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Keywords:

  • DNA methylation;
  • epigenetics;
  • histone modification;
  • microRNA

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biogenesis and RNAi functions of miRNAs
  5. Epigenetically-regulated miRNAs
  6. miRNAs regulating epigenetic pathway-related genes
  7. Materials and methods
  8. References
  9. Supporting Information

MicroRNAs (miRNAs) comprise species of short noncoding RNA that regulate gene expression post-transcriptionally. Recent studies have demonstrated that epigenetic mechanisms, including DNA methylation and histone modification, not only regulate the expression of protein-encoding genes, but also miRNAs, such as let-7a, miR-9, miR-34a, miR-124, miR-137, miR-148 and miR-203. Conversely, another subset of miRNAs controls the expression of important epigenetic regulators, including DNA methyltransferases, histone deacetylases and polycomb group genes. This complicated network of feedback between miRNAs and epigenetic pathways appears to form an epigenetics–miRNA regulatory circuit, and to organize the whole gene expression profile. When this regulatory circuit is disrupted, normal physiological functions are interfered with, contributing to various disease processes. The present minireview details recent discoveries involving the epigenetics–miRNA regulatory circuit, suggesting possible biological insights into gene-regulatory mechanisms that may underlie a variety of diseases.


Abbreviations
DGCR8

DiGeorge syndrome critical region gene 8

DNMT

DNA methyltransferase

EMT

epithelial–mesenchymal transition

HDAC

histone deacetylase

miRNA

microRNA

NF-κB

nuclear factor kappa B

PRC

polycomb repressor complex

RISC

RNA-induced silencer complex

RLC

RISC-loading complex

RNAi

RNA interference

SNP

single nucleotide polymorphism

TGIF2

TGFβ-inducing factor 2

VNTR

variable nucleotide tandem repeat

YY1

Yin Yang 1

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biogenesis and RNAi functions of miRNAs
  5. Epigenetically-regulated miRNAs
  6. miRNAs regulating epigenetic pathway-related genes
  7. Materials and methods
  8. References
  9. Supporting Information

MicroRNAs (miRNA) comprise a class of short noncoding RNAs with 18–25 nucleotides in length that are found in animal and plant cells. In 1993, the first miRNAs were recognized in Caenorhabditis elegans by Lee et al. [1]. In 2001, various small regulatory RNAs were discovered in plants and mammals [2–4] and designated ‘microRNA’ [5]. Currently, 1100 human miRNAs are registered in the miRBase database (release 16, September 2010) [6–9]. miRNAs are involved in RNA interference (RNAi) machinery to regulate gene expression post-transcriptionally, and they contribute to diverse physiological and pathophysiological functions, including the regulation of developmental timing and pattern formation [2], restriction of differentiation potential [10], cell signaling [11], cardiovascular diseases [12] and carcinogenesis [13]. The biogenesis and RNAi functions of miRNA (i.e. how miRNAs are generated and processed into a mature form, and how they regulate gene expression) have been intensively investigated and well-described [10]. Furthermore, developments in miRNA-related technologies, such as miRNA expression profiling and synthetic oligoRNA, have contributed to the identification of miRNAs involved in a number of physiological and pathological phenotypes. However, some questions remain largely unanswered, such as how miRNA expression is controlled and which genes are regulated by each miRNA. Recently, accumulating studies have shown that a subgroup of miRNAs is regulated epigenetically. Although epigenetics and miRNAs have been frequently reviewed [14–18], few reviews have focused upon the relationship between epigenetics and miRNA. In the present minireview, we illustrate the current knowledge regarding the epigenetics–miRNA regulatory networks aiming to provide biological insights for a wide range of biomedical researchers.

