Báo cáo khoa học: Function of microRNA-375 and microRNA-124a in pancreas and brain doc

REVIEW ARTICLE
Function of microRNA-375 and microRNA-124a in
pancreas and brain
Nadine N. Baroukh
1
and Emmanuel Van Obberghen
1,2
1 INSERM U907, Faculte
´
de Me
´
decine, Institut de Ge
´
ne
´
tique et Signalisation Mole
´
culaire (IFR50), Universite
´
de Nice Sophia-Antipolis, Nice,
France
2 Laboratoire de Biochimie, Ho
ˆ
pital Pasteur, CHU de Nice, France
Introduction
Completion of the sequencing of the human genome
has led to the identification and mapping of  25 000
protein-coding genes, which represent only 2–3%
of human genomic DNA. Approximately 45% of
the remaining DNA consists of repetitive sequences,
whereas the rest of the human genome harbours non-

coding functional elements and nonfunctional
sequences that have been referred to as ‘junk DNA’.
Increasing evidence supports the notion that the
majority of functional elements in the genome do not
Keywords
development; diabetes; gene regulation;
metabolism; microRNA; neurons; pancreatic
b-cell lines
Correspondence
N. Baroukh, INSERM U907, IFR50, Faculte
´
de Me
´
decine, Universite
´
de Nice Sophia-
Antipolis, 28 avenue de Valombrose, 06107
Nice Cedex 2, France
Fax: +33 4 93 81 54 32
Tel: +33 4 93 37 77 82
E-mail:
(Received 25 March 2009, revised 7 July
2009, accepted 3 September 2009)
doi:10.1111/j.1742-4658.2009.07353.x
In recent years, our understanding of how gene regulatory networks con-
trol cell physiology has improved dramatically. Studies have demonstrated
that transcription is regulated not only by protein factors, but also by small
RNA molecules, microRNAs (miRNAs). The first miRNA was discovered
in 1993 as a result of a genetic screen for mutations in Caenorhabditis
elegans. Since then, the use of sophisticated techniques and screening tools

has promoted a more definitive understanding of the role of miRNAs in
mammalian development and diseases. miRNAs have emerged as impor-
tant regulators of genes involved in many biological processes, including
development, cell proliferation and differentiation, apoptosis and metabo-
lism. Over the last few years, the number of reviews dealing with miRNAs
has increased at an impressive pace. In this review, we present general
information on miRNA biology and focus more closely on comparing the
expression, regulation and molecular functions of the two miRNAs, miR-
375 and miR-124a. miR-375 and miR-124a share similar features; they are
both specifically expressed in the pancreas and brain and directly bind a
common target gene transcript encoding myotrophin, which regulates exo-
cytosis and hormone release. Here, we summarize the available data
obtained by our group and other laboratories and provide an overview of
the specific molecular function of miR-375 and miR-124a in the pancreas
and the brain, revealing a potential functional overlap for these two
miRNAs and the emerging therapeutic potential of miRNAs in the treat-
ment of human metabolic diseases.
Abbreviations
DGCR8, DiGeorge syndrome critical region gene 8; Foxa2, Forkhead box a2; miRNA, microRNA; PDK-1, 3¢-phosphoinositide-dependent
protein kinase-1; Pdx-1, pancreas ⁄ duodenum homeobox protein 1; pre-miRNA, precursor miRNA; pri-miRNA, primary miRNA; REST,
response element silencing transcription factor; SCP1, C-terminal domain phosphatase 1.
FEBS Journal 276 (2009) 6509–6521 Journal compilation ª 2009 FEBS. No claim to original French government works 6509
code for proteins [1,2]. A major advance in under-
standing the regulation of genetic information came
with the discovery of microRNA (miRNA) molecules.
miRNAs are nonprotein-coding small RNAs,  19–23
nucleotides in length, that are implicated in the post-
transcriptional fine tuning of gene regulation. The first
miRNAs discovered were lin-4 and let-7, which are
crucial for regulating developmental timing in the nem-

