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

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-

Function of microRNA-375 and microRNA-124a in

pancreas and brain

Nadine N Baroukh1and Emmanuel Van Obberghen1,2

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, Hoˆpital 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: nadine.baroukh@unice.fr

(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-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.

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 miRpri-miR-NA (pre-miRpri-miR-NA), 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

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

Microproce

ssor

Drosha

Pasha-DGCR8

Ran+GTP

Exportin 5

pre-miRNA

Pol II miRNA gene

AAAAA-3’

Cytoplasm Nucleus

Dicer

Dicer

miRNA duplex

AAAAA mRNA degradation

mRNA target AAAAA

miRNA

Translational repression

miRNA ORF

AAAAA 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.

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-124a1on chromosome 14, miR-124a2 on

chromo-some 3 and miR-124a3 on chromochromo-some 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 increasregulat-ing 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 noncodidentify-ing 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 http://microrna.sanger.ac.uk) hsa, Homo sapiens

(human); mmu, Mus musculus (mouse).

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-124ais 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.

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 semaca-quences 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.

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 develdevel-opment 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 centnon-neu-ral 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

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 10gene, 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

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 secreproduc-tion 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.

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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