Mamm Genome (2009) 20:573580

DOI 10.1007/s00335-009-9206-5

Epigenetics, miRNAs, and human cancer: a new chapter

in human gene regulation
Nicola Valeri Ivan Vannini Francesca Fanini
Federica Calore Brett Adair Muller Fabbri

Received: 26 May 2009 / Accepted: 6 July 2009 / Published online: 22 August 2009
Springer Science+Business Media, LLC 2009

Abstract Cancer is a genetic and epigenetic disease.

MicroRNAs (miRNAs), a class of small noncoding RNAs,
have been shown to be deregulated in many diseases
including cancer. An intertwined connection between epigenetics and miRNAs has been supported by the recent
identification of a specific subgroup of miRNAs called
epi-miRNAs that can directly and indirectly modulate
the activity of the epigenetic machinery. The complexity of
this connection is enhanced by the epigenetic regulation of
miRNA expression that generates a fine regulatory feedback loop. This review focuses on how epigenetics affects
the miRNome and how the recently identified epi-miRNAs
regulate the epigenome in human cancers, ultimately contributing to human carcinogenesis.

Introduction
The term epigenetics includes all heritable changes in gene
expression not associated with concomitant alterations in
the DNA sequence. Epigenetic regulation usually includes
DNA methylation and histone modifications.
N. Valeri
F. Calore
B. Adair
M. Fabbri
Department of Molecular Virology, Immunology, and Medical
Genetics and Comprehensive Cancer Center, Ohio State
University, Columbus, OH 43210, USA
I. Vannini
F. Fanini
M. Fabbri
Istituto Scientifico Romagnolo per lo Studio e la Cura dei
Tumori, Meldola 47014, Italy
M. Fabbri (&)
Comprehensive Cancer Center, The Ohio State University,
Biomedical Research Tower, 460 West 12th Avenue, Room
1092, Columbus, OH 43210, USA
e-mail: muller.fabbri@osumc.edu

DNA methylation is a reversible reaction, catalyzed by

three major DNA methyltransferases: DNMT1 (maintenance DNMT), which preserves the methylation patterns
throughout each cell division (Li et al. 1992, 1993), and
DNMT3a and DNMT3b (de novo DNMTs), which transfer
a methyl group to previously unmethylated genomic
regions (Okano et al. 1999). Methylation occurs in specific
genomic areas called CpG islands (Herman and Baylin
2003; Weber et al. 2007), usually at the promoter region of
a gene, and prevents gene expression. In addition to DNA
methylation, histone modifications represent the other
important process involved in epigenetic gene regulation.
Histones are the main protein components of chromatin,
which can be classified into two forms: the accessible,
gene-dense, and early-replicating euchromatin and the
inaccessible, late-replicating, and gene-dense heterochromatin. Histones store epigenetic information in the form of
post-translational modifications, which define the so-called
histone code. They are globular proteins with protruding
N-terminal tails that can undergo several chemical reactions, favoring the switch versus eu- or heterochromatin.
For example, di- or trimethylation of lysine 9 of histone 3
(H3K9me) is a characteristic mark of heterochromatin,
highly conserved in fungi, plants, and animals, whereas
acetylation of histones H3 and H4 and methylation of
lysine 4 of histone H3 (H3K4me) are found in euchromatin
(Bernstein et al. 2007). Effectors of these changes in histone tails are histone deacetylases (HDACs) and histone
methyltransferases (HMTs). Finally, the polycomb repressive complex 2 (PRC2) can mediate epigenetic gene
silencing by trimethylating histone H3 lysine 27
(H3K27me3), a mark of heterochromatin.
MicroRNAs (miRNAs) are noncoding RNAs (ncRNAs)
with key regulatory functions and involved in many
biological processes spanning from development,

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differentiation, and cell cycle regulation to senescence