Biogenesis and RNAi functions of miRNAs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biogenesis and RNAi functions of miRNAs
  5. Epigenetically-regulated miRNAs
  6. miRNAs regulating epigenetic pathway-related genes
  7. Materials and methods
  8. References
  9. Supporting Information

As illustrated in Fig. 1, in the nucleus, mainly RNA polymerase II initially transcribes miRNAs into long segments of coding or noncoding RNA, known as pri-miRNAs, which are usually capped and polyadenylated. Portions in the pri-miRNAs measuring approximately 70–100 nucleotides in length and containing a stem-loop, are captured and extracted from pri-miRNAs by a complex containing RNase type III, Drosha and the dsRNA binding protein DiGeorge syndrome critical region gene 8 (DGCR8) (also called Pasha) [19]. These short stem-loop-shaped RNAs are called pre-miRNAs, and the protein complex of RNase, Drosha and DGCR8 is known as the microprocessor complex. Pre-miRNAs form a complex with exportin-5 and RAN-GTP, and are then exported from the nucleus to the cytoplasm. The pre-miRNAs are further processed to a double-stranded miRNA duplex by a dsRNase type III, Dicer. This double-stranded miRNA duplex is incorporated into a RNA-induced silencer complex (RISC)-loading complex (RLC) in an ATP-dependent manner [20]. Next, one strand (the passenger strand) of the miRNA is removed from the RLC, whereas the other strand (the guide strand) remains in the complex to form a mature RNA-induced silencer complex (mature RISC) and serves as a template for capturing target mRNAs. Under most conditions, the mature RISC represses gene expression post-transcriptionally. For highly complementary target mRNAs, the mature RISC complex cleaves target mRNAs via a catalytic domain (RNase III domain) of Argonaute proteins, a core component of the RISC complex, and degrades them by the SKI complex and XRN1 [21]. For partially complementary targets, the RISC complex decaps and deadenylates target mRNAs via the DCP1-DCP2 and CAF1-CCR4-NOT complexes, respectively, to reduce the stability of the target mRNAs [22]. In addition, the RISC complex also represses the translation of target genes under most conditions. However, not all miRNAs work in translational repression. Under serum-starved conditions, miR-369-3 activates translation of tumor necrosis factor-α by binding to AU-rich elements in the 3′ UTR of the transcript with fragile X mental retardation-related protein 1 [23]. Thus, molecular mechanisms of the RISC in translational regulation remain to be clarified. At the same time, turnover of miRNAs is mediated by the XRN2 gene in C. elegans [24]. However, the mechanisms underlying miRNA turnover in human cells also remain unclear.

image

Figure 1.  Epigenetics–miRNA regulatory circuit. Epigenetics and miRNAs regulate whole gene expression pattern transcriptionally and post-transcriptionally, respectively. At the same time, epigenetics and miRNAs controll each other to form a regulatory circuit and to maintain normal physiological functions. A disruption of this regulatory circuit may cause various diseases, such as cardiovascular diseases and cancers. PABP, poly(A) binding protein; TF, transcriptional factors; TRBP, Tar RNA binding protein.

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Epigenetically-regulated miRNAs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biogenesis and RNAi functions of miRNAs
  5. Epigenetically-regulated miRNAs
  6. miRNAs regulating epigenetic pathway-related genes
  7. Materials and methods
  8. References
  9. Supporting Information

As described above, the biogenesis of miRNA has been intensively studied and is well-described. However, the regulation of miRNA expression remains largely unclear. In early studies, promoter regions had been determined for only a small portion of miRNAs. Although several in silico studies attempted to predict the promoter regions of miRNAs [25–27], most of these predicted miRNA promoters were not confirmed in wet-laboratory experiments.

miRNAs can be classified as either ‘intragenic’ and ‘intergenic’, according to whether the miRNA is localized in a genome region transcribed by a gene, or not. Our in silico analysis (see Materials and methods) revealed that, among 939 miRNAs, 293 (31.2%) of miRNAs were intergenic, whereas 317 (44.4%), 119 (12.7%) and 110 (11.7%) were overlapped by RNA transcripts in the same, opposite and both directions, respectively. Localization of promoters for intergenic and inversely-directed intragenic miRNAs is largely unknown, whereas promoters for overlapping primary genes are considered to be promoters for the intragenic miRNAs that are localized in the same direction as the primary gene. However, some studies have identified that an independent promoter within the intron in which a miRNA is embedded can also regulate miRNA expression [28]. Additionally, as shown in one study [29], a single member of a miRNA cluster, although ordinarily expressed from the same pri-miRNA, can alternatively be regulated independently by its own promoter in certain scenarios. Furthermore, the total amount of miRNAs contained within a given quantity of total RNA can be reduced in cancer cells and rapidly proliferating cells [13,30], a finding for which the underlying mechanism is still unknown. Thus, the means by which miRNA expression is regulated appears somewhat complicated.

