MINIREVIEW
Minireview: MicroRNA Function in Pancreatic ␤ Cells
Sabire Özcan
Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky,
Lexington, Kentucky 40536
MicroRNAs are small noncoding ribonucleotides that regulate mRNA translation or degradation
and have major roles in cellular function. MicroRNA (miRNA) levels are deregulated or altered in
many diseases. There is overwhelming evidence that miRNAs also play an important role in the
regulation of glucose homeostasis and thereby may contribute to the establishment of diabetes.
MiRNAs have been shown to affect insulin levels by regulating insulin production, insulin exocytosis, and endocrine pancreas development. Although a large number of miRNAs have been
identified from pancreatic ␤-cells using various screens, functional studies that link most of the
identified miRNAs to regulation of pancreatic ␤-cell function are lacking. This review focuses on
miRNAs with important roles in regulation of insulin production, insulin secretion, and ␤-cell
development, and will discuss only miRNAs with established roles in ␤-cell function. (Molecular
Endocrinology 28: 1922–1933, 2014)
ature microRNAs (miRNAs) are small noncoding
ribonucleotides (⬃22 nt) capable of recognizing
and binding to partially complementary sequences within
the 3⬘ untranslated region (UTR) of specific mRNAs.
Most miRNAs function by mediating the degradation or
translational inhibition of mRNAs (1, 2). In rare cases,
miRNAs also can affect translation and gene expression
in a positive manner (3–5). They pair with the target
mRNA through their seed region at the 5⬘ end (6). MiRNAs are transcribed as pri-miRNAs and processed to premiRNAs by the enzyme complex Drosha and DGCR8
within the nucleus (6). After transport into the cytoplasm
pre-miRNAs are processed to mature miRNAs by the
Dicer complex, and the obtained double-stranded RNA
associates with the RNA-induced silencing complex that
mediates the interaction of miRNAs with target mRNAs.
Most of the initial studies on miRNA function used
deletions of Dicer (7), Drosha (8), DGCR8 (9), and Ago2
(10) genes. Although the homozygous deletion of Dicer is
embryonic lethal in mice (11) and zebrafish (12), tissuespecific deletions of Dicer have been used to study the role
of miRNAs in various cell types. The human genome contains more than 2500 mature miRNA sequences, which
constitute greater than 5% of all genes. Many miRNAs
M
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received September 19, 2014. Accepted November 11, 2014.
First Published Online November 14, 2014
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exist in miRNA families with identical seed sequences (6).
It is predicted that each miRNA family regulates more
than 300 different target mRNAs and close to 50% of
target mRNAs have binding sites for two or more
miRNAs (6, 13, 14). It is estimated that miRNAs regulate
greater than 75% of mRNAs in a cell (15). Thus, miRNAs
play a critical role in the regulation of entire protein networks and signaling pathways. They are involved in development, neuronal cell fate, apoptosis, and cell proliferation. The abundance of many miRNAs is altered in
various diseases including cancer, diabetes, neurological
disorders, autoimmune and cardiovascular diseases. Although miRNAs play important roles in diverse aspects of
signaling and metabolic control, the exact function and
targets of most of the identified miRNAs remain
unknown.
Maintaining normoglycemia requires the production
and secretion of insulin, which then acts on insulin-sensitive tissues, including muscle, liver, and adipocytes. The
first miRNA involved in glucose homeostasis was identiAbbreviations: Abca1, ATP-binding cassette transporter 1; Ago2, argonaute 2; E, embryonic day; GSIS, glucose-stimulated insulin secretion; FoxA2, Forkhead A2; Hnf1, hepatocyte nuclear factor-1; hPSC, human pluripotent stem cell; MCT1, monocarboxylate transporter-1; MEN1, multiple endocrine neoplasia-1; miR, microRNA; miRNA, microRNA;
MODY, maturity-onset diabetes of the young; mTOR, mammalian target of rapamycin;
Mtpn, myotrophin; NeuroD, neurogenic differentiation; Ngn3, neurogenin-3; NOD,
nonobese mouse model of autoimmune diabetes; Pax6, paired box transcription factor 6;
Pdcd4, programmed cell death 4; PDK-1, protein kinase-1; Rab, renin-angiotensin system
oncogene family; RIP, rat insulin promoter; Sirt1, sirtuin-1; STZ, streptozotocin; UTR,
untranlsated region; VAMP2, vesicle-associated membrane protein-2; ZEB, zinc finger
E-box binding homeobox.
Mol Endocrinol, December 2014, 28(12):1922–1933
doi: 10.1210/me.2014-1306
doi: 10.1210/me.2014-1306
fied in pancreatic islets as micro-RNA (miR)-375 (16).
Since then, many miRNAs have been identified with important functions in pancreatic ␤-cells (17, 18). This article focuses on miRNAs critical for ␤-cell function and
reviews the current state of knowledge about miRNAs
that regulate insulin gene expression, insulin secretion,
and endocrine pancreas development. Therefore, this review discusses only a selected number of miRNAs that are
abundant in pancreatic ␤ cells and have established roles
in modulation of ␤-cell function (Table 1).
Dicer1
Analysis of mice with ␤-cell specific deletion of Dicer1
provided the first evidence that miRNAs are important
for pancreatic ␤-cell function (11, 19). Dicer1 was initially deleted in the developing endocrine pancreas
around the embryonic day (E) 10.5 using the Pdx-1-Cre
mice (20). These Dicer1-deficient mice were defective in
all pancreatic lineages and displayed a significant loss of
pancreatic ␤-cells and died shortly after birth by postnatal
day 3. They had an abnormal islet structure and displayed
a significant reduction in the number of neurogenin-3
(Ngn3)-expressing endocrine progenitors (19). This was
Table 1.
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the first evidence demonstrating that miRNAs are critical
for endocrine pancreas development.
Although Dicer1 whole-body knockout mice are embryonic lethal (11), Dicer1-hypomorphic mice with 20%
of Dicer1 expression in all tissues were found to be viable
(21). Analysis of these Dicer1-hypomorphic mice revealed that all of the tissues of 8- to 10-week-old mice
were histologically normal except for the pancreas. Although the development of the pancreas at the fetal and
neonatal stages was normal, at 4 weeks of age, pancreas
morphology was abnormal with the presence of small
islets and cells that were double positive for insulin and
glucagon (21). Despite this abnormal islet morphology,
the mice displayed normal glucose tolerance and had normal insulin levels. It is not clear whether these mice developed some compensatory mechanisms or whether a
low expression of Dicer1 is sufficient for normal
development.