and metabolism (Bartel 2004; Boehm and Slack 2006;
Carleton et al. 2007; Harfe 2005; Pasquinelli et al. 2005).
The full spectrum of miRNAs expressed in a specific cell
type (the miRNome) varies between normal and pathologic tissues (Sevignani et al. 2006), and specific signatures of deregulated miRNAs harbor diagnostic and
prognostic implications (Calin et al. 2005). The first link
between miRNAs and cancer came from the discovery
that these ncRNAs are frequently located in cancerassociated genomic regions (CAGRs), which include
minimal regions of amplification, loss of heterozygosity,
common breakpoint regions in or near oncogenes (OGs)
and tumor suppressor genes (TSGs), and fragile sites
(preferential sites of chromatid exchange, deletion,
translocation, amplification, or integration of plasmid
DNA and tumor-associated viruses) (Calin et al. 2004).
Since then, a myriad of studies have investigated the
aberrancies of the miRNome in almost all kind of human
cancer (for reviews see Deng et al. 2008; Fabbri et al.
2008a, b, 2009; Garzon and Croce 2008; Nelson and
Weiss 2008). In particular, while some miRNAs act
mainly as TSGs, others are frequently overexpressed in
human tumors with respect to the normal tissue counterpart, target TSGs, and therefore exert a tumorigenic
function. The MiR-15a/16-1 cluster, for instance, is able
to control the expression of about 14% of all the genes in
the human genome (Calin et al. 2008) and acts as a TSG
in chronic lymphocytic leukemia by targeting the antiapoptotic gene BCL2 (Cimmino et al. 2005). MiRNAs,
with a well-established role as OGs, include the miR-1792 cluster which is transactivated by the c-MYC oncogene and dramatically accelerates lymphomagenesis in
murine models (He et al. 2005; Mendell 2008), miR-155
which induces leukemia in transgenic murine models
(Costinean et al. 2006) and has an important function as
regulator of inflammation and the immune response
(Rodriguez et al. 2007; Thai et al. 2007; Tili et al. 2007),
and miR-21 which targets important TSGs such as
PTEN1 (Cully et al. 2006) and PDCD4 in several neoplasms (Asangani et al. 2008; Frankel et al. 2008; Meng
et al. 2007). In an attempt to understand which mechanisms underlie the abnormal miRNA expression observed
in cancer, an increasing number of studies have investigated how miRNAs are regulated. It is now widely
accepted that miRNAs undergo the same regulatory
mechanisms of any other protein-coding gene (PCG),
including epigenetic regulation. Intriguingly, a subgroup
of these ncRNAs (epi-miRNAs) control, directly and
indirectly, the expression of epigenetic effectors such as
DNMTs, HDACs, and polycomb genes, casting a new
light on the functions of miRNAs as both genetic and
epigenetic regulators.

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Biogenesis of miRNAs
MiRNA genes are transcribed in the nucleus by an RNA
polymerase II. The first transcript is a long, capped, and
polyadenylated precursor (pri-miRNA) which is subsequently processed by a ribonuclease III (called Drosha), in
conjunction with its binding partner DGCR8 (DiGeorge
syndrome critical region gene 8, or Pasha), into a 70-100-nt
hairpin-shaped RNA called pre-miRNA. Translocated to
the cytoplasm by exportin 5, pre-miRNA is cleaved into a
18-24-nt miRNA duplex by a ribonucleic complex composed of a ribonuclease III (Dicer) and TRBP (HIV-1
transactivating response RNA binding protein). The duplex
binds to a large protein complex called RISC (RNAinduced silencing complex) which is composed of several
proteins, including members of the Argonaute family. One
strand of the miRNA duplex remains stably associated with
RISC and drives the other strand (the proper mature
miRNA) to its target mRNAs, which can undergo cleavage
(in case of perfect miRNA:mRNA complementarity) or
translational repression (in case of imperfect complementarity) (Bartel 2004; Brennecke et al. 2005; Gregory and
Shiekhattar 2005). Usually miRNAs exert their regulatory
function by binding to a seed sequence in the 30 untranslated region (30 -UTR) of their target mRNAs.
However, binding to the 50 -UTR and to the coding regions
of the messengers has also been described (Vasudevan
et al. 2007; Vatolin et al. 2006). The disruption of the
miRNA processing machinery contributes to impaired
miRNA production and carcinogenesis. Overall, miRNA
expression is reduced in tumors with respect to the normal
tissue counterpart (Calin and Croce 2006; Lu et al. 2005).
In 2005, Karube et al. (2005) described for the first time a
reduction of Dicer expression in lung cancer and showed
that lower levels of Dicer correlate with a poor prognosis in
lung cancer. More recently, Kumar et al. (2007) showed
that impaired miRNA processing (by targeting Drosha,
DGCR8, and Dicer in mouse models) enhanced cellular
transformation and tumorigenesis, whereas Melo et al.
(2009) described how TARBP2 (TAR RNA-binding protein 2) frameshift mutations reduce the levels of TRBP,
leading to a defect in miRNA processing and increased
carcinogenesis. Taken together, these works underline the
importance of miRNA biogenetic machinery in cell physiology and provide evidence that its disruption by mice
genetics, mutation, or loss of expression is involved in
human carcinogenesis.

Epigenetic regulation of miRNAs in human cancer

Epigenetics is responsible for aberrant miRNA expression
in several malignancies. In a breast cancer cell line, Scott