Recently, Saito et al. [29] established that the expression of miR-127 is regulated epigenetically. In their study, pharmacological unmasking of epigenetically silenced miRNAs activated 17 of 313 miRNAs investigated in the bladder cancer cell line T24 and the normal fibroblast cell line LD419. The gene for miR-127 was upregulated the most in epigenetically unmasked cancer cells. DNA methylation level and histone modification status at identified promoter regions of miR-127 correlated significantly with mature miR-127 expression. Subsequent to this initial report, the number of studies documenting the epigenetic regulation of miRNAs has increased dramatically (Table 1). We summarize the findings regarding some of the more intensively studied miRNAs for which expression is regulated by epigenetic mechanisms.

Table 1.   Epigenetically-regulated miRNAs. The numbers in the ‘binding sites’ column represent the distance (bp) between the stop codon and binding sites of seed sequences in the miRNAs. The letters ‘c’ and ‘p’ with respect to miRNA binding site numbers indicate that the miRNA binding sites are the ‘conserved region’ and ‘poorly conserved region’ among vertebrates, according to the TargetScan database (http://www.targetscan.org/).
miRNA genesInter-/intra genicLocusHost geneTarget genesBinding sitesReferences
  1. a SNPs are located within the miRNA binding sites (not only the seed sequence regions, but also an approximately 23 bp region), which may affect the affinity of miRNA with the binding sites.

let-7a-3Intergenic22q13.31 IGF2BP1-31632c, 1651c, 4269c, 4923c, 5568c[55,83]
miR-1-1Intragenic20q13.33C20orf166FoxP1772c, 819ca, 965p, 3447p[84]
MET499p, 811c
HDAC42333c, 3513ca,3546ca
miR-9-1Intragenic1q22C1orf61NFKB129pa[31–35]
miR-9-2Intragenic5q14.3CR612213
miR-9-3Intragenic15q26.1FLJ30369
miR-10aIntragenic21q21.32HOX3BHOXA3299c[31,85]
HOXD10276c
miR-34aIntragenic1p36.23EF570048CDK61087c, 6941p, 9172c[39]
miR-34b/cIntragenic Intragenic11q23.1BC021736CDK61087c, 6941p, 9172c[28,31,33,40,41]
MYC138pa
E2F32714c
CREB3259p, 3317c
miR-107Intragenic10q23.31PANK1CDK6308c, 1815p[86]
miR-124-1Intergenic8p23.1  1532p, 1647p, 7788p, 8004p[31,34,44–48]
miR-124-2Intragenic8q12.3AK124256/CDK6 
miR-124-3Intergenic20q13.33FLJ42262C/EBPα283c, 340c, 981c
VIM81c
SMYD343p
miR-126Intragenic9q34.3EGFL7  [87]
miR-127Intergenic14q32.31 BCL6584c[29,47]
miR-129-2Intragenic11p11.2EST  [32]
miR-132/212Intergenic17p13.2   [31]
miR-137Intragenic1p21.3AK311400CDK64214p, 7114p, 7133c[32,40,47]
E2F679c
NCOA21244c
miR-148aIntergenic7p15.2 TGIF2159c, 566pa, 2288c[33,34]
miR-152Intragenic17q21.32COPZ2  [34]
miR-181a/b-2Intragenic9q33.3NR6A1PLAG1391p, 3501c, 4389c[88]
miR-193aIntergenic17q11.2 E2F6127c[40,47]
PTK2545p
MCL1315ca
miR-196a-2Intragenic12q13.13EST  [89]
miR-196bIntragenic7p15.2EST  [31]
miR-199a*-1Intragenic19p13.2DNM2MET1425ca[90]
miR-199a*-2Intragenic1q24.3DNM3
miR-141/200cIntergenic12p13.31 ZEB2207c, 733p, 774c [51–53]
miR-200a/b/429Intergenic1p36.33 ZEB1369c, 463c
ZEB2391ca, 454ca, 812c, 897c, 1028c, 1362ca
SOX2477ca
KLF442ca
miR-203Intergenic14q32.33 ABL11074c[31,40,48,51,54]
BCR-ABL11074c
Bmi-11443c
miR-342Intragenic14q32.2EVL  [91]
miR-370Intragenic14q32.31ESTMAP3K8567p[92]
miR-512-5pIntergenic19q13.41 Mcl-11631p[93]
miR-663Intragenic20p11.1BC036544  [34]