atode, Caenorhabditis elegans [3,4]. Since these initial
reports, several hundred miRNAs have been identified
in various species. Many miRNAs are evolutionarily
maintained, suggesting a conservation of function. An
interesting study in zebrafish embryos showed that
most miRNAs are expressed during specific develop-
mental stages and in particular cell types, although
some are expressed ubiquitously [5]. These data sup-
port the notion of spatiotemporal- and cell type-spe-
cific miRNA expression [5,6]. In addition, microarray
analyses have shown that transient miRNA overex-
pression in cells leads to the downregulation of a large
number of transcripts [7]. Theoretically, one miRNA
could co-ordinate the regulation of hundreds of genes.
Comparative genomics has indeed predicted that one-
third of human genes could be miRNA targets [8].
Once identified, these miRNA molecules were depos-
ited for annotation in the miRNA catalogue estab-
lished by the Sanger Institute [9]. miRNAs are named
using the ‘miR’ prefix and a unique identifying number
[10]. Computational methods have been developed and
employed for the prediction of target genes for inverte-
brate and mammalian miRNAs, becoming an impor-
tant resource for the functional investigation of
individual miRNAs [11,12]. Our current knowledge
indicates that miRNAs govern a wide range of physio-
logical and developmental processes. They play an
important role in the control of cell survival, prolifera-
tion, differentiation and metabolism, whose dysfunc-
tion is a potential cause of disease [13–18]. For

example, single nucleotide polymorphisms that modify
miRNA-binding sites have been shown to alter pheno-
type [19] or cause disease [20]. We and others have
focused on the functions of miR-375 and miR-124a
and their respective target genes.
Biogenesis of miRNAs and their mode
of action on gene regulation
miRNAs are generated by a two step processing path-
way to yield RNA molecules of  22 nucleotides that
regulate target gene expression at the post-transcrip-
tional level [21]. Biogenesis of miRNAs starts with the
transcription of a long primary precursor product,
pri-miRNA, synthesized by RNA polymerase II. Like
other transcripts, pri-miRNA presents a 5¢cap struc-
ture and a 3¢poly(A) tail (Fig. 1). The pri-miRNA is
processed by a nuclear protein complex, Microproces-
sor, containing the RNaseIII-type protein Drosha and
its double-stranded RNA-binding partner protein
Pasha ⁄ DGCR8 (DiGeorge syndrome critical region
gene 8). The Microprocessor complex cleaves pri-miR-
NA to precursor miRNA (pre-miRNA), a 60–70 nucle-
otide RNA with a typical stem loop structure [22].
Pasha ⁄ DGCR8 acts together with the endonuclease
Drosha and plays a critical role in the biogenesis and
processing of miRNAs [23]. Pre-miRNAs are exported
into the cytoplasm by the nuclear exportin-5 trans-
porter [24,25]. Once in the cytoplasm, the pre-miRNA
is processed by another RNaseIII-type protein, Dicer,
which acts in concert with another double-stranded
RNA-binding protein (the HIV transactivating

response RNA-binding protein) and Argonaute pro-
teins to liberate the mature miRNA duplex (20–22
nucleotides) [26–29]. Processing by Dicer results in the
production of a small double-stranded miRNA duplex
containing two nucleotide-long 3¢ overhangs [30]. The
mature duplex miRNA is incorporated into an effector
complex referred to as the RNA-induced silencing
complex. On the basis of thermodynamic properties,
one strand is eliminated, whereas the other remains
integrated in the complex [31,32]. miRNAs mediate
their effect on gene expression by annealing to the
3¢-UTR of target genes. Functional miRNA-binding
sites in the coding region or 5¢-UTR of endogenous
mRNAs have not been clearly identified, because they
are less frequent and appear less effective than those in
the 3¢-UTR [7,8,33]. However, Lytle et al. [34] demon-
strated that introducing a target site for let-7a miRNA
into the 5¢-UTR of a luciferase reporter represses gene
expression by let-7a. In many cases, target recognition
by a miRNA only requires a continuous 6 bp ‘seed
match’ between the 5¢ end of the miRNA and its tar-
get. By binding to complementary sequences located at
the 3¢-UTR of target mRNAs and depending on par-
tial or complete sequence homology, miRNAs can
downregulate transcript levels in addition to suppress-
ing protein translation [35] (Fig. 1). It seems that
miRNAs might repress protein expression by multiple
means, although the exact mechanisms remain unclear.
miRNAs may interfere with translation at both the ini-
tiation and elongation stages, or translation may be