N. Valeri et al.: Epigenetics, miRNAs, and human cancer

et al. (2006) were able to demonstrate that 27 miRNA

expression levels are rapidly modified by treatment with
the HDAC inhibitor LAQ824, indicating that indeed epigenetic factors are involved in miRNA regulation. Similarly, by treating bladder cancer cells with both a DNA
demethylating agent (5-aza-20 -deoxycytidine) and an
HDAC inhibitor (4-phenylbutyric acid), Saito et al. (2006)
showed that about 5% of all human miRNAs increased
their expression levels. However, given the constantly
increasing number of newly identified miRNAs in the
human genome, the fraction of re-expressed miRNAs after
treatment with epigenetic drugs is significantly higher.
Among the re-expressed miRNAs there is miR-127, which
is embedded in a CpG island region and is epigenetically
silenced by both promoter hypermethylation and histone
modifications in cancer cells (Saito et al. 2006). This
miRNA belongs to a large miRNA cluster that includes
miR-136, -431, -432, and -433, but it is the only member of
the cluster whose re-expression was observed when cells
were treated with the two epigenetic drugs (Saito et al.
2006). Moreover, when each drug was used alone no variation in miR-127 expression was detected (Saito et al.
2006), suggesting that a combination of epigenetic processes (both DNA methylation and histone modifications)
rules miR-127 levels. Because the BCL6 oncogene is a
direct target of this miRNA (Saito et al. 2006), the epigenetic silencing of miR-127 unlocks the expression of the
oncogene and contributes to bladder carcinogenesis.
In the colorectal cancer cell line HCT-116, Lujambio
et al. (2007) created a double knockout (DKO) for DNMT1
and DNMT3b and compared the miRNA expression profile
of DKO and wild-type cells. About 6% of the 320 analyzed
miRNAs were upregulated in the DKO cells. Among the
deregulated miRNAs, only miR-124a was embedded in a
CpG island that is densely methylated in the cancer cell
line but not in normal tissue. This miRNA directly targets
CDK6, and its restoration reduces the levels of CDK6 and
has an impact on the phosphorylation status of the CDK6
downstream effector Rb protein (Lujambio et al. 2007).
Recently, Prospers group analyzed 353 acute lymphoblastic leukemia (ALL) patients and identified a signature
of 13 miRNAs embedded in CpG islands, with high heterochromatic markers (such as high levels of K9H3me2
and/or low levels of K4H3me3) (Agirre et al. 2009;
Roman-Gomez et al. 2009). Treatment with 5-aza-20 deoxycytidine upregulated at least one miRNA of the signature in 65% of ALL patients (Roman-Gomez et al.
2009). Among these, miR-124a was methylated in 59% of
ALL patients, and its promoter hypermethylation was
associated with a higher relapse rate and mortality rate
versus nonhypermethylated cases, since miR-124a promoter methylation status is an independent prognostic
factor for disease-free and overall survival (Agirre et al.

575

2009). Moreover, also in ALL the impact of miR-124a in

the CDK6-Rb pathway was demonstrated by showing that
miR-124a directly silences CDK6 (Agirre et al. 2009).
Recently, Ando et al. (2009) showed that hypermethylation
of miR-124a promoter is involved in the formation of
epigenetic field defect, a gastric cancer-predisposing condition characterized by accumulation of abnormal DNA
methylation in normal-appearing gastric mucosa and
induced mostly by Helicobacter pylori infection. These
findings reveal that miR-124a promoter hypermethylation
is an early event in gastric carcinogenesis. In addition to
miR-124a, miR-107, another epigenetically controlled
miRNA, targets CDK6 and impacts this oncogenic pathway in pancreatic cancer (Lee et al. 2009).
In HCT-116 cells deficient in DNMT1 and DNMT3B,
Brueckner et al. (2007) showed increased expression of let7a-3, a miRNA normally silenced by promoter hypermethylation in the wild-type cell line. In lung adenocarcinoma
primary tumors, let-7a-3 promoter was found hypomethylated with respect to the normal counterpart (Brueckner
et al. 2007), whereas hypermethylation of let-7a-3 promoter was described in epithelial ovarian cancer, paralleled
the low expression of insulin-like growth factor-II
expression, and was associated with a good prognosis (Lu
et al. 2007). Therefore, DNA methylation could act as a
protective mechanism by silencing miRNAs that have
oncogenic function.
Overall, the above-mentioned studies demonstrate that
epigenetic factors can control human carcinogenesis, not
only by directly affecting the expression of OGs and TSGs,
but also by affecting the expression of miRNAs involved in
oncogenic pathways. MiRNA epigenetic control might be
tissue-specific since no variation in miRNA expression was
observed in lung cancer cells treated with either demethylating agents or HDAC inhibitors or their combination
(Yanaihara et al. 2006).
Another miRNA which is under epigenetic control is
miR-1. In hepatocarcinoma miR-1 is frequently silenced by
promoter hypermethylation (Datta et al. 2008). However,
in DNMT1-null HCT-116 cells (but not in DNMT3B-null
cells), hypomethylation and re-expression of miR-1-1 were
observed (Datta et al. 2008), revealing a key role for the
maintenance DNMT in the regulation of this miRNA.
Several studies have demonstrated that miRNAs affect
the metastatic process by targeting metastasis-related genes
(Huang et al. 2008; Ma et al. 2007; Tavazoie et al. 2008).
Lujambio et al. (2008) investigated whether epigenetic
factors determine miRNA expression in metastatic cancer.
By treating three lymph node metastatic cell lines with
5-aza-20 -deoxycytidine and performing a miRNA microarray analysis, followed by CpG island analysis and
bisulfite genomic sequencing, the authors identified three
miRNAs that showed cancer-specific CpG island