miR-9

miR-9 is expressed from three genomic loci, miR-9-1, miR-9-2 and miR-9-3, all of which are associated with CpG islands. Hypermethylation of miR-9 loci is observed in various malignant tissues, including breast, lung, colon, head and neck cancers, melanoma and acute lymphoblastic leukemia [31–34]. In breast cancer, the miR-9-1 locus is highly methylated not only in invasive ductal carcinoma, but also in ductal carcinoma in situ and the intraductal component of invasive ductal carcinoma [34]. In addition, an in vitro experimental study showed that xenoestrogen exposure may induce aberrant epigenetic patterns at various miRNA gene loci, including miR-9-3 [35]. These findings suggest that epigenetic silencing of miR-9 loci constitutes an early event in breast carcinogenesis. Furthermore, the miR-9 DNA methylation signature is correlated with cancer metastasis [33]. Target genes of mature miR-9 responsible for carcinogenesis and cancer metastasis remain largely unknown. However, a recent study demonstrated that mature miR-9 targets nuclear factor kappa B (NF-κB), which is overexpressed in a number of different cancers [36].

miR-34 (a and b/c)

The net level of miR-34 reflects the expression of three separate genes for miR-34: miR-34a, miR-34b and miR-34c. miR-34a is monocistronic, whereas miRs-34b/c are polycistronic. Promoter regions of both loci contain p53-binding sites, and are regulated by the p53 signal. Likely as a result of this feature, the expression of mature miR-34a species is induced by DNA damage and oncogenic stress, as well as other p53-related events that control the cell cycle, induce apoptosis and suppress tumor formation [37,38]. The host or ‘mother’ gene (FLJ41150) of miR-34a is associated with a CpG island surrounding its transcriptional start site, which is frequently methylated in various malignancies [39]. The epigenetic mechanism underlying miR-34b/c transcriptional regulation was described in detail by Toyota et al. [28]. The miR-34b/c host gene (BC021736) contains a CpG island, not within its own promoter region, but also located at the first intron–second exon boundary. The latter CpG island also happens to lie within the promoter region of the oppositely-oriented BTG4 gene, thus exerting bidirectional promoter activity for both the BTG4 gene and the miR-34b/c polycistoron [28]. Thus, miR-34b/c expression may be regulated by both the promoter of the host gene and the promoter in the latter CpG island. The methylation levels of the CpG island are inversely correlated with mature miR-34b/c expression levels in various cancers [28,31,33,40,41]. In colorectal cancer cell lines, in which the miR-34b/c locus is epigenetically silenced, the p53 signal alone does not induce miR-34b/c expression [28]. This finding suggests that hypermethylation of the CpG island modulates p53-mediated miR-34b/c expression. In terms of the functions of miR-34 species, mature miR-34 miRNAs target various genes related to the cell cycle, oncogenesis and cancer metastasis, including MYC, CDK4, CDK6, E2F3, CREB and MET [33,37,41]. Ectopic expression of miR-34 species induces cell-cycle arrest and apoptosis and suppresses cell growth and metastasis, possibly by silencing these target genes [28,33,37,39–41].

