Epigenetics is defined as the heritable chances that affect gene expression without changing the DNA sequence. Epigenetic regulation of gene expression can be through different mechanisms such as DNA methylation, histone modifications and nucleosome positioning. MicroRNAs are short RNA molecules which do not code for a protein but have a role in post-transcriptional silencing of multiple target genes by binding to their 3′ UTRs (untranslated regions). Both epigenetic mechanisms, such as DNA methylation and histone modifications, and the microRNAs are crucial for normal differentiation, development and maintenance of tissue-specific gene expression. These mechanisms also explain how cells with the same DNA content can differentiate into cells with different functions. Changes in epigenetic processes can lead to changes in gene function, cancer formation and progression, as well as other diseases. In the present chapter we will mainly focus on microRNAs and methylation and their implications in human disease, mainly in cancer.
Introduction to microRNAs
miRNAs (microRNAs) are a class of short (approx. 22 nt) endogenous non-coding RNAs that act as post-transcriptional regulators of gene expression. According to the September 2009 release of the microRNA database mirbase (http://www.mirbase.org/) there are 10883 miRNA entries from vertebrates, insects, plants and viruses discovered either by cloning or bioinformatics . Among them 721 are detected in humans. The first member of the miRNA family, lin-4, was originally identified in Caenorhabditis elegans as a developmental timing regulator . miRNAs play fundamental roles in the control of many biological processes such as growth, development, differentiation, proliferation and cell death [2,3]. They perform these functions by repression of their target genes. Each miRNA may target several hundred mRNAs and more than 60% of the mRNAs are predicted to have a miRNA-binding site in their 3′ UTR (3′ untranslated region). The huge number of miRNAs identified and evidence accumulated over the years indicate that a vast number of normal and pathological mechanisms are controlled by miRNA-mediated regulatory networks (reviewed in [4,5]).
Genomic organization, biogenesis and function
miRNAs can be intergenic, intronic or exonic. Intergenic miRNAs have either their own promoters (monocistronic) or share the same promoter (polycistronic), whereas the intronic miRNAs are present either singly or in clusters using the promoter of their host gene. miRNAs are transcribed in the nucleus by RNA polymerase II. They are 5′ capped and 3′ polyadenylated. The maturation of miRNAs requires two endonucleolytic cleavage steps by RNase III-like enzymes: Drosha and Dicer. Following transcription, Drosha processes the primary miRNA transcript (pri-miRNA), which can be several kilobases long, into a 60–100 nt hairpin structure named the precursor-miRNA (pre-miRNA). Pre-miRNAs are folded into mini-helical structures to be recognized by exportin-5, the nuclear export factor carrying the pre-miRNAs from the nucleus to the cytoplasm. In the cytoplasm, the pre-miRNA hairpin is cleaved at the loop end by Dicer, thereby creating a 22 nt RNA duplex comprising the mature miRNA guide strand and the miRNA* passenger strand. The mature miRNA is loaded into the RISC (RNA-induced silencing complex), whereas the passenger strand is degraded (Figure 1). The exact details of the miRNA biogenesis mechanism are still to be investigated, and much less is known about the mechanisms regulating the expression of miRNAs. Recent studies point out that not all miRNAs are created by the same mechanisms. After being loaded into the RISC complex mature miRNAs are directed to their binding sites in their target mRNAs. In broad terms, this binding leads to repression of mRNA translation by one of the following mechanisms: translational block by folding the mRNA in an inactive steric conformation, deadenylation and destabilization of the mRNA, cleavage of the mRNA or sequestration in P-bodies (processing bodies) (reviewed in [4,6,7]).
miRNAs regulate important biological processes
For many biological functions it is very important to have the miRNA expression in balance. Developmental timing, differentiation, organogenesis, cell proliferation, apoptosis, differentiation of embryonic stem cells, limb development, synaptic development and plasticity, skin differentiation, cardiogenesis, normal immune function and regulation of insulin secretion in the pancreas are some of the biological functions where miRNAs play a crucial role (reviewed in ).
miRNA and disease
In the last decade it has become clear that aberrant miRNA deregulation and expression is observed in most human malignancies, although it is often not clear whether this deregulation is the cause or the effect of the disease. Some of the most investigated malignancies are cancers, dysfunctional heart conditions, metabolic diseases and viral infections. Very recently a knowledge-base on the aberrant expression of miRNAs in various diseases was introduced . In cancers, miRNAs can act either as oncogenes or tumour suppressors, and a multitude of papers have investigated the differential expression of miRNAs in tumour tissues and their function in cancer cells and metastatic potential (see Table 1 for an overview). Many miRNAs act as tumour suppressor genes and they are frequently silenced in cancers. However, the underlying mechanism for this is less clear. One explanation is epigenetic silencing of miRNA genes and this has now been described in various cancers for several miRNAs. Mechanisms such as change in turnover rate or DNA copy number could be other reasons for differential expression of miRNAs which need to be further investigated [9,10].