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hypermethylation: miR-148a, miR-34b/c, and miR-9 (Lujambio et al. 2008). The reintroduction of miR-148a and
miR-34b/c in cancer cells with epigenetic inactivation
inhibited the cells motility and their metastatic potential in
xenograft models and was associated with downregulation
of miRNA oncogenic target genes such as c-MYC, E2F3,
CDK6, and TGIF2 (Lujambio et al. 2008). Finally, promoter hypermethylation of these three miRNAs was significantly associated with metastasis in human
malignancies (Lujambio et al. 2008). The miR-34b/c
cluster is epigenetically regulated in colorectal cancer
(promoter hypermethylation in 90% of primary colorectal
cancer tumors versus normal colon mucosa) (Toyota et al.
2008), whereas epigenetic silencing of miR-9 and miR148a (together with miR-152, -124a, and -663) was
described in breast cancer. In breast cancer cell lines
treated with 5-aza-20 -deoxycytidine, a reactivation of miR9-1 occurred, with no changes in the levels of the other
aberrantly methylated miRNAs (Lehmann et al. 2008),
suggesting that different epigenetic processes can control
epigenetically regulated miRNAs in different types of
cancer.
The relationship between miRNA and cognate host gene
epigenetic regulation was addressed by Grady et al. (2008)
by studying miR-342, located in an intron of the EVL (Ena/
Vasp-like) gene. EVL promoter hypermethylation occurs
in 86% of colorectal cancers and is already present in 67%
of adenomas, suggesting that it is an early event in colon
carcinogenesis. The combined treatment of 5-aza-20 -deoxycytidine with the HDAC inhibitor trichostatin A restores
the synchronized expression of EVL and miR-342. The
EGFL7 gene, frequently downregulated in several cancer
cell lines and in primary bladder and prostate tumors, hosts
miR-126 in one of its introns. While the mature miR-126
can be encoded by three different transcripts of the cognate
host gene, each with its own promoter, miR-126 is concomitantly upregulated with one of EGFL7 transcripts,
which has a CpG island promoter, when cancer cell lines
are treated with inhibitors of DNA methylation and histone
deacetylation, indicating that silencing of intronic miRNAs
in cancer may occur by means of epigenetic changes of
cognate host genes (Saito et al. 2009).
Finally, Fazi et al. (2007) showed that transcription
factors can recruit epigenetic effectors at miRNA promoter
regions and contribute to the regulation of their expression.
The AML1/ETO fusion oncoprotein is the aberrant product
of t(8;21) translocation in acute myeloid leukemia (AML)
and can bind to the pre-miR-223 region. The oncoprotein
recruits epigenetic effectors (i.e., DNMTs, HDAC1, and
MeCP2), leading to aberrant hypermethylation of the CpG
in close proximity to the AML1/ETO binding site and H3H4 deacetylation of the same chromatin region.

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In summary, a myriad of studies show that miRNAs

undergo epigenetic regulation, similar to any other PCG.
Table 1 summarizes those that have been identified at the
moment. MiRNAs represent an indirect mechanism
through which epigenetics affects the expression of OGs
and TSGs and ultimately impacts on human carcinogenesis. The complexity of the miRNA-epigenetics relationship
is enriched by the recent findings that a subset of miRNAs
(which we call epi-miRNAs) can also regulate the
expression levels of effectors of the epigenetic machinery.

Epi-miRNAs: regulators of epigenetic effectors

It has been estimated that miRNAs regulate the expression
of about 30% of all human genes (Lewis et al. 2005; Xie
et al. 2005). The term epi-miRNAs refers to a subgroup of
miRNAs that target, directly or indirectly, effectors of the
epigenetic machinery. Table 2 lists currently identified epimiRNAs. The first evidence of the existence of epi-miRNAs was obtained in lung cancer cell lines in which a
family of miRNAs (namely, miR-29a, -29b, and -29c)
directly targets DNMT3a and DNMT3b (Fabbri et al.
2007). Re-expression of miR-29s induces disruption of de
novo DNA methylation and leads to a global DNA hypomethylation of cancer cells. Moreover, TSGs that are
epigenetically silenced in lung cancer by promoter hypermethylation (such as FHIT and WWOX) were re-expressed
as a consequence of their promoter CpG island demethylation occurring after miR-29s treatment, which led to
cancer cell apoptosis and inhibition of lung cancer growth
in xenograft models (Fabbri et al. 2007). This discovery
revealed a previously unknown mechanism: miRNAs can
indirectly regulate gene expression by directly regulating
epigenetic processes. Since DNMT1 might compensate the
lack of de novo DNMTs, the global hypomethylation
observed in cells treated by miR-29s suggests a possible
impact of this miRNA family on DNMT1 activity as well.
Indeed, Garzon et al. (2009) were able to demonstrate in
acute myeloid leukemia (AML) cells that in addition to
directly targeting both DNMT3a and DNMT3b, miR-29b is
also able to indirectly silence DNMT1 by directly targeting
SP1, a transactivator of the DNMT1 gene. DNMT3b
expression is also under the control of miR-148a and miR148b. By binding to an unusual site in the coding region
(not the 30 -UTR) of DNMT3b mRNA, the miR-148 family
regulates this de novo DNMT and might be responsible for
the several different splice variants of DNMT3b (Duursma
et al. 2008). Interestingly, since miR-148a is also epigenetically regulated and its promoter is frequently hypermethylated in different tumors (Lehmann et al. 2008;
Lujambio et al. 2008), a self-amplified epigenetic loop for