miR-124

Many studies have shown that mature miR-124 is the most abundant miRNA in the adult brain, and that it plays a key role in neurogenesis [42]. Conversely, epigenetic silencing of three miR-124 loci (miR-124-1 to -3) is frequently observed not only in brain tumors, but also in a variety of other cancer types [43–48], such as colon (prevalence: 75%), breast (32–50%), lung (48%), leukemia (36%) and lymphoma (41%). miR-124 loci are also hypermethylated in precancerous lesions. Methylation levels at miR-124 loci in the gastric mucosae of healthy volunteers infected by Helicobacter pylori are markedly elevated compared to healthy individuals without H. pylori infection [47]. Thus, H. pylori infection appears to induce aberrant epigenetic patterns at miRNA loci in normal gastric mucosae, which may contribute to gastric carcinogenesis as a ‘field effect’. Targets of mature miR-124 include the 3′ UTR of CDK6, an oncogene. Epigenetically masking of miR-124 induces activation of CDK6 and consequent phosphorylation of Rb at serine residues 807 and 811, the targets of CDK6, resulting in an acceleration of cell growth. Notably, in acute lymphoblastic leukemia, epigenetic silencing of miR-124 loci is linked to both disease-free and overall survival [31].

miR-137

Physiologically, miR-137 is involved in neurogenesis by targeting CDK6, analogous to miR-124 [43], as well as in melanocyte function by targeting microphthalmia-associated transcription factor [49]. miR-137 is an intragenic miRNA that is directly overlapped by a CpG island. The CpG island is specifically hypermethylated in cancer tissues [32,40,47]. Overexpression of miR-137 in cancer cells induces cell cycle G1 arrest and apoptosis [40]. Furthermore, a 15 nucleotide variable tandem repeat (VNTR) (5′-TAGCAGCGGCAGCGG-3′) is located just 5′ to pre-miR-137, and extending the length of this VNTR impairs the maturation of miR-137. Specifically, pri-miR-137 with three VNTRs is more efficiently processed to mature miR-137 than is pri-miR-137 with 12 VNTRs. Thus, both genomic and epigenetic variations affect mature miR-137 expression levels and may contribute to disease formation.

miR-148

Lujambio et al. [33] screened cancer metastasis-related miRNAs that are epigenetically inactivated, using a pharmacological epigenetic reversal technique in metastatic cancer cell lines, which identified three miRNAs, one of which is miR-148. The miR-148 locus is more heavily methylated in metastatic than in non-metastatic cancer tissues. Cancer cells that stably express exogenous miR-148 exhibit reduced invasiveness, cell motility and metastatic propensity in an in vivo model [33]. In addition, miR-148 targets TGFβ-inducing factor 2 (TGIF2), which is overexpressed in highly malignant ovarian cancers [50]. Thus, epigenetic inactivation of miR-148 would be expected to enhance TGIF2 activation. In addition, several isoforms of DNA methytransferase (DNMT)3b are targeted by miR-148 within their coding region (described in detail below). Therefore, although being targeted epigenetically, miR-148 may itself exert effects on DNA methylation in cells.

The miR-200 family

The miR-200 family consists of miR-141, 200a/b/c and 429, which share similar seed sequences. miRs-141/200c and miRs-200a/b/429 comprise multicistronic miRs whose genomic loci are located in close proximity to each other. Several studies have established that the miR-200 family is involved in epithelial–mesenchymal transition (EMT). EMT occurrence in cancer cells comprises a phenomenon in which these cells obtain phenotypes characteristic of mesenchymal cells, such as spindle-shaped morphology, activated cell motility and invasiveness. Therefore, EMT research is important for understanding the molecular mechanisms underlying the malignant potential of cancer cells. Recently, Wellner et al. [51] demonstrated that an EMT activator, ZEB1, suppresses miR-200c, whereas miR-200c targets ZEB1. This finding suggests that miR200c and ZEB1 form a feedback loop regulatory mechanism that maintains EMT [51]. Additional studies showed that both the miR-141/200c [52,53] and miR-200a/b/429 [53] clusters are epigenetically regulated. Thus, EMT could conceivably be regulated by epigenetic events targeting the miR-200 family. Table 1 shows that miR-200a/b/429 binding sites in the 3′ UTR of ZEB2 have several single nucleotide polymorphism (SNP) sites. However, to date, no study is available demonstrating the clinical significance of these SNPs.