A different study found that mice with ablation of Dicer1 in adult endocrine pancreas using an inducible
CreER transgene driven by the rat insulin promoter (RIP)
(22) developed hyperglycemia and diabetes as adults (23).
This was due to reduced insulin consent and insulin
List of miRs Important for Pancreatic ␤-Cell Function
miRNA
miR-7
Function
Endocrine pancreas development,
Adult ␤-cell replication
Insulin secretion
Targets
GATA6, Pax6
S6k1, eIF4E, Mknk1, Mknk2, Mapkap1
Snca, Cplx1, Pfne2, Wipf2, Basp1
References
31–35, 40
36
40
miR-9
Insulin secretion
Onecut-2, Sirt1
41, 45
miR-15a/b
Endocrine pancreas regeneration
Ngn3
48
miR-21
Insulin secretion
␤-Cell apoptosis
Pdcd4
51
56
miR-24
␤-Cell proliferation
Insulin biosynthesis
␤-Cell lipotoxicity
Men1
Sox6
NeuroD, HNFa
63
23
61
miR-29
Insulin secretion
␤-Cell apoptosis
Mct1, Onecut2
Mcl1
63, 66
66
miR-30
␤-Cell differentiation
Insulin secretion
Insulin biosynthesis
Vimentin, Snail1, Rfx6
NeuroD
Map4k4
32, 76
77
75
miR-33a/miR-145
Insulin secretion
Abca1
78,79
miR-124a
Endocrine pancreas development
Insulin secretion
Islet amyloid polypeptide synthesis
FoxA2, Ngn3
Rap27
FoxA2
48, 81
83
84
miR-184
Compensatory ␤-cell expansion
Ago2
29
miR-200
␤-Cell specification
c-Maf, Fog2, Zeb1, Zeb2, Sox17
76, 85
miR-375
Pancreas development/compensatory ␤-cell expansion
Insulin biosynthesis
Insulin secretion
Cadm1
Pdk-1
Mtpn, HuD, Gephyrin, Ywhaz, Aifm1
88, 89
44
16, 30, 89
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␤-Cell MicroRNAs
mRNA in Dicer1-deficient ␤-cells. However, ␤-cell mass
and islet size were normal, whereas insulin1 and insulin2
mRNA levels were decreased by 70% (23). The reduction
in insulin gene expression was associated with an increased expression of the transcriptional repressors
Bhlhe22 and Sox6 (23).
The importance of miRNAs for normal ␤-cell function
was further confirmed by two other studies that used
RIP-Cre to delete Dicer1 (24, 25). The first study found
that 80% of the Dicer1-deficient animals developed diabetes by 12 weeks of age. The development of the endocrine pancreas in these animals was normal; however,
they were defective in insulin gene expression and glucose-stimulated insulin secretion (GSIS) (24). Immunostaining of the pancreas revealed that these mice had altered islet morphology and decreased ␤-cell mass.
Furthermore, the number of insulin granules, including
the docked granules, were decreased (24). The second
study also found that RIP-Cre Dicer1 animals have reduced ␤-cell mass, decreased insulin biosynthesis, and impaired glucose tolerance (25). Thus, both studies confirmed that miRNAs are important for ␤-cell survival and
for insulin biosynthesis in adult endocrine pancreas.
In a recent study, Dicer1 was deleted in NGN3⫹ endocrine progenitor cells (26). Although pancreas development in these mice was normal, they developed diabetes
due to diminished insulin gene expression and ␤-cell
mass. Interestingly, Dicer1-deficient mice displayed increased levels of neuronal transcripts preceding the onset
of diabetes (26). The up-regulated neuronal genes were
targets of the neuronal transcriptional repressor, RE1silencing transcription factor REST. This suggests that
miRNAs play a critical role in maintaining islet cell identity by suppressing neuronal gene expression via a direct
targeting of the repressor, RE1-silencing transcription
factor (26). In summary, the ablation of Dicer1 at various
stages of endocrine pancreas development suggests that
miRNAs play critical roles in insulin biosynthesis, insulin
secretion, and ␤-cell development and survival.
Argonaute 2 (Ago2)
Ago2 is one of the four members of the argonaute
family involved in mediating the interaction of miRNAs
with their target mRNAs (27, 28). It also is the most
abundant one in many tissues, including pancreatic
␤-cells. Ago2 has been recently shown to play an important role in insulin secretion and ␤-cell compensatory expansion (29, 30). The first study on Ago2 function in
pancreatic ␤-cells found that miR-375 was the most enriched miRNA in Ago2-associated complexes in MIN6
cells. Using RNA interference experiments, it was shown
that silencing of Ago2 in MIN6 cells enhances insulin
Mol Endocrinol, December 2014, 28(12):1922–1933
secretion by increasing the expression of many of the
miR-375 target genes, including Myotrophin, Gephryn,
Ywhaz, and Aif1 (30). The same group later investigated
Ago2 function in more detail using Ago2 knockout mice
(29). Mice lacking Ago2 were unable to compensate for
insulin resistance by expanding ␤-cell mass. This defect in
compensatory ␤-cell expansion was due to the up-regulation of miR-375 target genes, including Cadm1, which is
a negative regulator of proliferation (29). Furthermore, it
was discovered that Ago2 levels are elevated during insulin resistance, which may be essential for the compensatory ␤-cell expansion observed in insulin-resistant state.
miR-7
Mir-7 is one of the evolutionarily conserved miRNAs
that is encoded by three different genomic loci in mice and
humans. MiR-7 is the most abundant and differentially
expressed miRNA in both rodent and human islets (31–
34). It also is highly expressed during human fetal pancreas development (31, 32). In the human developing
pancreas, miR-7 expression was first detected at approximately 9 weeks of gestational age, and its expression
peaked at approximately 14 and 18 weeks of gestational
age in endocrine cells with rising hormone levels (31).
Immunostaining data demonstrated that miR-7 colocalizes with insulin and glucagon in differentiating endocrine pancreas and is not expressed in acinar or ductal cells (31, 34).