N. Valeri et al.: Epigenetics, miRNAs, and human cancer

577

Table 1 Epigenetically regulated miRNAs

miRNA

Location

Cancer type

Target

References

miR-127

14q32.31

Bladder

BCL6

Saito et al.(2006)

miR-124a

8p23.1 (miR-124a-1)

Colorectal

CDK6

8q12.3 (miR-124a-2)

ALL

20q13.33 (miR-124a-3)

Gastric

Lujambio et al.(2007)
Agirre et al. (2009)
Roman-Gomez et al. (2009)
Ando et al. (2009)

miR-107

10q23.31

Pancreatic

CDK6

Lee et al. (2009)

let-7a-3

22q13.31

Colorectal

RAS

Brueckner et al. (2007)

Lung

IGF2

Lu et al. (2007)
Datta et al. (2008)

Ovarian
miR-1

20q13.33 (miR-1-1)

Colorectal

HDAC4

miR-148a

18q11.2 (miR-1-2)
7p15.2

HCC
Metastases

TGIF2

Breast
miR-34b/c

11q23.1

miR-9

1q22 (miR-9-1)
5q14.3 (miR-9-2)

Breast

Lujambio et al. (2008)

Lehmann et al. (2008)

Metastases

CDK6, c-MYC

Lujambio et al. (2008)

Colorectal

E2F3

Toyota et al. (2008)

Metastases

N/A

Lujambio et al. (2008)

Lehmann et al. (2008)

15q26.1 (miR-9-3)
miR-342

14q32.2

miR-126

9q34.3

Colorectal

N/A

Grady et al. (2008)

Bladder

N/A

Saito et al. (2009)

NFI-A, MEF2C

Fazi et al. (2007)

Prostatic
miR-223

Xq12

AML

ALL acute lymphoblastic leukemia, HCC hepatocellular carcinoma, AML acute myeloid leukemia

Table 2 Epi-miRNAs regulating effectors of the epigenetic machinery

miRNA
miR-29a, -29b, -29c

Location

Cancer or tissue type

Target

7q32.3 (miR-29a)

Lung

DNMT3a, DNMT3b, DNMT1

7q32.3 (miR-29b-1)

AML

References
Fabbri et al. (2007)
Garzon et al. (2009)

1q32.2 (miR-29b-2)
miR-148a, -148b

1q32.2 (miR-29c)
7p15.2 (miR-148a)

Cervical

DNMT3b

Mouse ES cells

RBL2

Duursma et al. (2008)

12q13.13 (miR-148b)
miR-290 cluster

7qA1

Benetti et al. (2008)

Sinkkonen et al. (2008)

miR-1

20q13.33 (miR-1-1)

Skeletal muscle tissue

HDAC4

Chen et al. (2006)

18q11.2 (miR-1-2)
miR-140

8qD3

Mouse cartilage tissue

HDAC4

Tuddenham et al. (2006)

miR-449a

5q11.2

Prostatic

HDAC1

Noonan et al. (2009)

miR-101

1p31.3 (miR-101-1)

Prostatic

EZH2

Varambally et al. (2008)

9p24.1 (miR-101-2)

Bladder

this epi-miRNA can be hypothesized, although more

experimental evidence is needed to confirm this model.
In mouse embryonic stem cells (ES), two independent
groups have shown that members of miR-290 cluster

Friedman et al. (2009)

directly target RBL2, an inhibitor of DNMT3 genes (Benetti et al. 2008; Sinkkonen et al. 2008). The miR-290
cluster is not expressed in ES Dicer-null cells, whereas
overexpression of RBL2 and disruption of the de novo

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methylation pathway were observed, leading to increased

telomere recombination and aberrant telomere elongation.
These effects were reversed by miR-290 cluster reexpression (Benetti et al. 2008; Sinkkonen et al. 2008).
Interestingly, the regulatory effect of the miR-290 cluster
on de novo DNMTs was not observed in human embryonic
kidney (HEK) 293 cells following Dicer knockdown,
suggesting that the miR-290 targeting effect on DNMT3s
might be cell- and/or species-specific (Sinkkonen et al.
2008).
Epi-miRNAs also regulate the expression of histone
deacetylases (HDACs) and polycomb repressive complex
genes (PRC). HDAC4 is a direct target of both miR-1 and
miR-140 (Chen et al. 2006; Tuddenham et al. 2006),
whereas miR-449a binds to the 30 -UTR region of HDAC1
(Noonan et al. 2009). HDAC1 is upregulated in several
kinds of cancer, and miR-449a re-expression in prostate
cancer cells induces cell-cycle arrest, apoptosis, and a
senescent-like phenotype by reducing the levels of HDAC1
(Noonan et al. 2009). EZH2 is the catalytic subunit of the
polycomb repressive complex 2 (PRC2) and is responsible
for heterochromatin formation by trimethylating histone
H3 lysine 27 (H3K27me3), leading to the silencing of
several TSGs. Varambally et al. (2008) showed that in
prostate cancer cell lines and primary tumors the expression of miR-101 decreases during cancer progression,
inversely correlating with an increase of EZH2. These
findings suggest a role for miR-101 as epi-miRNA, an
hypothesis that was tested and confirmed by showing that
miR-101 directly targets EZH2 in both prostate and bladder
cancer models (Friedman et al. 2009; Varambally et al.
2008). Moreover, miR-101-mediated suppression of EZH2
inhibits cancer cell proliferation and colony formation,
revealing a TSG role for miR-101, mediated by its modulatory effects on the cancer epigenome (Friedman et al.
2009).