unaffected, with nascent polypeptides being degraded.
Alternatively, target mRNAs may be repressed transla-
tionally, because they are sequestered physically from
ribosomes and accumulate in P-bodies [36–38].
P-bodies are cytoplasmic subcompartments involved in
mRNA metabolism, degradation and translation
Function of miR-375 and 124a in pancreas and brain N. N. Baroukh and E. Van Obberghen
6510 FEBS Journal 276 (2009) 6509–6521 Journal compilation ª 2009 FEBS. No claim to original French government works
control. These trafficking components are an essential
feature of the pathway [39]. Initially, miRNAs were
only thought to suppress gene expression, but recently
it has been shown that they can also have the opposite
effect of inducing gene expression by activating tran-
scription [40,41] or upregulating translation [42,43].
Given the known modes of action of miRNAs, the
temporal and spatial expression profiles of miRNAs
and their specificity for protein targets, miRNAs have
opened up research on their potential role in the devel-
opment and maintenance of cell phenotypes.
Specific genomic features for miR-375
and miR-124a
Several hundred miRNAs have been identified
and sequenced in mammalian species, with  700 in
human, 500 in mouse and macaque and 300 in rat
(from Rfam database, [9]). Generally, most miRNA
genes are located far away from any annotated gene,
implying independent transcription with their own pro-
moters. However, some miRNAs lie within predicted
introns of genes encoding proteins. In  80% of these
cases, the introns have the same orientation as the

miRNAs, indicating that the protein-coding genes serve
as host genes for coexpressed miRNAs. Some miRNAs
are located in close genomic proximity to each other
and others are transcribed as polycistronic units [21].
To date, little is known about the transcriptional regu-
lation of miRNA genes and studies have mostly con-
centrated on miRNAs located within the intergenic
region of the genome. However, a sequence motif
GANNNNGA has been found to display a conserved
distribution in nematodes. It was observed to be most
RISC/target silencing
pri-miRNA
Microprocessor
Drosha
Pasha-
DGCR8
Ran+GTP
Exportin
5
pre-miRNA
Pol II
miRNA gene
AAAAA-3’
Cytoplasm
Nucleus
Dicer
Dicer
miRNA
duplex
AAAAA

AAAAA
mRNA degradation
mRNA target
mRNA target
AAAAA
AAAAA
miRNA
ORF
RISC
Translational repression
miRNA
ORF
STOP
RISC
AAAAA
AAAAA
mRNA target
mRNA target
RISC
Partial homology
High homology
mRNA binding
miRNA
degradation
P-bodies
5’
Fig. 1. Overview of the miRNA biogenesis
pathway. miRNAs are generated as primary
transcripts termed pri-miRNA. After two
ribonuclease cleavage steps, the mature

miRNA of  22 nucleotides is produced.
Mature miRNA is incorporated into the RNA
interference (RNAi) effector complex RISC
(RNA-induced silencing complex), which
drives mature miRNA to homologous
mRNAs for direct translational suppression
and mRNA degradation. For simplicity, not
all cellular factors involved in miRNA
processing are shown.
N. N. Baroukh and E. Van Obberghen Function of miR-375 and 124a in pancreas and brain
FEBS Journal 276 (2009) 6509–6521 Journal compilation ª 2009 FEBS. No claim to original French government works 6511
abundant in the upstream sequences of two important
miRNAs, miR-1 and miR-124 [44].
The miR-375 gene is found on chromosome 2 in
humans and chromosome 1 in mice (Table 1). miR-375
is located in an intergenic region between the cryba2
(b-A2 crystallin, an eye lens component) and Ccdc108
(coiled-coil domain-containing protein 108) genes;
a genomic region conserving the synteny between
humans and mice (see Ensembl, which provides gen-
ome sequences for vertebrates). Moreover, the
sequences of pre-miR-375 in both species present a
100% homology (Fig. 2A), highlighting the high
degree of conservation for this specific miRNA.
Recently, a study revealed that pancreas ⁄ duodenum
homeobox protein 1 (Pdx-1) and neurogenic differenti-
ation factor 1, two critical components of pancreatic
endocrine cell functions, control gene expression of
miR-375 in a combinatorial manner [45]. Two regula-
tory modules have been described in the vicinity of

miR-375; the first is located 500 bp upstream of the
miRNA 5¢ end and the second 1700 bp downstream.
The first domain may correspond to the proximal pro-
moter, whereas the second domain may correspond to
a distal enhancer [45]. Taken together, these sequence
features indicate that the miR-375 gene is transcribed
from its own promoter.
miR-124 was first identified by cloning studies in
mice [6]. There are three precursor hairpin sequences;
miR-124a1 on chromosome 14, miR-124a2 on chromo-
some 3 and miR-124a3 on chromosome 2 (Table 1).
Each miR-124a locus is associated with either
expressed sequence tags or annotated mRNAs.
However, these mRNAs do not code for any known
proteins, suggesting that they may be part of the pri-
miRNA transcript. All three miR-124a genes have
closely related predicted human homologues (Fig. 2B).
Lagos-Quintana et al. [6] also reported a mature
miRNA sequence, miR-124b, with a G insertion at
position 12. However, miR-124b has not been found in
either the mouse or human genome. miR-124a expres-
sion is negatively regulated by the transcriptional
repressor, response element silencing transcription fac-
tor (REST), in non-neuronal cells and neural progeni-
tors. Indeed, REST functions as a negative regulator
of miR-124a via response element (RE1) sites in three
miR-124a genomic loci [46]. Additionally, comparative
sequence analysis indicates the presence of evolution-
ary conserved cAMP response elements recognized by
cAMP response element-binding protein, a basic leu-

