27ac, H3K27me3, H3K36me3, and H2A.Z; two DNA modifications—5-mC and 5-hmC; and transcribed mRNAs and noncoding RNAs (ncRNAs). Genomic sequences were clustered based on the spatiotemporal epigenomic information. These clusters not only clearly distinguished gene bodies, promoters, and enhancers, but also were predictive of bidirectional promoters, miRNA promoters, and piRNAs. This suggests specific epigenomic patterns exist on piRNA genes much earlier than germ cell development. Temporal changes of H3K4me2, unmethylated CpG, and H2A.Z were predictive of 5-hmC changes, suggesting unmethylated CpG and H3K4me2 as potential upstream signals guiding TETs to specific sequences. Several rules on combinatorial epigenomic changes and their effects on mRNA expression and ncRNA expression were derived, including a simple rule governing the relationship between 5-hmC and gene expression levels. A Sox17 enhancer containing a FOXA2 binding site and a Foxa2 enhancer containing a SOX17 binding site were identified, suggesting a positive feedback loop between the two mesendoderm transcription factors. These data illustrate the power of using epigenome dynamics to investigate regulatory functions.

Involvement of microRNA-Mediated Gene Expression Regulation in the Pathological Development of Stem Canker Disease in Populus trichocarpa

MicroRNAs (miRNAs), a type of short (21–23 nucleotides), non-coding RNA molecule, mediate repressive gene regulation through RNA silencing at the post-transcriptional level, and play an important role in defense and response to abiotic and biotic stresses. In the present study, Affymetrix® miRNA Array, real-time quantitative PCR (qPCR) for miRNAs and their targets, and miRNA promoter analysis were used to validate the gene expression patterns of miRNAs in Populus trichocarpa plantlets induced with the poplar stem canker pathogen, Botryosphaeria dothidea. Twelve miRNAs (miR156, miR159, miR160, miR164, miR166, miR168, miR172, miR319, miR398, miR408, miR1448, and miR1450) were upregulated in the stem bark of P. trichocarpa, but no downregulated miRNAs were found. Based on analysis of the miRNAs and their targets, a potential co-regulatory network was developed to describe post-transcriptional regulation in the pathological development of poplar stem canker. There was highly complex cross-talk between diverse miRNA pathway responses to biotic and abiotic stresses. The results suggest that miR156 is probably an integral component of the miRNA response to all environmental stresses in plants. Cis-regulatory elements were binding sites for the transcription factors (TFs) on DNA. Promoter analysis revealed that TC-rich repeats and a W1-box motif were both tightly related disease response motifs in Populus. Promoter analysis and target analysis of miRNAs also revealed that some TFs regulate their activation/repression. Furthermore, a feedback regulatory network in the pathological development of poplar stem canker is provided. The results confirm that miRNA pathways regulate gene expression during the pathological development of plant disease, and provide new insights into understanding the onset and development of poplar stem canker.

Development of a novel microRNA promoter microarray for ChIP-on-chip assay to identify epigenetically regulated microRNAs

To gain a global view of epigenetic alterations around microRNA (miRNA) promoter regions, and to identify epigenetically regulated miRNAs, we developed a novel miRNA promoter microarray for chromatin immunoprecipitation (ChIP)-on-chip assay. We designed a custom oligo microarray covering regions spanning −10 to +2.5kb of precursor miRNAs in the human genome. This microarray covers 541 miRNAs, each of which is covered by approximately 100 probes (60-mer) over its 12.5-kb genomic position, that includes predicted transcription start sites. Using this custom-made miRNA promoter microarray, we successfully performed ChIP-on-chip assay to identify miRNAs regulated by histone modification. Fifty-three miRNAs (9.8%) showed increased levels of both histone H3 acetylation and histone H3-K4 methylation in AGS gastric cancer cells treated with the DNA-methylation inhibitor 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor 4-phenylbutyric acid. One of these miRNAs, miR-9, is downregulated in gastric cancer tissues and is activated by chromatin-modifying drugs, suggesting that it may be a potential target for epigenetic therapy of gastric cancer.

Extensive Promoter DNA Hypermethylation and Hypomethylation Is Associated with Aberrant MicroRNA Expression in Chronic Lymphocytic Leukemia

Dysregulated microRNA (miRNA) expression contributes to the pathogenesis of hematopoietic malignancies, including chronic lymphocytic leukemia (CLL). However, an understanding of the mechanisms that cause aberrant miRNA transcriptional control is lacking. In this study, we comprehensively investigated the role and extent of miRNA epigenetic regulation in CLL. Genome-wide profiling conducted on 24 CLL and 10 healthy B cell samples revealed global DNA methylation patterns upstream of miRNA sequences that distinguished malignant from healthy cells and identified putative miRNA promoters. Integration of DNA methylation and miRNA promoter data led to the identification of 128 recurrent miRNA targets for aberrant promoter DNA methylation. DNA hypomethylation accounted for more than 60% of all aberrant promoter-associated DNA methylation in CLL, and promoter DNA hypomethylation was restricted to well-defined regions. Individual hyper- and hypomethylated promoters allowed discrimination of CLL samples from healthy controls. Promoter DNA methylation patterns were confirmed in an independent patient cohort, with 11 miRNAs consistently showing an inverse correlation between DNA methylation status and expression level. Together, our findings characterize the role of epigenetic changes in the regulation of miRNA transcription and create a repository of disease-specific promoter regions that may provide additional insights into the pathogenesis of CLL.