Initial studies on miR-7 function have established a
critical role for this miRNA in the regulation of differentiation of ␣- and ␤-cells in early endocrine pancreas development (34). MiR-7 is expressed in endocrine precursors in which it targets the paired box transcription factor
6 (Pax6), which is important for endocrine pancreas differentiation and acts downstream of Ngn3 (35). Consistent with the idea that miR-7 is an endocrine-specific
miRNA, its expression is significantly reduced in Ngn3
knockout mice (34). Overexpression of miR-7 in the developing pancreas explants (E12.5 pancreatic buds) resulted in decreased insulin and Pax6 mRNA and protein
levels. The effect of miR-7 overexpression on endocrine
pancreas development was further studied using a
knock-in model, in which the miR-7a-1 genomic sequence was inserted into the Rosa26 locus. To induce the
expression of miR-7 in the pancreatic lineage, the Rosa26-miR-7 animals were crossed with Pdx-1-Cre mice.
Overexpression of miR-7 in E13.5 pancreatic cells resulted in a significant down-regulation of insulin and glucagon mRNA levels. Furthermore, the Pax6 levels were
decreased by 49%, but the expression of Cpa1 and Ptf1a,
which are markers of exocrine lineage were not changed
(34). Consistent with this, knockdown of miR-7 led to
doi: 10.1210/me.2014-1306
Figure 1. MiRNAs involved in the regulation of insulin biosynthesis
and insulin secretion in pancreatic ␤-cells. Insulin biosynthesis is
regulated by miR-24, miR-30, and miR-375, whereas insulin exocytosis
is modulated by miR-7, miR-29, miR-30, miR-33a, miR-124a, miR-145,
and miR-375.
increased Pax6 levels, which was associated with increased insulin and glucagon RNA and protein levels
(34). In conclusion, miR-7 functions as an inhibitor of ␣and ␤-cell differentiation during endocrine pancreas development by acting upstream of Pax6.
Recent data suggest that miR-7a is the major mature
isoform out of the two miR-7a and miR-7b isoforms, and
mir-7a-2 is the major precursor that is expressed in adult
pancreatic ␤-cells (36). Using mouse and human primary
islets, it was demonstrated that miR-7a negatively regulates adult ␤-cell proliferation (36). Inhibition of miR-7a
activated mammalian target of rapamycin (mTOR) signaling and promoted the replication of adult ␤-cells both
in mice and human primary islets (36). Activation of
mTOR signaling by inhibition of miR-7a in ␤-cells was
blocked by rapamycin, an inhibitor of mTOR. This confirmed that the effect of miR-7 on ␤-cell proliferation is by
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targeting mTOR signaling. The mTOR signaling pathway regulates cell proliferation and metabolism in response to various stimuli (37). Surprisingly, it was shown
that miR-7a targets five of the mTOR signaling pathway
components at the translational level, which include
S6k1, eIF4E, Mknk1, Mknk2, and Mapkap1 (36). Previous data suggest that the activation of mTOR signaling
using various strategies induces ␤-cell replication and expansion (38, 39). Thus, the modulation of miR-7a levels
could be used as a new strategy to promote ␤-cell replication to replace the lost ␤-cells in persons with type 2
diabetes. Taken together, these data suggest that miR-7
negatively regulates endocrine pancreas development by
targeting Pax6 (34) and adult ␤-cell replication by inhibiting mTOR signaling (36).
A very recent study in which miR-7a2 was either deleted or overexpressed in mice implicates miR-7a2 as a
negative regulator of GSIS (Figure 1) (40). MiR-7a2-deficient mice displayed increased insulin secretion and the
up-regulation of proteins that mediate the fusion of insulin granules with the plasma membrane in pancreatic
␤-cells. This included proteins involved in vesicle fusion (Snca, Cspa, and Cplx1), cytoskeleton dynamics
(Pfne2, Wipf2, and Phactr1), and membrane targeting
(Zdhhc9), which have been validated as direct targets
of miR-7 (40). Overexpression of miR-7a2 in mice led
to the inhibition of insulin secretion and ␤-cell dedifferentiation, which resulted in the development of diabetes. Interestingly, miR-7a levels were decreased in
human islets of obese and diabetic patients. A decrease
in miR-7a levels also was confirmed in islets of obese
and diabetic mice (40). Thus, these data suggest that
miR-7a is critical for GSIS and for ␤-cell adaptation to
obesity and diabetes (Figure 2).
miR-9
Analysis of miR-9 expression revealed that it is restricted to brain and pancreatic islets (41), and it is highly
expressed during human pancreatic
islet development (32). Detailed
studies on miR-9 function demonstrated that the overexpression of
miR-9 inhibits insulin secretion in
response to glucose or potassium
(KCl) in pancreatic INS-1E ␤-cells
(41). The impaired insulin secretion
in response to KCl by overexpression of miR-9 suggested that it regulates the insulin exocytosis machinFigure 2. MiRNAs important for endocrine pancreas development and ␤-cell differentiation and
ery. Further analysis revealed that
compensatory expansion. MiR-7, miR15a/b, miR124a, and miR-375 have been shown to regulate
overexpression of miR-9 causes a
endocrine pancreas development. MiRNAs important for ␤-cell differentiation or expansion are
14-fold increase in granuphilin
miR-7, miR-24, miR-30, miR-184, miR-200, and miR-375.
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␤-Cell MicroRNAs
mRNA levels, whereas the levels of other Rab effectors
were unchanged (41). Granuphilin is a Rab3/Rab27 GTPase effector that is associated with dense-core secretory
granules and negatively regulates insulin exocytosis by
controlling the docking of insulin granules (42).
Granuphilin-deficient mice display abnormally increased
insulin secretion (43). Using the TargetScan algorithm,
the transcription factor Onecut2 was identified as a potential miR-9 target with three binding sites at 3⬘ UTR.
Consistent with the idea that Onecut2 is a miR-9 target,
its expression was reduced by the overexpression of
miR-9. Furthermore, silencing of Onecut2 increased
granuphilin expression and diminished insulin secretion
by glucose (41). These data suggest that miR-9 negatively
regulates insulin secretion mainly by inhibiting the translation of Onecut2 mRNA and by increasing the level of
the Rab effector Granuphilin.