cine zipper transcription factor, within the proximal
regulatory region of miR-124a, implicating the role of
cAMP response element-binding protein in the positive
regulation of this miRNA [47]. Despite the importance
of characterizing functional DNA activity, few specific
transcription elements have been described as regulat-
ing miRNA gene expression. However, the increasing
amount of sequence information from multiple organ-
isms has enabled biologists to use sequence compari-
sons in gene regulation studies [48–50]. The rationale
for using interspecies sequence comparisons in identify-
ing noncoding regulatory elements is based on the
observation that sequences that perform fundamental
functions are frequently conserved between species.
Thus, one possible alternative is to use these available
tools for multiple sequence alignments among species
to identify conserved regulatory elements regulating
miRNA genes. Using software for sequence compari-
sons (i.e. evolutionary conserved region browser)
[51], we examined the sequence homology among ani-
mal species to search for conserved regions near the
miR-124a2 gene that may affect its gene regulation.
Our preliminary interspecies analysis of the miR-124a2
gene revealed the presence of a 177 bp sequence
with  75% identity between human and zebrafish,
 1.8 kbp upstream of miR-124a2 (Fig. 3). On the
basis of its high level of sequence conservation (and
lacking the characteristics of coding regions), one may
propose that this element plays a role in regulating the
expression of the miR-124a2 gene. It is crucial to verify

this prediction by characterizing this element through
in vitro studies and to explore its effect on miR-124a
expression.
Tissue expression of miR-375 and
miR-124a
The miR-375 sequence was first cloned from a mouse
insulinoma pancreatic b-cell line (MIN6 cells) and iden-
tified as the most abundant, evolutionarily conserved,
islet-specific miRNA [52]. miR-375 is expressed in islet
b-cells as well as in non-b-cells of the pancreas [53,54].
Table 1. Identification and chromosome (chr) localization of
human ⁄ mouse miR-375 and miR-124a (adapted from Rfam miR
registry at ). hsa, Homo sapiens
(human); mmu, Mus musculus (mouse).
miR-ID Accession Chr Start End
hsa-miR-375 MI0000783 2 219574611 219574674
mmu-miR-375 MI0000792 1 74947232 74947295
hsa-miR-124a1 MI0000443 8 9798308 9798392
hsa-miR-124a2 MI0000444 8 65454260 65454368
hsa-miR-124a3 MI0000445 20 61280297 61280383
mmu-miR-124a1 MI0000716 14 65209494 65209578
mmu-miR-124a2 MI0000717 3 17695662 176957770
mmu-miR-124a3 MI0000150 2 180828745 180828812
Function of miR-375 and 124a in pancreas and brain N. N. Baroukh and E. Van Obberghen
6512 FEBS Journal 276 (2009) 6509–6521 Journal compilation ª 2009 FEBS. No claim to original French government works
Other identified islet-specific miRNAs are miR-7, miR-9
and miR-376 [54–56]. Overall, data show that miRNAs
are necessary for islet cell genesis in mice [57]. Inhibition
of miR-375 in zebrafish has a profound deleterious
effect on pancreatic development, particularly in endo-

crine cells [58]. miR-375 was first thought to be
restricted to pancreatic cells, but evidence shows that it
is also expressed within the brain, exclusively in the pitu-
itary and at a lower level in hypothalamic cells [59].
Several miRNAs identified during the mouse pancreatic
b-cell line MIN6 cloning were also identified in the
brain, indicating an overlap in function of these particu-
lar miRNA sequences [52]. Furthermore, the pituitary
gland and pancreatic cells share similarities in terms of
specialized biological functions, such as exocytosis, the
final step in the secretory pathway. At this point, it is
tempting to speculate that miR-375 has a common
function in both tissues and may regulate exocytosis
through similar target genes.
miR-124a is preferentially expressed in the brain (the
most abundant miRNA in embryonic and adult central
nervous systems) and the retina. The brain is an organ
with complex cell type composition, among which
neurons and glial cells are predominant. miRNA
expression analysis in human, mouse and rat brain
demonstrates that miR-124, miR-9, miR-128a and
miR-128b are highly and specifically expressed in all
brain regions, except for the pituitary gland, which
shows abundant expression of miR-7, miR-375 and
clusters of miR-141 and miR-200a [54,60,61]. During
neurogenesis, miR-124a is present at very low levels in
neural progenitors, but is highly expressed in differen-
tiating and mature neurons [62]. Because of its absence
from proliferative cells and its wide expression in
differentiated neurons, miR-124a is not assumed to be