Introduction to miRNA and epigenetics
Epigenetic phenomenons such as DNA methylation of CpG islands in promoter regions of genes and histone modifications are well known to regulate gene expression. The major epigenetic changes in cancer are aberrant DNA hypermethylation of tumour suppressor genes, global genomic DNA hypomethylation and disruption of the histone modification patterns.
Like classical protein coding genes, miRNA genes are also subject to epigenetic regulation and miRNAs can also regulate various components of the epigenetic machinery. For a detailed review, see .
Epigenetic regulation of miRNA expression in cancer
Several miRNAs are down-regulated in cancer and act as bona fide tumour suppressor genes, and therefore these miRNAs are obvious candidates for epigenetic silencing. High-quality papers have been published on the impact of epigenetic regulation of miRNA genes with regard to cell proliferation, apoptosis, tumour suppressor or oncogenic effects both in cell culture systems, in vivo models and in various human primary tumours. The epigenetic status has also been correlated with metastatic status and with survival of cancer patients, which will be the focus of this part of the chapter. An up-to-date overview is presented in Table 2.
The concept of controlling miRNA expression by epigenetic mechanisms may be as widespread as for protein coding genes, since half of the miRNA genes are associated with CpG islands and miRNA gene methylation is detected with high frequency in normal and malignant cells .
A common approach to identifying epigenetically regulated miRNAs has been to treat cancer cells with inhibitors of DNA methylation (e.g. 5-azacytidine) and/or histone deacetylases [e.g. PBA (4-phenylbutyric acid) or TSA (trichostatin A)] and compare miRNA expression to that in untreated cells.
It has turned out that epigenetic regulation of miRNA expression is a common hallmark in human cancers and that epigenetic tags are associated with metastatic status and clinically relevant endpoints, such as disease-free survival and overall survival, thereby suggesting their use as biomarkers in cancer detection, prognosis, monitoring and predicting response to treatment.
In CRC (colorectal cancer), epigenetically regulated miRNAs have been indentified in both cell lines and in tumour tissues. The expression of miR-342 was found to be regulated by CpG island methylation. Interestingly, methylation of miR-342 may be specific to CRC, since in vitro studies employing 40 non-CRC cell lines only found partial methylation in a single cell line. Analysis of tissue indicated that methylation of miR-342 may be an early event in CRC since methylation was detected in 86% of CRC adenocarcinomas and in 67% of adenomas . Comparison of normal and colon cancer tissues has shown that miR-124a is hypermethylated in 75% of the tumours (n=56) . Methylation of miR-124a was also found in tumours from the lungs (48%, n=27) and breast (32%, n=22), but not in neuroblastomas or sarcomas . Analysis of primary CRC tumours (n=111) and adjacent normal colon (n=17) found that miR-34b/c was methylated in 90% of the primary CRC tissues and very limited methylation was found in the normal mucosa .
In primary breast cancer specimens, aberrant hypermethylation has been shown for miR-9-1, miR-124a3, miR-148, miR-152 and miR-663 in 34–86% of cases in a series of 71 primary human breast cancer specimens . The miR-9-1 gene is hypermethylated in pre-invasive intraductal lesions, suggesting that hypermethylation of miR-9-1 is an early and frequent event in breast cancer development.
Two reports have associated methylation of miRNA genes with metastatic status of cancer patients.
Bandres et al.  identified five down-regulated miRNAs in primary CRC, which were located in the vicinity (<1000 bp) of a CpG island. Methylation status for three of these were analysed in primary CRC samples and adjacent normal tissue, and miR-9-1, miR-129-2 and miR-137 were methylated in 56% (n=36), 91% (n=34) and 100% (n=31) of primary CRC cases respectively. Methylation of miR-9-1 was totally absent in histological normal mucosa and methylation was more frequent in stage III and IV compared with stage I and II. Importantly, methylation status of miR-9-1 was associated with regional nodal invasion, vascular invasion and metastasis in a group of 32 patients (16 non-methylated and 20 methylated).