MiR-9 is encoded from three different genomic loci in
humans as well as in mice that result in the same mature
miR-9. Previous data suggest that miR-9 levels are induced by high glucose in INS-1E ␤-cells (44). This finding
was recently confirmed in mouse pancreatic islets after
the ip administration of glucose. Interestingly, mature
miR-9 as well as miR-9 –1 and miR-9 –2 precursor levels
were increased significantly only 60 minutes after the glucose injection, coinciding with the decline of insulin levels
(45). Based on online prediction tools, sirtuin-1 (Sirt1)
emerged as a potential miR-9 target. This was confirmed
in the insulinoma ␤-TC-6 line, in which the transfection
with pre-miR-9 caused a decrease in Sirt1 protein levels
(45). This finding is consistent with a previous report that
identified Sirt1 as miR-9 target in mouse embryonic stem
cells (46). Sirt1 is a nuclear protein that deacetylates histones, transcription factors, and other proteins in a NADdependent manner. Sirt1 has been implicated in the regulation of GSIS and mice that overexpress Sirt1 in ␤-cells
display enhanced GSIS (47). In summary, studies on
miR-9 function in ␤-cells suggest that it negatively controls insulin secretion in part by targeting the transcription factor Onecut2 (41) as well as the NAD-dependent
deacetylase Sirt1 (45).
miR-15
MiR-15a and miR-15b were identified in a screen for
miRNAs that are up-regulated during pancreas regeneration and shown to be 200-fold up-regulated in the regenerating compared with the developing pancreas (48).
Furthermore, it was shown that miR-15a/b targets Ngn3,
which is normally expressed in all proendocrine cells during pancreas development (20, 48). Ngn3 is present in
pancreatic endocrine progenitor cells but is not expressed
in the mature islets or during pancreas regeneration (49),
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and Ngn3 null mice lack islets (50). Although Ngn3 transcript was detectable during endocrine pancreas regeneration after partial pancreatectomy, there was no Ngn3
protein present as previously reported (49). This suggested a posttranscriptional inhibition of Ngn3 levels during pancreas regeneration. The microRNAs, miR-15a
and miR-15b, were highly expressed at day 3 of postpancreatectomy and shown to target Ngn3 (48). Inhibition of
miR-15 increased Ngn3 levels in regenerating pancreatic
endocrine cells and also increased the levels of (NeuroD)
and Nkx2.2, two downstream targets of Ngn3. Furthermore, the overexpression of miR-15a/b in E12.5 pancreatic buds reduced the number of insulin- and glucagonpositive cells (48). Based on these data, it was concluded
that increased expression of miR15a/b during pancreas
regeneration down-regulates Ngn3 levels to induce the
regeneration process.
miR-21
A screen for miRNAs that are up-regulated by exposure to proinflammatory cytokines or in prediabetic
(NOD) mice led to the identification of miR-21 (51, 52).
Treatment of MIN6 ␤-cells with either IL-1␤ or a cytokine mixture containing IL-1␤, TNF-␣, and IFN-␥ led to
an approximately 3-fold increase in miR-21 levels (51,
52). After testing various combinations of cytokines,
IL-1␤ alone was the most potent inducer of miR-21. Proinflammatory cytokines such as IL-1␤ and TNF-␣ are
produced by infiltrating leukocytes and islets and contribute to ␤-cell failure and establishment of diabetes (53).
Analysis of miR-21 levels in prediabetic female NOD
mice with normal blood glucose levels revealed a significant increase of miR-21 levels in islets of 8- and 13-week
animals compared with 4-week animals. Overexpression
of miR-21 in MIN6 ␤-cells did not have a significant
effect on proinsulin mRNA levels or insulin content but
decreased GSIS (51). MiR-21 overexpression also was
associated with a decrease in (VAMP2), a SNARE protein
essential for ␤-cell exocytosis and of the Rab3a GTPase
(54 –56). Treatment of MIN6 ␤-cells with 1 ng/mL IL-1␤
for 24 hours also resulted in decreased GSIS and VAMP2
and Rab3a levels. However, the inhibition of miR-21 in
MIN6 ␤-cells treated with IL-1␤ prevented the decrease in
the GSIS and VAMP2 but not Rab3a levels (51). The 3⬘
UTR of VAMP2 does not contain miR-21 binding sites,
suggesting an indirect regulation of VAMP2 by miR-21.
In summary, miR-21 plays a negative role in the regulation of ␤-cell function by reducing insulin exocytosis in
response to cytokines.
MiR-21 also is critical in the regulation of ␤-cell apoptosis during type 1 diabetes (57). Expression of miR-21
is induced by members of the NF-␬B family, c-Rel and p65
doi: 10.1210/me.2014-1306
(58), and this increase in miR-21 levels results in the suppression of ␤-cell death by reducing the levels of the tumor
suppressor protein programmed cell death 4 (Pdcd4) (52,
57, 59). Pdcd4 stimulates cell death by activating the apoptotic Bax family of proteins. The inactivation of Pdcd4
in pancreatic ␤-cells protects them from cell death in
NOD and in streptozotocin (STZ)-treated C57BL/6 mice
(57). These data suggest that the NF-␬B-miR-21-Pdcd4
axis regulates ␤-cell death during type 1 diabetes. Thus,
both studies found that miR-21 is up-regulated in response to inflammation in pancreatic ␤-cells and inhibits
insulin exocytosis and ␤-cell death.
miR-24
Conditional deletion of Dicer1 in adult ␤-cells using
tamoxifen-inducible RIP Cre-recombinase led to the development of diabetes due to significant decreases in insulin mRNA and insulin content (23). MiR-24 was identified as one of the four miRNAs (miR-26, miR-182, and
miR-148) in ␤-cell Dicer1-deficient mice that are involved
in regulation of insulin biosynthesis (23). Deletion of Dicer1 in adult ␤-cells resulted in a 70% reduction of insulin
mRNA levels that was associated with elevated levels of
the transcriptional repressors Sox6 and Bhlhe22 (60, 61).
Inhibition of miR-24 in primary islet cultures resulted in a
down-regulation of insulin mRNA and increased the levels of Sox6. Thus, conditional inactivation of Dicer1 decreases miR-24 levels and thereby increases Sox6 protein
levels, which then represses insulin gene expression (23).