Conclusion
The unbalanced expression of OGs and TSGs is the common pathogenetic mechanism of human cancer. Therefore,
elucidation of which processes regulate gene expression
and ultimately lead to this unbalance has the potential to
reveal how this aberration occurs and how it is involved in
human carcinogenesis. MiRNAs are frequently deregulated
in human tumors compared with the normal tissue counterpart. The discovery that miRNAs undergo epigenetic
regulation, similar to any other PCG, and that epi-miRNAs
can regulate effectors of the epigenetic machinery introduces new layers of complexity in gene regulation. A better
understanding of these mechanisms and of the intertwined
relationship between miRNAs and epigenetics is necessary

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N. Valeri et al.: Epigenetics, miRNAs, and human cancer

to understand how the human genome is regulated and how

gene expression aberrations that contribute to human carcinogenesis can be therapeutically corrected.
Acknowledgments Nicola Valeri is supported by an AmericanItalian Cancer Foundation Post-Doctoral Research Fellowship. Muller
Fabbri is supported by a 2009 Kimmel Scholar Award.

References
Agirre X, Vilas-Zornoza A, Jimenez-Velasco A, Martin-Subero JI,
Cordeu L et al (2009) Epigenetic silencing of the tumor
suppressor microRNA Hsa-miR-124a regulates CDK6 expression and confers a poor prognosis in acute lymphoblastic
leukemia. Cancer Res 69:44434453
Ando T, Yoshida T, Enomoto S, Asada K, Tatematsu M et al (2009)
DNA methylation of microRNA genes in gastric mucosae of
gastric cancer patients: its possible involvement in the formation
of epigenetic field defect. Int J Cancer 124:23672374
Asangani IA, Rasheed SA, Nikolova DA, Leupold JH, Colburn NH
et al (2008) MicroRNA-21 (miR-21) post-transcriptionally
downregulates tumor suppressor Pdcd4 and stimulates invasion,
intravasation and metastasis in colorectal cancer. Oncogene
27:21282136
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and
function. Cell 116:281297
Benetti R, Gonzalo S, Jaco I, Munoz P, Gonzalez S et al (2008) A
mammalian microRNA cluster controls DNA methylation and
telomere recombination via Rbl2-dependent regulation of DNA
methyltransferases. Nat Struct Mol Biol 15:268279
Bernstein BE, Meissner A, Lander ES (2007) The mammalian
epigenome. Cell 128:669681
Boehm M, Slack FJ (2006) MicroRNA control of lifespan and
metabolism. Cell Cycle 5:837840
Brennecke J, Stark A, Russell RB, Cohen SM (2005) Principles of
microRNA-target recognition. PLoS Biol 3:e85
Brueckner B, Stresemann C, Kuner R, Mund C, Musch T et al (2007)
The human let-7a-3 locus contains an epigenetically regulated
microRNA gene with oncogenic function. Cancer Res 67:1419
1423
Calin GA, Croce CM (2006) MicroRNA signatures in human cancers.
Nat Rev 6:857866
Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E et al (2004)
Human microRNA genes are frequently located at fragile sites
and genomic regions involved in cancers. Proc Natl Acad Sci
USA 101:29993004
Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M et al
(2005) A microRNA signature associated with prognosis and
progression in chronic lymphocytic leukemia. New Engl J Med
353:17931801
Calin GA, Cimmino A, Fabbri M, Ferracin M, Wojcik SE et al (2008)
MiR-15a and miR-16-1 cluster functions in human leukemia.
Proc Natl Acad Sci USA 105:51665171
Carleton M, Cleary MA, Linsley PS (2007) MicroRNAs and cell
cycle regulation. Cell Cycle 6:21272132
Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE et al (2006)
The role of microRNA-1 and microRNA-133 in skeletal muscle
proliferation and differentiation. Nat Genet 38:228233
Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M et al (2005)
miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc
Natl Acad Sci USA 102:1394413949
Costinean S, Zanesi N, Pekarsky Y, Tili E, Volinia S et al (2006) PreB cell proliferation and lymphoblastic leukemia/high-grade