Chronic Ethanol Feeding Alters miRNA Expression Dynamics During Liver Regeneration

Adaptation to chronic ethanol (EtOH) treatment of rats results in a changed functional state of the liver and greatly inhibits its regenerative ability, which may contribute to the progression of alcoholic liver disease. In this study, we investigated the effect of chronic EtOH intake on hepatic microRNA (miRNA) expression in male Sprague-Dawley rats during the initial 24 hours of liver regeneration following 70% partial hepatectomy (PHx) using miRNA microarrays. miRNA expression during adaptation to EtOH was investigated using RT-qPCR. Nuclear factor kappa B (NFκB) binding at target miRNA promoters was investigated with chromatin immunoprecipitation. Unsupervised clustering of miRNA expression profiles suggested that miRNA expression was more affected by chronic EtOH feeding than by the acute challenge of liver regeneration after PHx. Several miRNAs that were significantly altered by chronic EtOH feeding, including miR-34a, miR-103, miR-107, and miR-122 have been reported to play a role in regulating hepatic metabolism and the onset of these miRNA changes occurred gradually during the time course of EtOH feeding. Chronic EtOH feeding also altered the dynamic miRNA profile during liver regeneration. Promoter analysis predicted a role for NFκB in the immediate-early miRNA response to PHx. NFκB binding at target miRNA promoters in the chronic EtOH-fed group was significantly altered and these changes directly correlated with the observed expression dynamics of the target miRNA. Chronic EtOH consumption alters the hepatic miRNA expression profile such that the response of the metabolism-associated miRNAs occurs during long-term adaptation to EtOH rather than as an acute transient response to EtOH metabolism. Additionally, the dynamic miRNA program during liver regeneration in response to PHx is altered in the chronically EtOH-fed liver and these differences reflect, in part, differences in miRNA expression between the EtOH-adapted and control livers at the baseline state prior to PHx.

Genome-Wide Analysis of DNA Methylation and Expression of MicroRNAs in Breast Cancer Cells

DNA methylation of promoters is linked to transcriptional silencing of protein-coding genes, and its alteration plays important roles in cancer formation. For example, hypermethylation of tumor suppressor genes has been seen in some cancers. Alteration of methylation in the promoters of microRNAs (miRNAs) has also been linked to transcriptional changes in cancers; however, no systematic studies of methylation and transcription of miRNAs have been reported. In the present study, to clarify the relation between DNA methylation and transcription of miRNAs, next-generation sequencing and microarrays were used to analyze the methylation and expression of miRNAs, protein-coding genes, other non-coding RNAs (ncRNAs), and pseudogenes in the human breast cancer cell lines MCF7 and the adriamycin (ADR) resistant cell line MCF7/ADR. DNA methylation in the proximal promoter of miRNAs is tightly linked to transcriptional silencing, as it is with protein-coding genes. In protein-coding genes, highly expressed genes have CpG-rich proximal promoters whereas weakly expressed genes do not. This is only rarely observed in other gene categories, including miRNAs. The present study highlights the epigenetic similarities and differences between miRNA and protein-coding genes.

Abstract 123: DNA methylation of miRNA promoters in breast cancer

Abstract MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression and deregulation of their expression is linked to human diseases including cancer. Epigenetic mechanisms including DNA methylation play an important role in the regulation of miRNA expression, and a number of reports describe silencing of miRNA expression linked to the aberrant DNA methylation of individual miRNA genes in cancer. In this study, we have used a comprehensive miRNA custom tiling microarray to evaluate aberrant DNA methylation of miRNA gene promoters in human breast cancer, with selected hypermethylated promoters being validated by Sequenom MassARRAY technology. We found more than 25% of miRNA gene promoters analyzed have significant hypermethylated regions in breast cancer cell lines (CCL) relative to logarithmically growing normal pre-stasis finite lifespan human mammary epithelial cells isolated from healthy individuals. In addition to in vitro grown breast cancer cell lines, we analyzed DNA methylation of miRNA promoters in a dozen breast tumor tissue specimens and five normal breast specimens. Aberrant DNA hypermethylation of miRNA promoters in breast cancer specimens displayed a perfect overlap with those hypermethylated in breast cancer cell lines, albeit the number of miRNAs promoters was smaller in the heterogeneous tissue samples. These aberrantly methylated miRNA promoters readily clustered breast cancer samples and cell lines together, and separate from normal beast tissue and mammary cell strains. Integration of our data with publically-available H3K27me3 ChIP-Seq data from stem cells reveals that miRNA promoters targeted by aberrant DNA methylation in cancer cells significantly overlap with promoters targeted by polycomb repression complexes in stem cells, similar to the results other have seen with protein coding genes promoters in cancer. We have found previously that the proportion of miRNA genes targeted by polycomb repression in normal mammary cell types is about 27% - three times higher than seen in protein coding genes, perhaps explaining why the proportion of miRNA genes with aberrant DNA methylation in our analysis of breast cancer is so high. In summary, our data show that a large fraction of miRNA gene promoters are targets of aberrant DNA methylation in human breast cancer, and that the miRNA promoters aberrantly methylated in breast cancer cell lines are also targeted in tumor tissue samples, indicating that these changes in DNA methylation are not artifacts of cell culture and might thus have clinical relevance. Similar to protein coding genes, miRNA gene promoters targeted by polycomb complexes in stem cells are more prone to hypermethylation in cancer. 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 123. doi:1538-7445.AM2012-123