A different study implicated miR-24 in the suppression
of two maturity-onset diabetes of the young (MODY)
genes, HNF1␣ and NeuroD (62). MiR-24 levels were
found to be up-regulated in the obese and diabetic db/db
mice compared with the control mice and in HFD-fed
mice. Additionally, miR-24 levels were increased by oxidative stress and high glucose. Overexpression of miR-24
in the MIN6 ␤-cell line reduced cell proliferation and
decreased GSIS. These findings suggest that the up-regulation of miR-24 as seen in db/db or HFD-fed mice is
likely via the ROS/PKC pathway and results in pancreatic
␤-cell dysfunction and thereby links glucolipotoxicity to
type 2 diabetes (62). In this case, miR-24 has been shown
to down-regulate the MODY proteins NeuroD1 and
Hnf1-␣.
Recently miR-24 also has been implicated in regulation of ␤-cell proliferation by controlling the availability
of the tumor suppressor protein menin or multiple endocrine neoplasia-1 (MEN1) (63). Menin negatively regulates islet expansion and adaptation by increasing the production of cell cycle inhibitors (64). Ablation of MEN1 in
mice results in pancreatic islet hyperplasia (65, 66),
whereas its overexpression leads to the inability of islets
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to undergo proliferation and increase insulin production
during pregnancy (67). Overexpression of miR-24 in the
human ␤-cell line ␤lox5 decreased menin mRNA as well
as protein levels. Furthermore, it decreased the expression
of the cell cycle inhibitor p27, which is transcriptionally
regulated by menin (63). Transfection of the ␤lox5 cell
line with pre-miR-24 led to increased cell viability and
proliferation. MiR-24 is encoded by two different loci,
and menin has been shown to regulate miR-24 expression
from both loci, suggesting the presence of a feedback loop
between menin and miR-24 (63). These data suggest that
miR-24 promotes ␤-cell proliferation in the endocrine
pancreas by decreasing the levels of cell cycle inhibitors by
directly targeting menin. In summary, miR-24 has multiple targets in ␤-cells and regulates ␤-cell proliferation and
insulin production as well as glucolipotoxicity.
miR-29
There are three isoforms of miR-29 and all three are
highly expressed in primary mouse islets (68). MiR-29
levels are increased in many tissues of diabetic animals,
and recent data suggest that miR-29 negatively regulates
GSIS (69). Studies in mouse islets have shown that
miR-29 targets the plasma membrane monocarboxylate
transporter-1 (MCT1; SLC16A1) and thereby affects insulin secretion (68). Although MCT1 is widely expressed
in other tissues, its levels in ␤-cells are tightly regulated
and kept low to avoid the uptake of circulating pyruvate
or its export by MCT1, which would interfere with insulin release (68, 70, 71). Thus, this study suggests that
miR-29 may control insulin release via the regulation of
MCT1 transporter levels.
Increased miR-29 levels also have been observed in the
islets of prediabetic NOD mice and in isolated mouse and
human islets in response to exposure to proinflammatory
cytokines (72). In this study, overexpression of miR-29
isoforms in MIN6 ␤-cells and islets interfered with GSIS,
which was due to the suppression of the transcription
factor Onecut2 expression. This led to an increase in
granuphilin levels, which inhibits insulin release. Overproduction of miR-29 also promoted apoptosis by diminishing the levels of the antiapoptotic protein Mcl1 (72).
Collectively these data suggest that miR-29 plays a critical
role in cytokine-induced ␤-cell dysfunction during
prediabetes.
A recent analysis of 5⬘-shifted isomiRs in ␤-cells identified miR-29 as one of most significant miRNA families
that regulates ␤-cell function and is associated with type 2
diabetes (73). MiR-29a levels are up-regulated in skeletal
muscle, liver, and white adipose tissue, and this up-regulation is associated with insulin resistance in GK rats (74 –
77). In conclusion, the up-regulation of miR-29 levels
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may contribute to type 2 diabetes via decreased GSIS as
well as by mediating insulin resistance in peripheral tissues. In agreement with this idea, miR-29a levels also are
increased in the serum of type 2 diabetes patients (78),
suggesting that miR-29 family of miRNAs may serve as
potential biomarkers for diabetes.
miR-30
The members of the miR-30 family are highly expressed in human fetal pancreas and are involved in the
regulation of epithelial-mesenchymal transition (32, 79).
They inhibit the translation of mesenchymal mRNAs,
such as vimentin and Snail1, and thereby enable the differentiation of pancreatic mesenchymal cells into insulinproducing islet-like cells (32).
The miR-30 family members have different functions
and targets. MiR-30d has been previously shown to be
high glucose induced and to regulate the levels of the
␤-cell transcription factor MafA but has no effect on
Pdx-1 and NeuroD1 expression (80, 81). MafA is not a
direct target of miR-30d. Instead, miR-30d suppresses the
levels of the TNF-␣-activated kinase Map4k4 and thereby
leads to increased MafA levels and insulin gene transcription (81). However, how the regulation of Map4k4 levels
by miR-30d in the end leads to increased expression of
MafA remains unknown. In a different study, miR-30d
was identified as one of the miRNAs that is up-regulated
during differentiation of human pluripotent stem cells
(hPSCs) to insulin-producing ␤ islet-like cells and shown
to target the transcription factor Rfx6 (82). Interestingly,
in both studies miR-30d regulates the levels of ␤-cell transcription factors.