In a recent report, direct relation of miRNA hypermethylation and metastasis was explored . Cell lines established from lymph node metastasis were treated with 5-azacytidine and the miRNA expression relative to untreated cells was investigated. These experiments identified 16 hypermethylated and up-regulated miRNAs, located in the proximity of a CpG island. Comparison with methylation status of non-cancerous tissues further reduced the number of miRNAs displaying cancer-specific CpG island hypermethylation. The selected miRs – miR-148a, miR-34b/c, and miR-9-1/2/3 – were tested in vitro and in vivo for their potential involvement in metastasis. Re-introduction of miR-34b/c and miR-148 into a metastatic carcinoma cell line, which is hypermethylated and silenced for miR-34b/c and miR-148 expression, reduced the migratory capability of the cancer cells. Likewise, experiments with nude mice showed that re-introduction of miR-34b/c and miR-148 caused reduced tumour growth and diminished metastatic potential of the metastatic carcinoma cell line. A collection of primary tumour samples (n=278) from various tumour types were analysed and hypermethylation was undetectable in the corresponding normal tissue. Notably, hypermethylation of miR-34b/c, miR-148 and miR-9-3 in primary tumours was significantly associated with those tumours that were positive for metastatic cancer cells in the corresponding lymph nodes (n=207).
In a report focusing on ovarian cancer, eight miRNAs (miR-337, miR-368, miR-376a/b, miR-377, miR-410, miR-432, miR-495) located in the chromosome 14 miRNA cluster (Dlk1-Gtl2 domain) were identified as potential tumour suppressor genes regulated by DNA methylation . An expression signature separated late-stage ovarian cancers (n=73) into two distinct clusters. Patients belonging to the cluster with low expression of the eight miRNAs displayed higher tumour proliferation and had shorter 5-year survival. Analysis of other cancer types indicated that down-regulation of the chromosome 14 miRNA cluster may be an event common to many human epithelial tumours.
The let-7a-3 gene is located in a CpG island and its methylation status was analysed in 214 malignant tumours: no correlation between disease stage and tumour grade was detected . Although the disease-free survival was not associated with methylation of let-7a-3, the patients with low let-7a-3 methylation (n=138) had significantly worse overall survival than those with high methylation (n=67).
Two reports from the same laboratory have analysed epigenetic regulation of miRNA expression in ALL (acute lymphoblastic leukaemia) and associated it with clinical outcome.
In ALL-derived cell lines, analysis of histone modifications around CpG islands located in the 5′ UTR of miRNA genes identified 13 miRNA candidate genes for epigenetic silencing: miR-9-1/2/3, miR-10b, miR-34b/c, miR-124a1/a2/a3, miR-132, miR-196b, miR-203 and miR-212 . Methylation of at least one in 13 miRNA was found in 65% (n=353) of the ALL human tumours and was a strong and independent negative prognostic marker for disease-free survival and overall survival.
In ALL patients, miR-124 is regulated by CpG island hypermethylation and histone modifications, and re-introduction of miR-124a severely reduced tumorigenicity of ALL cells in a xenograft mouse model . The miR-124a methylation status was analysed in 353 ALL patients and hypermethylation was found in 59%; this correlated with decreased expression of miR-124a. Furthermore, hypermethylation significantly correlated with higher relapse and mortality rates, and multivariate analysis showed that miR-124a is an independent prognostic factor for both disease-free survival and overall survival.
Taken together, these results show that DNA demethylation and HDAC (histone deacetylase) inhibition can activate expression of miRNAs and further large-scale clinical investigations are clearly warranted.
miRNAs as regulators of epigenetic processes
As well as being regulated by epigenetic mechanisms, miRNAs also play a role in controlling the chromatin structure by post-transcriptional regulation of chromatin-modifying enzymes (reviewed in [11,25]). Among the predicted human miRNA target genes there are a number of genes involved in epigenetic regulation, such as the methyl CpG-binding proteins, HMTs (histone methyltransferases), chromodomain-containing proteins and HDACs . This subset of miRNAs, which directly or indirectly regulate the expression levels of effectors of the epigenetic processes, have been termed ‘epi-miRNAs’ . Aberrant regulation of miRNA expression plays an important and direct role in the aberrant epigenetic silencing of tumour suppressor genes by DNA methylation in human cancers (see Table 3).
DNA methylation patterns are laid down during development by DNMT3a and DNMT3b (where DNMT is DNA methyltransferase), whereas maintenance during replication is facilitated by DNMT1. The first direct link between a miRNA and the DNMTs was established between the miR-29 family (miR-29a/b/c) and DNMT3a and DNMT3b, and other miRNAs such as miR-148 and miR-143 have also been indicated as regulators of the methylation enzymes. In non-small cell carcinoma of the lung, miR-29 is down-regulated, whereas the DNMT3A and DNMT3B expression is increased. Re-expression of miR-29 is shown to disrupt the de novo DNA methylation and caused general hypomethylation, leading to expression of tumour suppressor genes that are silenced by methylation, which resulted in apoptosis in cancer cells both in vitro and in vivo. This study indicated that miR-29 regulates the DNMT3 genes in lung cancer and revealed a new mechanism whereby the miRNAs indirectly regulate the gene expression through direct regulation of epigenetic mechanisms . Another group has shown that overexpression of miR-29b in AML (acute myeloid leukaemia) cells resulted in marked reduction of DNMT1, DNMT3A and DNMT3B at both the RNA and protein levels. They concluded that the expression of miR-29b promoted DNA hypomethylation not only through direct targeting of DNMT3a and DNMT3b, but also by decreasing the DNMT1 expression indirectly via down-regulation of Sp1, a known transactivating factor of the DNMT1 gene .