associated with a transition in the differentiation
states. In addition, this expression pattern is highly
specific and consistent with the hypothesis that
miR-124a targets genes expressed at differentiation
phases [59]. Furthermore, miR-124a overexpression in
cultured HeLa cells leads to a decrease in transcript
levels of a brain-specific set of genes, and shifts HeLa
gene expression towards that of cerebral cortex-like
gene expression [7]. Initially described as a brain-spe-
cific miRNA in mammals, miR-124a, like miR-375, is
also well represented in the mouse pancreatic MIN6
b-cell line [52]. Further data from our laboratory have
recently demonstrated that the miR-124a expression
level is increased in mouse pancreas at embryonic (e)
stage e18.5 compared with stage e14.5, indicating a
A
B
Fig. 2. Human (hsa) and mouse (mmu) miR-375 (A) and miR-124a isoform (B) CLUSTALW stem loop precursor sequence alignments. Mature
miRNA sequences are underlined. Asterisks indicate conserved nucleotides.
N. N. Baroukh and E. Van Obberghen Function of miR-375 and 124a in pancreas and brain
FEBS Journal 276 (2009) 6509–6521 Journal compilation ª 2009 FEBS. No claim to original French government works 6513
Fig. 3. An adapted representation showing the human miR-124a2 genomic region (human localization; chromosome 8: 65450636–
65457988) compared with fugu (fr2), zebrafish (danRer5), chicken (galGal3), opossum (monDom4), mouse (mm9), rat (rn4) and rhesus maca-
que (rheMac2) orthologous sequences. Using the
EVOLUTIONARY CONSERVED REGION BROWSER the 5¢–3¢ region adjacent to the human miR-124a2
gene was compared with their orthologous interval sequences in vertebrate species. Human and rat or mouse sequence comparisons
showed a similar genomic structure within this region (high degree of conservation). To identify ECRs (red) with a greater likelihood of con-
taining potential biological activity, we determined which conserved sequences were also present in distant vertebrates, including opossum,
chicken, zebrafish and fugu. The multiple alignments revealed the presence of a conserved sequence (177 bp in length, indicated by an
arrow), with 75.1% identity between human and zebrafish (Danio rerio). Sequence conservation between human (chromosome 8:

65452286–65452462) and zebrafish (chromosome 24: 23035090–23035260) is shown in sequence alignment.
Function of miR-375 and 124a in pancreas and brain N. N. Baroukh and E. Van Obberghen
6514 FEBS Journal 276 (2009) 6509–6521 Journal compilation ª 2009 FEBS. No claim to original French government works
role in development [63]. miR-124a expression in both
tissues (pancreas and brain) may play a role in the
acquisition and maintenance of tissue identity, which is
assumed to be a general function of miRNA in devel-
opment [5]. Organ development is a highly orches-
trated process that entails precise control of gene
expression (coding or noncoding genes). Interestingly,
all tissues maintain a unique miRNA expression
profile, indicating their contribution to regulating a
unique set of target genes that is specific for an organ’s
development and function.
Functional studies implicating miR-375
and miR-124a
The biological functions of most miRNAs need to be
defined and one challenge is to experimentally identify
and validate their mRNA targets. Some miRNAs,
including miR-375 and miR-124a, have been character-
ized for their functional effects.
Focusing on miR-375, Poy et al. [52] elucidated the
role of this pancreatic islet-specific miRNA in cell
lines. Overexpression of miR-375 in pancreatic cells
impaired glucose-stimulated secretion of insulin with
no alteration in glucose-mediated production of ATP
or rise in intracellular calcium. In addition, a loss of
function of miR-375 revealed an increase in glucose-
stimulated insulin secretion. These results show that
miR-375 is implicated in the regulation of insulin