MiR-30a-5p has the same seed sequence as miR-30d
and has been implicated in glucotoxicity-induced ␤-cell
dysfunction. MiR-30a-5p levels increase in response to
glucotoxic conditions, and the overexpression of mir30a-5p decreases insulin production and GSIS by suppressing the expression of the transcription factor Beta2/
NeuroD (83). MiR-30a-5p levels also were increased in
db/db mice and injection of anti-miR-30a-5p into db/db
animals led to a significant reduction of nonfasting blood
glucose levels (83). It is interesting that despite the fact
that miR-30d and miR-30a-5p have the same seed region
and differ only in one nucleotide, they target different
genes in ␤-cells. In summary, the existing data suggest
that the miR-30 family regulates the expression of various
transcription factors in ␤-cells.
miR-33a and miR-145
MicroRNAs involved in regulation of cholesterol homeostasis in pancreatic ␤-cells have been shown to negatively regulate GSIS. Increased expression of the miRNAs
Mol Endocrinol, December 2014, 28(12):1922–1933
miR-33a and miR-145 in murine islets cause a reduction
in GSIS by increasing cholesterol levels by targeting the
expression of the ATP-binding cassette transporter 1
(Abca1) (84, 85). Abca1 is important for the regulation of
cholesterol homeostasis and regulates plasma HDL levels
by causing cholesterol efflux. ␤-Cell-specific deletion of
Abca1 leads to impaired glucose tolerance and ␤-cell
function (86). On the other hand, inhibition of miR-33a
or miR-145 expression in isolated islets increases Abca1
and decreases cholesterol levels, leading to enhanced GSIS
(84, 85). Interestingly, miR-145 levels are decreased in
response to high levels of glucose, which stimulates GSIS
(85). These findings suggest that miR-33a and miR-145
may negatively impact GSIS by suppressing the levels of
the Abca1 transporter and thereby reducing cholesterol
efflux.
miR-124a
miR-124a consists of three isoforms (miR-124a1,
miR-124a2, and miR-124a3) encoded by different genes
and has been implicated in the regulation of insulin secretion and expression of ␤-cell-specific genes as well as pancreas development (48, 87, 88). The first study that implicated miR-124a in the regulation of ␤-cell function
showed that the expression of miR-124a2 isoform increases during pancreas development. Furthermore, it
was shown that it targets FoxA2, a transcription factor
that plays an important role in ␤-cell differentiation (87).
Consistent with this finding, the FoxA2 target genes
Pdx-1, Kir6.2, and Sur-1 also were regulated by miR124a2 (87). A role for miR-124a in endocrine pancreas
development was confirmed by another group that
showed that Ngn3, a transcription factor essential for
␤-cell differentiation, is a target of miR-124a (48). In this
study miR-124a and other miRNAs were found to be
expressed after a partial pancreatectomy in mice and to
inhibit Ngn3 expression to allow ␤-cell regeneration.
Later in a separate study, miR-124a was identified as
one of several miRNAs that regulate basal insulin secretion by increasing the levels of insulin exocytosis proteins
SNAP25 Rab3A, and synapsin-1A and by decreasing
Rab27A and Noc2 levels. Of these proteins, only Rab27A
was shown to be a direct target of miR-124a (89). It is
likely that the effect of miR-124a on insulin exocytosis
may be partly mediated by directly targeting FoxA2,
which regulates the expression of the channel subunits,
Kir6.2 and Sur1. A recent publication suggests that the
thioredoxin-interacting protein negatively regulates miR124a expression (90). Inhibition of miR-124a by TXNIP
stimulates the expression of the IAPP by blocking the
suppression of FoxA2 (90). Based on these published
data, miR-124a has important functions in ␤-cell devel-
doi: 10.1210/me.2014-1306
opment as well as insulin secretion and ␤-cell gene expression. All of these functions of miR-124a may be due to its
ability to target FoxA2 in pancreatic ␤-cells.
miR-184
MiR-184 is important for the regulation of ␤-cell expansion during the insulin-resistant state (29). In pancreatic islets, miR-184 targets Ago2, which is part of the
RNA-induced silencing complex required for targeting of
mRNAs by miRNAs. Loss of Ago2 blocks the compensatory expansion of ␤-cells in response to insulin resistance
by causing an increased expression of miR-375 targets
(29, 30). Interestingly, miR-184 is silenced in the islets of
insulin-resistant mice and humans, which results in an
increased expression of Ago2 and in compensatory ␤-cell
expansion (29). Forced expression of miR-184 in ob/ob
diabetic mice decreases Ago2 levels and blocks the compensatory expansion of ␤-cells. However, the exact mechanisms that regulate miR-184 levels during normal or
insulin-resistant states remain to be determined. In summary, miR-184 plays a key role in the regulation ␤-cell
expansion by targeting Ago2 during the insulin-resistant
state.
miR-200
The miR-200 family members (miR200a, miR-200b,
miR-200c, miR-141, and miR-429) have been shown to
play a role in ␤-cell specification. A recently published
study shows that miR-200c is highly abundant in ␤-cells
and functions to repress glucagon expression by targeting
the transcription factors cMaf and Fog2, which normally
stimulate glucagon gene expression (91). As expected, the
level of miR-200c in ␣-cells was barely detectable. Forced
expression of miR-200c in the ␣-cell line ␣TC6 led to a
decreased expression of glucagon by the down-regulation
of cMaf and Fog2 levels (91).
MiR-200a was found to be induced during the differentiation of hPSCs to insulin-producing ␤-islet-like cells
(82). In this system, miR-200a was shown to target
ZEB 1 and ZEB2 transcription factors involved in epithelial-mesenchymal transition by repressing E-cadherin (CDH1) expression (82, 92). Furthermore, miR200a also has been shown to target Sox17 in hPSCs, a
transcription factor critical for endoderm specification
of definitive endoderm. Altogether these findings implicate the members of miR-200 family in the regulation of ␤-cell specification and development.
miR-375
MiR-375 was the first miRNA identified from pancreatic ␤-cells because of its high abundance in the endocrine
pancreas. Initial studies on miR-375 demonstrated that it
mend.endojournals.org
1929
as a negative regulator of insulin secretion and targets
myotrophin (Mtpn) expression (16). Mtpn is an important regulator of actin depolymerization and granule fusion (16, 93). Thus, in mature ␤-cells, miR-375 regulates
insulin secretion at the level of vesicle fusion with the
plasma membrane (16). A recent follow-up of miR-375
function in MIN6 cells suggests that miR-375 globally
affects glucose-induced insulin secretion by suppressing
the expression of Gephyrin, Ywhaz, Aifm1, and Mtpn
(30). This study also demonstrated that miR-375 is one of
the most enriched miRNAs present in Ago2-associated
complexes. Interestingly, similar to the inhibition of miR375, the loss of Ago2 in MIN6 cells results in enhanced
insulin release and increased expression of miR-375 target genes (30). In conclusion, miR-375 and Ago2 appear
to have similar roles in pancreatic ␤-cells and negatively
regulate GSIS.