miR-203

In hematopoietic malignancies, 12% of miRNAs are located in fragile genomic regions that encompass only seven megabases (0.2% of whole genome). miR-203 is one of these regions, and it targets ABL1 and BCR-ABL1, an oncogenic fusion gene generated by the Philadelphia translocation [54]. Epigenetic silencing of miR-203 enhances activation of the BCR-ABL1 fusion gene, resulting in an elevation of tumor cell growth rate. Epigenetic inactivation of miR-203 is frequently observed in other types of malignancies, including oral cancer, hepatocellular carcinoma, etc. [40,48]. Another candidate target gene of miR-203 is Bmi-1, a member of the polycomb repressor complex 1 [51], which is a histone modifier complex regulating gene expression. Introduction of ectopic miR-203 into cancer cells induces apoptosis and represses cell growth [48], possibly as a result of polycomb-mediated modification in epigenetic patterns.

let-7a-3

Epigenetic control of let-7a-3 expression was discovered by a comparison between parent and DNMT1-3B double-knockout HCT116 colon cancer cells [55]. The let-7a-3 locus is generally methylated in normal tissues but hypomethylated in some types of cancers, such as colon and lung cancer [55]. Methylation levels of let-7a-3 correlate inversely with let-7a-3 pri-miRNA expression levels [55]. However, the effect of let-7a-3 methylation status on mature let-7a expression level is unclear because levels of mature let-7a reflect the expression of three let-7a genes, let-7a-1, let-7a-2 and let-7a-3. Indeed, let-7a-3 methylation levels in ovarian cancer correlate with mature let-7a levels. In the context of miRNA function, let-7a-3 has oncogenic potential. The introduction of let-7a-3 enhanced the colony-forming ability of A549 lung adenocarcinoma cells. In addition, let-7a may regulate IGF-II via targeting of IGF2-binding proteins (IMP-1 and 2). Methylation levels at the let-7a-3 locus correlate inversely with IGF-II levels, and are also linked to the survival of ovarian cancer patients. In general, the let-7 family is considered to comprise tumor suppressor miRNAs [56–58]. Diversity in functions among let-7 family members may cause apparently contradictory observations.

Imprinting and miRNAs

Genomic imprinting is an epigenetic process by which a small proportion of genes (< 1% of all genes in mammals) are expressed in a parent-of-origin-specific manner [59]. In genomic imprinting, DNA methylation and histone modification regulate monoallelic expression. These epigenetic patterns are established in germline cells, and are inherited through somatic cells. For example, at the well-investigated IGF2/H19 locus, the IGF2 gene is expressed from the paternal allele, whereas the H19 gene is expressed from the maternal allele. Abnormal genomic imprinting is associated with several diseases. Some inheritable disorders, such as Prader–Willi syndrome and Angelman syndrome, are caused by aberrant imprinting. Furthermore, the phenomenon known as loss of imprinting, in which the normally inactivated allele becomes reactivated as a result of hypomethylation or histone abnormalities, is frequently observed in cancers [60].

Several miRNAs are located within imprinting-associated regions, including miR-296 and miR-298 at the GNAS/NESP locus, miR-483 and miR-675 at the IGF2/H19 locus, and miR-335, miR-29a and miR-29b at the MEST/KLF14 locus [61]. However, the imprinting and expression status of such miRNAs remains largely unknown.

miRNAs regulating epigenetic pathway-related genes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biogenesis and RNAi functions of miRNAs
  5. Epigenetically-regulated miRNAs
  6. miRNAs regulating epigenetic pathway-related genes
  7. Materials and methods
  8. References
  9. Supporting Information

miRNAs themselves are capable of targeting genes that control epigenetic pathways. As shown in Table 2, various miRNAs may control chromatin structure by regulating histone modifier molecules, such as polycomb group-related genes and histone deacetylase (HDAC). The polycomb group proteins are transcriptional repressors that regulate lineage choices occurring during development and differentiation. There are two polycomb repressor complexes (PRCs), PRC1 and PRC2. The PRC1 core complex contains Cbx, Mph, Ring, Bmi-1 and Me118, whereas the PCR2 core complex consists of Ezh2, Suz12 and Eed [62]. In an initial step, PRC2 initiates silencing by catalyzing histone H3 Lysine-27 (H3K27) methylation. Recent studies have advanced our understanding of the means by which epigenomic dysregulation potentially contributes to various diseases.