secretion, which is a key determinant of blood glucose
homeostasis. The authors demonstrated that myotro-
phin, a gene described originally in neuronal vesicle
transport, is a direct target of miR-375. An interaction
between miR-375 and the 3¢-UTR of myotrophin
mRNA was shown to repress myotrophin translation
and result in the inhibition of insulin secretion. In
addition to its role in exocytosis control, myotrophin
is also known as a transcription factor, regulating
nuclear factor-kappa B in cardiomyocytes [64]. Nuclear
factor-kappa B activity was shown to improve
cytoskeleton organization and regulate glucose-induced
insulin secretion [65,66]. These findings represent
another interesting aspect of the action of myotrophin
in cells and may explain the mechanism by which
miR-375 also mediates insulin exocytosis. Of course,
more work needs to be carried out to confirm this
hypothesis. miR-375 target gene regulation is not
limited to its action on mytrophin, as described by El
Ouaamari et al. [67], who demonstrated that miR-375
negatively regulates 3¢-phosphoinositide-dependent
protein kinase-1 (PDK-1) [67]. PDK-1 is a key mole-
cule in the phosphatidylinositol-3-kinase cascade
stimulated by insulin and it is known to activate, by
phosphorylation, a series of substrates involved in cell
physiology [68]. Consequently, in response to insulin,
miR-375 regulates phosphorylation states of proteins
functioning downstream of PDK-1, such as protein
kinase B and glycogen synthase kinase. Moreover, our
group has shown that miR-375, through its action on

phosphatidylinositol-3-kinase ⁄ PDK-1 ⁄ protein kinase B
signalling reduces the glucose stimulatory effect on
insulin gene expression and attenuates the viability and
the proliferation of pancreatic b-cells [67]. Similar to
our observations, others have demonstrated a down-
regulation of miR-375 in pancreatic cancer, pointing
to an antiproliferative effect of miR-375 [69–71].
Recently, mice lacking miR-375 (375KO) were gener-
ated. Using these mice, Poy et al. [53] demonstrated
that miR-375 is required for normal glucose homeo-
stasis and influences pancreatic a- and b-cell mass by
regulating a cluster of genes controlling cellular growth
and proliferation. Taken together, these data demon-
strate multiple implications of miR-375 on various cell
functions. This is in agreement with the concept that
one miRNA may target many transcripts, which may
confer just as many cell functions [72].
Another example is miR-124a, which was shown
to knockdown transcript levels for over 174 genes in
HeLa cells, and its introduction in cells promotes a
neuronal-like transcript profile [7]. Blocking miR-124a
activity in mature neurons selectively increases levels
of some non-neuronal transcripts. Thus, it has been
proposed that miR-124a suppresses non-neural genes
in mammalian neurons and contributes to the acquisi-
tion and maintenance of neuronal identity [46]. Specifi-
cally, one miR-124a target is the mRNA of the
antineural function protein small C-terminal domain
phosphatase 1 (SCP1), a protein expressed in non-neu-
ral tissues during central nervous system development

and whose downregulation induces neurogenesis [73].
Interestingly, SCP1 was found among the 174 down-
regulated genes by miR-124a in HeLa cells [7] and
among upregulated genes in miR-124a-depleted corti-
cal neurons [46]. Computational approaches also
uncovered miR-124-binding sites in the 3¢-UTRs of
MeCP2 and CoREST, encoding two components of
the REST complex [47]. Together, these data indicate
that neurogenesis requires the functions of the
REST ⁄ SCP1 system as well as the post-transcriptional
downregulation of non-neuronal transcripts by
miR-124a (also under REST control) [46]. REST and
miRNA are repressor components that participate in a
double-negative feedback loop resulting in the stabil-
ization and maintenance of neuronal gene expression
[46,47]. More recently, Cheng et al. [74] found that
miR-124 is an important regulator of the temporal
N. N. Baroukh and E. Van Obberghen Function of miR-375 and 124a in pancreas and brain
FEBS Journal 276 (2009) 6509–6521 Journal compilation ª 2009 FEBS. No claim to original French government works 6515
progression of neurogenesis in the subventricular zone
in brains of adult mice. Consistent with another study
[73], their observations provide evidence that miR-124
promotes neuronal differentiation and cell cycle exit in
the subventricular zone stem cell lineage by targeting
the mRNA of Sox9, whose extinction abolishes the
production of neurons in this system [74]. In addition,
miR-124a plays an important role in the differentiation
of progenitor cells to mature neurons by directly
regulating polypyrimidine tract-binding protein 1, which
is involved in alternative pre-mRNA splicing in non-