Another target of miR-375 is 3⬘-phosphoinositide-dependent kinase-1 or Pdk1 in pancreatic ␤-cells (44). By
targeting PDK-1, miR-375 causes a decrease in the glucose regulation of insulin gene expression and DNA synthesis in INS-1E ␤-cells. Interestingly, miR-375 precursor
levels were decreased by glucose itself in INS-1E cells as
well as in primary rat islets (44). Moreover, miR-375
levels were decreased in the islets of the diabetic GK rats.
A different group found that miR-375 is highly expressed in the developing endocrine pancreas at E14.5, in
which it was colocalized with Pdx-1. This suggested that
miR-375 plays an important role during endocrine pancreas development (19, 32). Studies conducted in zebrafish demonstrated that miR-375 indeed plays a critical
role in endocrine pancreas development and is essential
for normal islet formation (94). These data were later
confirmed in mice by the generation of miR-375 knockout mice, which had an increased number of ␣-cells but a
reduced number of ␤-cells, causing excess glucagon production relative to insulin (95). The miR-375 knockout
animals had hyperglycemia caused by hyperactivation of
the glucagon axis and decreased ␤-cell proliferation. Ablation of miR-375 in the obese ob/ob mice blocked the
ability of these mice to expand their ␤-cell mass in response to insulin resistance (95). Moreover, DNA microarray screens combined with a bioinformatics analysis
revealed several potential miR-375 targets that are involved in the negative regulation of cell proliferation (95).
It also is likely that miR-375 may promote ␤-cell proliferation by suppressing the proliferation of non-␤-cells
within the islet. Consistent with this idea, the expression
of miR-375 together with miR-7 was induced during the
differentiation of human embryonic stem cells to insulinproducing cells. In this study, the overexpression of miR375 caused a decreased expression of gut-endoderm/pan-
1930
Özcan
␤-Cell MicroRNAs
creatic progenitor-specific markers, Hnf1␤ and Sox9
(96).
Interestingly, miR-375 has the potential to serve as a
marker for ␤-cell death and diabetes (97). MiR-375 was
present at high levels in the circulation of STZ-treated and
nonobese diabetic NOD mice. In the STZ-administered
mice, miR-375 levels dramatically increased prior to the
onset of hyperglycemia. In the NOD mice, miR-375 in the
circulation increased 2 weeks prior to diabetes onset (97).
Thus, circulating levels of miR-375 may serve as a potential biomarker for diagnosis of prediabetes.
Concluding remarks
Recent studies clearly established a role for miRNAs in
regulation of pancreatic ␤-cell function. Although the human genome encodes for more than 2500 mature miRNAs, only a few of these miRNAs have been implicated to
have a role in ␤-cell function. Thus, the role of most
miRNAs in ␤-cells is unknown and requires further
investigation.
Analysis of miRNA function in ␤-cells and other tissues is complicated by the fact that the manipulation of a
single miRNA may not have a drastic effect because miRNAs exist in families that contain the same seed region.
Furthermore, almost 50% of miRNA targets contain
binding sites for two or more miRNAs. Thus, miRNAs
can synergize or antagonize the expression of target mRNAs, complicating studies on single miRNA function.
Another complexity associated with miRNAs is that there
are many miRNA isomers that have varied seed regions
and target a different set of genes than the original
miRNA (73). It is interesting that many miRNAs are encoded by different genomic loci that result in the same
mature miRNA and target mainly the same set of genes.
Both miR-7 and miR-9 are encoded by three different
genomic loci, leading to the same mature miRNA. This
raises the question of whether pre- or pri-miRNAs also
may have a function that is different from that of the
mature miRNA.
The complex action of miRNAs is demonstrated by the
fact that they may regulate different processes within the
same cell. For example, miRNA-375 has been shown to
be involved in ␤-cell development as well as in insulin
secretion by targeting different mRNAs (16, 30, 95). The
same is true for miR-24, which causes glucolipotoxicity
when up-regulated by targeting the Hnf1␣ and NeuroD1
MODY mRNAs (62, 92). However, it also can regulate
insulin gene transcription by targeting the transcriptional
repressors Sox6 and Bhlhe22 (62, 92) and ␤-cell proliferation by altering the levels of the transcriptional regulator
menin (63).
Mol Endocrinol, December 2014, 28(12):1922–1933
MiRNAs have been shown to be abundant in circulation, released either from dead cells or the exosomes, and
represent important biomarkers for the progression of
many diseases. The number of circulating miRNAs that
are associated with diabetes are steadily increasing. Some
of these miRNAs including miR-375 may be useful biomarkers for the prognosis of prediabetes (97).
One future challenge for the miRNA field will be to
determine the mechanisms that regulate miRNA levels in
pancreatic ␤-cells during development and in mature
␤-cells. The abundance of many miRNAs is altered during
diabetes, including miR-184, which is silenced during insulin resistance to allow for compensatory ␤-cell expansion (29). However, the mechanisms that lead to the silencing of miR-184 during the insulin-resistant state
remain unknown. A detailed understanding of the regulation of miRNA levels during physiological as well as
pathophysiological conditions will be critical in the development of novel strategies for combating miRNA-related
diseases. Furthermore, the development of new techniques that allows the analysis of genome-wide transcription and the proteome in a single cell will accelerate our
understanding of metabolic regulation by miRNAs. Advances in miRNA-based therapies can be extremely powerful in combating diseases, such as diabetes, especially if
they are cell type specific because most miRNAs are ubiquitously expressed (98). There are already several ongoing clinical trials using miRNA-based therapies, indicating that the manipulation of miRNA levels in human
patients is a feasible strategy to treat various diseases.
Acknowledgments
We apologize to all authors whose original publications could
not be cited or discussed in this review due to space limitations.
Address all correspondence and requests for reprints to:
Sabire Özcan, PhD, Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, 741
South Limestone Street, BBSRB-155, Lexington, KY 40536.
E-mail: [email protected].
This work was supported by Grant R01DK067581 from the
National Institutes of Health/National Institute of Diabetes and
Digestive and Kidney Diseases; Grant P20RR020171 from the
National Institutes of Health/National Center for Research Resources; Grant 1-05-CD-15 from the American Diabetes Association; and Grant UL1TR000117 from the National Institutes
of Health Clinical and Translational Science Awards.