Furthermore, miR-143 regulates DNMT3a in CRC cells, whereas miR-148a and miR-148b represses Dnmt3b expression in mouse cells through binding to a highly conserved sequence in its coding region rather than the 3′ UTR [29,30]. Benetti et al.  proposed a new regulatory pathway for DNA methylation involving the mammalian miR-290 cluster (miR-290, miR-291-3p, miR-291-5p, miR-292-3p, miR-292-5p, miR-293, miR-294 and miR-295) as an important regulator of Rbl2, which in turn acts as a transcriptional repressor of Dnmt3a and Dnmt3b causing hypomethylation in the genome, especially in the telomeres. The whole cluster is shown to be down-regulated in Dicer-null cells in mouse, whereas Rbl2 is increased in expression leading to repression of Dnmt3a and Dnmt3b causing DNA methylation defects . However, the regulatory effect of the miR-290 cluster on methylation cannot be shown in DICER-knockdown human embryonic kidney cells. This indicates that the miR-290 clusters’ effect on DNMTs could be cell-type- or species-specific . Recently, a completely new mechanism was suggested for regulation of gene expression by miRNAs in moss. Khraiwesh et al. propose that initiation of epigenetic silencing by DNA methylation is regulated according to the ratio of the miRNA and its target mRNA .
miRNAs also regulate the expression of HDACs and HMTs. HDAC4 is shown to be a direct target of miR-1 and miR-140 [34,35]. A new miRNA HDAC4 regulatory mechanism has been revealed in ALS (amyotrophic lateral sclerosis) which is the most common adult motor neuron disease. miR-206 is shown to delay the progression of ALS, and HDAC4 is both computationally and experimentally shown to be a target of miR-206. Interestingly, in miR-206−/− animals the HDAC4 protein expression is increased in skeletal muscles, whereas Hdac4 mRNA levels were not changed. This indicated that miR-206 acts upon Hdac4 by translational inhibition rather than at the transcription level . miR-1 and miR-499 are indicated in differentiation of cardiomyocytes, possibly by repression of HDAC4 and SOX6 genes .
miR-449a targets HDAC1, which is up-regulated in many cancer forms. miR-449a is down-regulated in cancer, but introduction to prostate cancer cells resulted in cell-cycle arrest and apoptosis . Similarly re-expression of miR-101 in cancer models also resulted in inhibition of cancer formation. miR-101 targets EZH2, the catalytic subunit of the Polycomb repressive complex 2 responsible for histone H3 Lys27 trimethylation, a mark of epigenetic repression, and can alter the chromatin structure globally [39,40]. Li et al.  identified a new miRNA (miR-2861) in primary mouse osteoblasts that promotes osteoblast differentiation by repressing HDAC5 expression at the post-transcriptional level.
Recently, the molecular mechanisms of epigenetic regulation of miRNA expression and miRNA-mediated control of the epigenetic machinery have attracted much attention, especially in cancer research. By now it is apparent that some miRNA genes are regulated by DNA CpG island hypermethylation and chromatin modifications. Interestingly, these epigenetic marks are potential biomarkers since significant correlations with survival of cancer patients have been found. Likewise, it is also clear that miRNAs regulate various components of the epigenetic machinery and thereby contribute to the regulation of the expression of other genes.
It is essential to explore in more detail this new layer of complexity in gene regulation to improve our understanding of the regulation of the human genome. Importantly, these new insights on the intertwined relationship between miRNA and epigenetics are likely to lead to novel revolutionary anti-cancer therapeutic approaches. Such approaches may be targeting components of the epigenetic network to cause re-expression of miRNA tumour suppressor genes or directly targeting mature miRNAs or re-expressing miRNAs in order to directly affect target genes and regulate epigenetic feedback loops.
• miRNas are small non-protein coding molecules that regulate more than 30% of the protein coding genes.
• miRNAs play an important role in many biological processes such as differentiation, organ development and proliferation.
• In cancer and some other diseases such as diabetes, neurological and cardiac diseases, a perturbed miRNA expression is found in the relevant tissues.
• Some miRNAs are regulated by epigenetic mechanisms, especially by methylation.
• Methylation status of some miRNA genes correlates with survival of cancer patients.
• miRNAs may regulate the epigenetic machinery directly or indirectly by targeting enzymes such as DNMTs or HDACs.
The Wilhelm Johannsen Centre for Functional Genome Research is established by the Danish National Research Foundation.
- © The Authors Journal compilation © 2010 Biochemical Society