neural cells [75]. For this miRNA the scenario may be
even more complex, as investigations carried out on
chick neural tubes have identified two other endogenous
targets of miR-124a, laminin c1 and integrin b1, both
highly expressed by neural progenitors, but repressed
upon neural differentiation [76]. The observation that
miR-124a is expressed by mature neurons throughout
the brain strongly suggests that miR-124a has, in
addition to its described role in neurogenesis, other
physiological functions in mature neurons.
In the retina, miR-124a regulates the retinol dehydro-
genase 10 gene, which is known to be relevant to retinal
disease [77]. Several predicted targets of miR-124a are
genes involved in organ development and may act in a
similar manner during retinal development. One may
hypothesize that miR-124a or mutations affecting its
expression would probably be detrimental for the brain
and the retina and contribute to organ abnormalities.
miR-124a, abundantly expressed in the pancreas,
also represses the myotrophin gene, demonstrating,
together with miR-375, a converging translational con-
trol of a single protein. In fact, multiple targeting of a
transcript may ensure sequential miRNA actions and
fine tuning of gene expression [72,78]. Recently, we
identified the Forkhead box a2 (Foxa2) gene product as
a direct miR-124a target. Our work revealed that
increasing the level of miR-124a reduced the level of
the Foxa2 protein. This subsequently decreased the
level of Foxa2 downstream target genes, including
Pdx-1, inward rectifier potassium channel member 6.2

(Kir6.2) and sulfonylurea receptor 1 (Sur1). These
changes were associated with an increase in basal free
calcium, but did not change glucose- or potassium-
stimulated hormone secretion [63]. Another group
showed that miR-124a modulates the expression of
proteins involved in the insulin exocytosis machinery
[miR-124a increases the levels of synaptosomal-
associated protein 25 (SNAP25), Ras-related protein
Rab-3A (Rab3A) and synapsin-1A and decreases those
of Rab27A and nuclear complex protein 2 homolog
(Noc2)], affecting b-cell secretion [79]. These results
demonstrate once again that changes in expression of a
single miRNA can have an impact on the expression of
many genes by direct and ⁄ or indirect mechanisms and
can lead to alterations in cell functions [63,79]. Similar
to miR-375, miR-124a is a key regulator of a transcrip-
tional protein network in b-cells. Changes in miR-124a
levels may complement the previously described actions
of miR-375 by modulating the apparent sensitivity of
the exocytotic machinery. miR-124a and miR-375, and
other pancreas-specific miRNAs, seem to downregulate
a greater number of targets than previously
appreciated, thereby helping to define pancreas-specific
functions. Assigning a function to a miRNA might
only reveal the tip of the iceberg, as miR-124a
overexpression in the HepG2 cell line led to a signifi-
cant downregulation of many genes in categories
related to cell cycle ⁄ proliferation, indicating that
miR-124a is also involved in cell growth control [80].
An increasing number of functions is associated with

miR-124a and one of the most recently identified dem-
onstrates its involvement in glucocorticoid responsive-
ness in the brain [81]. The functional roles of miR-375
and miR-124a in the pancreas and the brain are
summarized in Fig. 4.
Concluding remarks
miRNAs are a fascinating new class of molecules that
are powerful regulators of gene expression and control
many biological processes. Although our knowledge of
these tiny molecules is growing each day, their particu-
lar characteristics (size, temporal and tissue-specific
expression, mode of action) pose a real challenge to
studying and elucidating miRNAs functions. On the
one hand, hundreds of genes are predicted to be regu-
lated by a single miRNA. On the other hand, the bind-
ing of multiple miRNAs to one target gene increases
the complexity of predictions [72,82]. However, scien-
tists have widely used computational target predictions
to orient lines of investigations and experimental data
tend to validate such orientation.
miR-375 and miR-124a share similar features; they
are both specifically expressed in the pancreas and the
brain, albeit at different levels. miR-375 is more abun-
dant in islets and miR-124a is more represented in the
brain. This tissue-specific coexpression suggests an
overlap of function (redundancy effect or co-ordinate
action). miR-375 inhibition has a dramatic effect on
pancreas development [58], whereas miR-124a is upreg-
ulated during pancreas development [63] and neuro-
genesis [46]. Together, these findings highlight the