Table 2.   miRNAs targeting genes that are involved in epigenetic regulatory pathways. The letters ‘c’ and ‘p’ with respect to miRNA binding site numbers indicate that the miRNA binding sites are the ‘conserved region’ and ‘poorly conserved region’ among vertebrates, according to the TargetScan database (http://www.targetscan.org/).
Target genesmiRNAsBinding sitesReferences
  1. a SNPs are located within the miRNA binding sites (not only the seed sequence regions, but also an approximately 23 bp region), which may affect the affinity of miRNA with the binding sites.

EZH2miR-26a249c[64–66,68]
miR10158p, 113ca
miR-214172p
Bmi1miR-128481c[51,71]
miR-2031443c
YY1miR-29b774c[73]
HDAC1miR-449459p[94]
HDAC4miR-12333c, 3513ca, 3546ca[95]
DNMT3AmiR-29855c[79,80]
DNMT3BmiR-291202c[79–81]
miR-1481424c and 2384c in coding region
MeCP2miR-1326886c[96]

EZH2

Expression levels of EZH2, a conserved catalytic subunit within PRC2, are elevated in cancers relative to corresponding normal tissues, with the highest EZH2 levels correlating with advanced disease stages and poor prognosis. In some cases, EZH2 overabundance is paralleled by DNA amplification of the gene [63]. A second mechanism of EZH2 overexpression is post-transcriptional regulation by miRNAs. EZH2 expression is controlled by miR-26a, miR-101, miR-205 and miR-214 [64–68]. Cancer-specific downregulation of these miRNAs results in overexpression of EZH2.

Bmi-1

In a subsequent step, PRC2 and the H3K27 methylation recruit PRC1 binding to chromatin to maintain stable gene silencing. PRC1 catalyzes ubiquitinylation of histone H2A and remains anchored to chromatin after its modification by the cooperation between PRC2 and PRC1. Bmi-1, a component of PRC1, plays an important role in gene silencing and is overexpressed in several cancers, including nonsmall cell lung cancer and colorectal cancer. Bmi-1 overexpression contributes to self-renewal in some types of cancer stem cells, including those of the pancreas [69], breast [70], brain [71] and white blood cell lineage [72]. Downregulation of miR-128 in glioma tissue causes elevated expression of Bmi-1, which consequently enhances self-renewal of the cancer stem cell population via chromatin remodeling [71]. In addition, recently, Wellner et al. [51] recently demonstrated that an EMT-related miRNA, miR-203, targets Bmi-1. This finding suggests that EMT mechanisms include the regulation of epigenetic regulators by miRNAs.

Yin Yang 1 (YY1)

YY1 is a transcription factor that contributes to various biological processes, including embryogenesis, the cell cycle, apoptosis, inflammation, carcinogenesis and epigenetics. In the epigenetic context, YY1 is a PRC-binding protein that recruits PRC2 and HDAC to a specific genome locus to induce chromatin remodeling. NF-κB-mediated miR-29b/c repression reactivates YY1 protein expression from post-transcriptional silencing induced by these two miRs. In addition, YY1 also represses miR-29b/c. This NF-κB-miR-29-YY1 regulatory circuit is also involved in myogenesis and tumorigenesis, probably via chromatin remodeling [73].

HDACs

In human cells, PRC2 physically associates with HDACs 1 and 2 [74]. If H3K27 is pre-acetylated, methylation at an H3K27 residue by PRC2 may require deacetylation by HDACs. Thus, both acetylation and deacetylation of histones is involved in the transcriptional regulation of target genes. In addition, recent studies have demonstrated that HDACs target not only histone proteins, but also nonhistone proteins: p53 and Myo-D are targeted by HDAC-1, whereas Bcl-6, Stat3 and YY1 are targeted by HDAC-2. By regulating both histone and nonhistone proteins, HDACs 1 and 2, classified as class I HDACs, are implicated in cell proliferation, apoptosis and chemoresistance. The expression of HDACs 1 and 2 is elevated in various cancers [75]. However, the mechanism of HDAC overexpression remains unclear. Dysregulation of miRNAs may contribute to the overexpression of HDACs observed in cancer cells. In prostate cancer, HDAC-1 is a direct target of miR-449a, and downregulation of miR-449a causes overexpression of HDAC-1. Thus, aberrant expression of miR-449a may contribute to the abnormal epigenetic patterns occurring in prostate cancer.