N. Valeri et al.: Epigenetics, miRNAs, and human cancer

lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad
Sci USA 103:70247029
Cully M, You H, Levine AJ, Mak TW (2006) Beyond PTEN
mutations: the PI3K pathway as an integrator of multiple inputs
during tumorigenesis. Nat Rev Cancer 6:184192
Datta J, Kutay H, Nasser MW, Nuovo GJ, Wang B et al (2008)
Methylation mediated silencing of microRNA-1 gene and its role
in hepatocellular carcinogenesis. Cancer Res 68:50495058
Deng S, Calin GA, Croce CM, Coukos G, Zhang L (2008)
Mechanisms of microRNA deregulation in human cancer. Cell
Cycle 7:26432646
Duursma AM, Kedde M, Schrier M, le Sage C, Agami R (2008) miR148 targets human DNMT3b protein coding region. RNA
14:872877
Fabbri M, Garzon R, Cimmino A, Liu Z, Zanesi N et al (2007)
MicroRNA-29 family reverts aberrant methylation in lung
cancer by targeting DNA methyltransferases 3A and 3B. Proc
Natl Acad Sci USA 104:1580515810
Fabbri M, Croce CM, Calin GA (2008a) MicroRNAs. Cancer J 14:16
Fabbri M, Garzon R, Andreeff M, Kantarjian HM, Garcia-Manero G
et al (2008b) MicroRNAs and noncoding RNAs in hematological
malignancies: molecular, clinical and therapeutic implications.
Leukemia 22:10951105
Fabbri M, Croce CM, Calin GA (2009) MicroRNAs in the ontogeny of
leukemias and lymphomas. Leukemia Lymphoma 50:160170
Fazi F, Racanicchi S, Zardo G, Starnes LM, Mancini M et al (2007)
Epigenetic silencing of the myelopoiesis regulator microRNA223 by the AML1/ETO oncoprotein. Cancer Cell 12:457466
Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A et al
(2008) Programmed cell death 4 (PDCD4) is an important
functional target of the microRNA miR-21 in breast cancer cells.
J Biol Chem 283:10261033
Friedman JM, Liang G, Liu CC, Wolff EM, Tsai YC et al (2009) The
putative tumor suppressor microRNA-101 modulates the cancer
epigenome by repressing the polycomb group protein EZH2.
Cancer Res 69:26232629
Garzon R, Croce CM (2008) MicroRNAs in normal and malignant
hematopoiesis. Curr Opin Hematol 15:352358
Garzon R, Liu S, Fabbri M, Liu Z, Heaphy CE et al (2009)
MicroRNA-29b induces global DNA hypomethylation and
tumor suppressor gene re-expression in acute myeloid leukemia
by targeting directly DNMT3A and 3B and indirectly DNMT1.
Blood 113:64116418
Grady WM, Parkin RK, Mitchell PS, Lee JH, Kim YH et al (2008)
Epigenetic silencing of the intronic microRNA hsa-miR-342 and
its host gene EVL in colorectal cancer. Oncogene 27:38803888
Gregory RI, Shiekhattar R (2005) MicroRNA biogenesis and cancer.
Cancer Res 65:35093512
Harfe BD (2005) MicroRNAs in vertebrate development. Curr Opin
Genet Dev 15:410415
He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D et al
(2005) A microRNA polycistron as a potential human oncogene.
Nature 435:828833
Herman JG, Baylin SB (2003) Gene silencing in cancer in association
with promoter hypermethylation. N Engl J Med 349:20422054
Huang Q, Gumireddy K, Schrier M, le Sage C, Nagel R et al (2008)
The microRNAs miR-373 and miR-520c promote tumour
invasion and metastasis. Nature Cell Biol 10:202210
Karube Y, Tanaka H, Osada H, Tomida S, Tatematsu Y et al (2005)
Reduced expression of Dicer associated with poor prognosis in
lung cancer patients. Cancer Sci 96:111115
Kumar MS, Lu J, Mercer KL, Golub TR, Jacks T (2007) Impaired
microRNA processing enhances cellular transformation and
tumorigenesis. Nat Genet 39:673677
Lee KH, Lotterman C, Karikari C, Omura N, Feldmann G et al (2009)
Epigenetic silencing of microRNA miR-107 regulates cyclin-