involvement of miR-375 and miR-124a in development
and their role in the establishment of organ identity.
In addition, several studies have demonstrated that
Function of miR-375 and 124a in pancreas and brain N. N. Baroukh and E. Van Obberghen
6516 FEBS Journal 276 (2009) 6509–6521 Journal compilation ª 2009 FEBS. No claim to original French government works
pancreatic b-cells display patterns of gene expression
overlapping with those of neuronal cells [83,84]. More-
over, it has been shown that miR-375 and miR-124a
directly bind a common target, the myotrophin gene
transcript, which encodes a cytoplasmic protein that
induces exocytosis and hormone secretion [52,72]. The
regulation of myotrophin protein by multiple miRNAs
provides evidence of a co-ordinated regulation. Both
miRNAs show an important role in endocrine function
and highlight the consequences of their dysregulation
on hormone release.
Another interesting observation of the action of
miRNAs is that miRNA tissue-specific expression is
regulated by tissue-specific transcription factors.
The islet-specific miR-375 is controlled by multiple
transcription factors, such as Pdx-1 and neurogenic
differentiation factor 1, both critical for b-cell devel-
opment. On the basis of this observation, it is tempt-
ing to speculate that miR-375 is involved in
b-cell development and that it is temporally con-
trolled during embryogenesis by these two transcrip-
tion factors. In a similar manner, the brain-specific
miR-124a is under the control of REST factor, a neu-
ronal repressor and a regulator of glucose-induced
insulin secretion [85], suggesting that a balance

between endocrine- and neuron-specific components
needs to be reached to exhibit adequate secretory cell
functions. Furthermore, like other genes, miRNAs
are regulated by effectors at a transcriptional level.
miR-375 gene expression is negatively regulated by
glucose in INS-1E cells and freshly isolated pancreatic
islets of Goto-Kakizaki diabetic rats (model of type 2
diabetes); whereas miR-124a expression is increased in
freshly isolated diabetic Goto-Kakizaki islets [67]. It
is interesting to note that miR-375 and miR-124a regu-
late insulin gene expression in pancreatic b-cell lines
[63,67], probably affecting a final retro-control loop
of regulation. miR-375 and miR-124a are expressed in
the same tissues, target a common protein, both show
glucose sensitivity; yet, they are regulated differen-
tially. They are both involved in pancreatic b-cell
development and in the regulation of insulin produc-
tion and secretion. It seems that miRNA acts at mul-
tiple hierarchical levels of gene regulatory networks
affecting cell functions, and that they are themselves
regulated by environmental and ⁄ or genetic factors.
This multilevel regulation may allow individual
miRNAs to affect the gene expression programme of
cells profoundly. It is clear that miRNA is involved
in organ development, but also in the whole process
of an organism’s development. Growing evidence
demonstrates the vast roles played by miRNAs in
biological systems and how the alterations of their
expression participate in the pathogenesis of human
diseases. In the pancreas, b-cells are highly specialized

and characterized by the exclusive ability to synthe-
size and release insulin according to fluctuations in
circulating glucose levels. The important roles of
miR-375, together with miR-124a, in regulating glu-
cose-stimulated insulin production and secretion, and
cell growth ⁄ proliferation, highlight miRNAs as targets
for developing novel strategies to correct defective
insulin secretion in some forms of type 2 diabetes.
The identification of a role for miRNA molecules in
controlling b-cell gene expression and ⁄ or b-cell func-
tions may lead to the identification of novel pharma-
Fig. 4. Schematic representation of the
functional and common implications of
miR-375 and miR-124a in pancreas and
brain.
N. N. Baroukh and E. Van Obberghen Function of miR-375 and 124a in pancreas and brain
FEBS Journal 276 (2009) 6509–6521 Journal compilation ª 2009 FEBS. No claim to original French government works 6517
cological targets for the treatment of b-cell failure
observed in diabetes.
Given the increasing number of miRNA sequences
identified, it is interesting to investigate their implication
and functional roles in metabolic disorders in vivo.A
more precise picture should be given with the generation
of genetically engineered animal models. Disrupting or
overexpressing an miRNA gene will allow roles in
mammalian physiology to be assigned to each sequence
[53,86–88]. Moreover, an interesting report has under-
lined the possible unintentional deletion of miRNA
during conventional gene disruption in mouse models
[89]. The authors found approximately 200 cases in

which miRNAs may have been disturbed in mouse gene
targeting models. These observations should be used to
re-examine gene knockout interpretation and to
investigate whether an miRNA may contribute to or be
responsible for the phenotype observed in vivo.
Acknowledgements
The authors would like to acknowledge J. Neels,
I. Mothe-Satney and P. Grimaldi for their critical
reading of the manuscript, suggestions and advice.
There is no conflict of interest.
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