DNMT 3A and 3B

DNMTs 1, 3A, and 3B are key DNA methylation enzymes. Recent studies in human cells have demonstrated that PRC2 and DNMTs are physically and functionally linked [76], and that DNMT-mediated DNA methylation lies downstream of PRC2-mediated H3K27 methylation [76,77]. Thus, these two key epigenetic repression systems cooperate in the silencing of target genes. Dysregulation of DNMTs has been linked to various disease processes, including cancer and congenital disorders. These DNMTs are predicted to be potential targets of miRNAs [78]. Fabbri et al. [79] showed that members of the miR-29 family directly target DNMTs 3A and 3B, and that exogenous miR-29 species can reactivate methylation-silenced tumor suppressor genes by restoring normal patterns of DNA methylation in nonsmall cell lung cancer cells. Another study reported similar findings in acute myeloid leukemia [80]. Thus, miRNAs may be involved in the establishment and/or maintenance of DNA methylation. In addition, some isoforms of DNMT3B are targeted at the penultimate exon of their coding regions by miR-148 [81]. DNMT3B exhibits several splicing isoforms, of which DNMT3B-1 and -3 are the most abundant. DNMT3B-1 possesses a catalytic domain and a miR-148 target site. Thus, DNMT3B-1 is a miR-148-sensitive isoform. By contrast, DNMT3B-3 lacks a catalytic domain and the miR-148 target site, and remains miR-148 resistant. The biological roles of different DNMT3B isoforms are not yet fully understood. However, this finding indicates that miRNAs can regulate gene expression uniquely among different gene isoforms by targeting a coding exon.

As described above and illustrated in Fig. 1, a number of miRNAs are regulated epigenetically. At the same time, a variety of miRNAs regulate epigenetic pathway-related molecules, most notably polycomb group proteins, HDACs and DNA methyltransferases. Taken together, post-transcriptional regulation by miRNAs and transcriptional control machinery by epigenetics cooperate with each other to organize the whole gene expression profile and to maintain physiological functions in cells. Once this miRNA–epigenetics regulatory circuit is disrupted, normal physiological functions are interfered with, contributing to various disease processes. A comprehensive elucidation of this regulatory network still remains to be completed. Therefore, continual studies on dysregulation of the miRNA–epigenetics regulatory circuitry would be highly beneficial for deepening our understanding of diseases.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biogenesis and RNAi functions of miRNAs
  5. Epigenetically-regulated miRNAs
  6. miRNAs regulating epigenetic pathway-related genes
  7. Materials and methods
  8. References
  9. Supporting Information

Typing of miRNAs by positional relationship to mRNA transcripts

Information about the localization and strand direction of 939 miRNAs, 35245 Refseq genes and 283708 mRNAs was retrieved from the genome browser of University of California Santa Cruz [82] on 31 January 2011. Because the original data table of refseq genes included miRNA genes, these miRNA data were excluded from the Refseq data set. Using matlab, version 2011a (Mathworks, Natick, MA, USA), we compared localization and strand direction between miRNAs and transcripts (Refseq genes and mRNAs). Intragenic and intergenic miRNAs were defined by whether the miRNAs are overlapped by transcripts, or not, respectively. In addition, intragenic miRNAs were divided into three different types, which are overlapped by transcripts only in the same strand direction, only in opposite direction, or in both directions, respectively. The complete results of this typing analysis are provided in Table S1.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biogenesis and RNAi functions of miRNAs
  5. Epigenetically-regulated miRNAs
  6. miRNAs regulating epigenetic pathway-related genes
  7. Materials and methods
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Biogenesis and RNAi functions of miRNAs
  5. Epigenetically-regulated miRNAs
  6. miRNAs regulating epigenetic pathway-related genes
  7. Materials and methods
  8. References
  9. Supporting Information

Table S1. miRNAs and overlapping transcripts.

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