579
dependent kinase 6 expression in pancreatic cancer. Pancreatology 9:293301
Lehmann U, Hasemeier B, Christgen M, Muller M, Romermann D
et al (2008) Epigenetic inactivation of microRNA gene hsa-mir9-1 in human breast cancer. J Pathol 214:1724
Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often
flanked by adenosines, indicates that thousands of human genes
are microRNA targets. Cell 120:1520
Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA
methyltransferase gene results in embryonic lethality. Cell
69:915926
Li E, Beard C, Jaenisch R (1993) Role for DNA methylation in
genomic imprinting. Nature 366:362365
Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J et al (2005)
MicroRNA expression profiles classify human cancers. Nature
435:834838
Lu L, Katsaros D, de la Longrais IA, Sochirca O, Yu H (2007)
Hypermethylation of let-7a-3 in epithelial ovarian cancer is
associated with low insulin-like growth factor-II expression and
favorable prognosis. Cancer Res 67:1011710122
Lujambio A, Ropero S, Ballestar E, Fraga MF, Cerrato C et al (2007)
Genetic unmasking of an epigenetically silenced microRNA in
human cancer cells. Cancer Res 67:14241429
Lujambio A, Calin GA, Villanueva A, Ropero S, Sanchez-Cespedes
M et al (2008) A microRNA DNA methylation signature for
human cancer metastasis. Proc Natl Acad Sci USA 105:13556
13561
Ma L, Teruya-Feldstein J, Weinberg RA (2007) Tumour invasion and
metastasis initiated by microRNA-10b in breast cancer. Nature
449:682688
Melo SA, Ropero S, Moutinho C, Aaltonen LA, Yamamoto H et al
(2009) A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function. Nat Genet 41:365370
Mendell JT (2008) miRiad roles for the miR-17-92 cluster in
development and disease. Cell 133:217222
Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST et al
(2007) MicroRNA-21 regulates expression of the PTEN tumor
suppressor gene in human hepatocellular cancer. Gastroenterology 133:647658
Nelson KM, Weiss GJ (2008) MicroRNAs and cancer: past, present,
and potential future. Mol Cancer Ther 7:36553660
Noonan EJ, Place RF, Pookot D, Basak S, Whitson JM et al (2009)
miR-449a targets HDAC-1 and induces growth arrest in prostate
cancer. Oncogene 28:17141724
Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases
Dnmt3a and Dnmt3b are essential for de novo methylation and
mammalian development. Cell 99:247257
Pasquinelli AE, Hunter S, Bracht J (2005) MicroRNAs: a developing
story. Curr Opin Genet Dev 15:200205
Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P et al (2007)
Requirement of bic/microRNA-155 for normal immune function. Science 316:608611
Roman-Gomez J, Agirre X, Jimenez-Velasco A, Arqueros V, VilasZornoza A et al (2009) Epigenetic regulation of microRNAs in
acute lymphoblastic leukemia. J Clin Oncol 27:13161322
Saito Y, Liang G, Egger G, Friedman JM, Chuang JC et al (2006)
Specific activation of microRNA-127 with downregulation of the
proto-oncogene BCL6 by chromatin-modifying drugs in human
cancer cells. Cancer Cell 9:435443
Saito Y, Friedman JM, Chihara Y, Egger G, Chuang JC et al (2009)
Epigenetic therapy upregulates the tumor suppressor microRNA126 and its host gene EGFL7 in human cancer cells. Biochem
Biophys Res Commun 379:726731
Scott GK, Mattie MD, Berger CE, Benz SC, Benz CC (2006) Rapid
alteration of microRNA levels by histone deacetylase inhibition.
Cancer Res 66:12771281

123

580
Sevignani C, Calin GA, Siracusa LD, Croce CM (2006) Mammalian
microRNAs: a small world for fine-tuning gene expression.
Mamm Genome 17:189202
Sinkkonen L, Hugenschmidt T, Berninger P, Gaidatzis D, Mohn F
et al (2008) MicroRNAs control de novo DNA methylation
through regulation of transcriptional repressors in mouse
embryonic stem cells. Nat Struct Mol Biol 15:259267
Tavazoie SF, Alarcon C, Oskarsson T, Padua D, Wang Q et al (2008)
Endogenous human microRNAs that suppress breast cancer
metastasis. Nature 451:147152
Thai TH, Calado DP, Casola S, Ansel KM, Xiao C et al (2007)
Regulation of the germinal center response by microRNA-155.
Science 316:604608
Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD et al (2007)
Modulation of miR-155 and miR-125b levels following
lipopolysaccharide/TNF-alpha stimulation and their possible
roles in regulating the response to endotoxin shock. J Immunol
179:50825089
Toyota M, Suzuki H, Sasaki Y, Maruyama R, Imai K et al (2008)
Epigenetic silencing of microRNA-34b/c and B-cell translocation gene 4 is associated with CpG island methylation in
colorectal cancer. Cancer Res 68:41234132
Tuddenham L, Wheeler G, Ntounia-Fousara S, Waters J, Hajihosseini MK et al (2006) The cartilage specific microRNA-140

N. Valeri et al.: Epigenetics, miRNAs, and human cancer

targets histone deacetylase 4 in mouse cells. FEBS Lett
580:42144217
Varambally S, Cao Q, Mani RS, Shankar S, Wang X et al (2008)
Genomic loss of microRNA-101 leads to overexpression of
histone methyltransferase EZH2 in cancer. Science 322:1695
1699
Vasudevan S, Tong Y, Steitz JA (2007) Switching from repression to
activation: microRNAs can up-regulate translation. Science
318:19311934
Vatolin S, Navaratne K, Weil RJ (2006) A novel method to detect
functional microRNA targets. J Mol Biol 358:983996
Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S et al (2007)
Distribution, silencing potential and evolutionary impact of
promoter DNA methylation in the human genome. Nat Genet
39:457466
Xie X, Lu J, Kulbokas EJ, Golub TR, Mootha V et al (2005)
Systematic discovery of regulatory motifs in human promoters
and 30 UTRs by comparison of several mammals. Nature
434:338345
Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K et al
(2006) Unique microRNA molecular profiles in lung cancer
diagnosis and prognosis. Cancer Cell 9:189198

123
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