Disclosure Summary: The author has nothing to disclose.
References
1. Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of
tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science. 2001;294(5543):858 – 862.
doi: 10.1210/me.2014-1306
2. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science. 2001;
294(5543):853– 858.
3. Orom UA, Nielsen FC, Lund AH. MicroRNA-10a binds the 5⬘UTR
of ribosomal protein mRNAs and enhances their translation. Mol
Cell. 2008;30(4):460 – 471.
4. Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R. MicroRNA-373
induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci USA. 2008;105(5):1608 –1613.
5. Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation. Science. 2007;
318(5858):1931–1934.
6. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215–233.
7. Harfe BD, McManus MT, Mansfield JH, Hornstein E, Tabin CJ.
The RNaseIII enzyme Dicer is required for morphogenesis but not
patterning of the vertebrate limb. Proc Natl Acad Sci USA. 2005;
102(31):10898 –10903.
8. Chong MM, Rasmussen JP, Rudensky AY, Littman DR. The RNAseIII enzyme Drosha is critical in T cells for preventing lethal inflammatory disease. J Exp Med. 2008;205(9):2005–2017.
9. Wang Y, Medvid R, Melton C, Jaenisch R, Blelloch R. DGCR8 is
essential for microRNA biogenesis and silencing of embryonic stem
cell self-renewal. Nat Genet. 2007;39(3):380 –385.
10. O’Carroll D, Mecklenbrauker I, Das PP, et al. A Slicer-independent
role for Argonaute 2 in hematopoiesis and the microRNA pathway.
Genes Dev. 2007;21(16):1999 –2004.
11. Bernstein E, Kim SY, Carmell MA, et al. Dicer is essential for mouse
development. Nat Genet. 2003;35(3):215–217.
12. Wienholds E, Koudijs MJ, van Eeden FJ, Cuppen E, Plasterk RH.
The microRNA-producing enzyme Dicer1 is essential for zebrafish
development. Nat Genet. 2003;35(3):217–218.
13. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R,
Rajewsky N. Widespread changes in protein synthesis induced by
microRNAs. Nature. 2008;455(7209):58 – 63.
14. Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows
that some microRNAs downregulate large numbers of target mRNAs. Nature. 2005;433(7027):769 –773.
15. Miranda KC, Huynh T, Tay Y, et al. A pattern-based method for
the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell. 2006;126(6):1203–1217.
16. Poy MN, Eliasson L, Krutzfeldt J, et al. A pancreatic islet-specific
microRNA regulates insulin secretion. Nature. 2004;432(7014):
226 –230.
17. Dumortier O, Van Obberghen E. MicroRNAs in pancreas development. Diabetes Obes Metab. 2012;14(suppl 3):22–28.
18. Guay C, Jacovetti C, Nesca V, Motterle A, Tugay K, Regazzi R.
Emerging roles of non-coding RNAs in pancreatic ␤-cell function
and dysfunction. Diabetes Obes Metab. 2012;14(suppl 3):12–21.
19. Lynn FC, Skewes-Cox P, Kosaka Y, McManus MT, Harfe BD,
German MS. MicroRNA expression is required for pancreatic islet
cell genesis in the mouse. Diabetes. 2007;56(12):2938 –2945.
20. Gu G, Dubauskaite J, Melton DA. Direct evidence for the pancreatic lineage: NGN3⫹ cells are islet progenitors and are distinct from
duct progenitors. Development. 2002;129(10):2447–2457.
21. Morita S, Hara A, Kojima I, et al. Dicer is required for maintaining
adult pancreas. PLoS One. 2009;4(1):e4212.
22. Dor Y, Brown J, Martinez OI, Melton DA. Adult pancreatic ␤-cells
are formed by self-duplication rather than stem-cell differentiation.
Nature. 2004;429(6987):41– 46.
23. Melkman-Zehavi T, Oren R, Kredo-Russo S, et al. miRNAs control
insulin content in pancreatic ␤-cells via downregulation of transcriptional repressors. EMBO J. 2011;30(5):835– 845.
24. Kalis M, Bolmeson C, Esguerra JL, et al. ␤-Cell specific deletion of
Dicer1 leads to defective insulin secretion and diabetes mellitus.
PLoS One. 2011;6(12):e29166.
25. Mandelbaum AD, Melkman-Zehavi T, Oren R, et al. Dysregula-
mend.endojournals.org
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
1931
tion of Dicer1 in ␤ cells impairs islet architecture and glucose metabolism. Exp Diabetes Res. 2012;2012:470302.
Kanji MS, Martin MG, Bhushan A. Dicer1 is required to repress
neuronal fate during endocrine cell maturation. Diabetes. 2013;
62(5):1602–1611.
Dueck A, Meister G. Assembly and function of small RNA-argonaute protein complexes. Biol Chem. 2014;395(6):611– 629.
Meister G. Argonaute proteins: functional insights and emerging
roles. Nat Rev Genet. 2013;14(7):447– 459.
Tattikota SG, Rathjen T, McAnulty SJ, et al. Argonaute2 mediates
compensatory expansion of the pancreatic ␤ cell. Cell Metab. 2014;
19(1):122–134.
Tattikota SG, Sury MD, Rathjen T, et al. Argonaute2 regulates the
pancreatic ␤-cell secretome. Mol Cell Proteomics. 2013;12(5):
1214 –1225.
Correa-Medina M, Bravo-Egana V, Rosero S, et al. MicroRNA
miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas. Gene Expr Patterns. 2009;9(4):
193–199.
Joglekar MV, Patil D, Joglekar VM, et al. The miR-30 family of
microRNAs confer epithelial phenotype to human pancreatic cells.
Islets. 2009;1(2):137–147.
Bravo-Egana V, Rosero S, Molano RD, et al. Quantitative differential expression analysis reveals miR-7 as major islet microRNA.
Biochem Biophys Res Commun. 2008;366(4):922–926.
Kredo-Russo S, Mandelbaum AD, Lennox KA, Behlke MA, et al.
Pancreas-enriched miRNA refines endocrine cell differentiation.
Development. 2012;139(16):3021–3031.
Yasuda T, Kajimoto Y, Fujitani Y, et al. PAX6 mutation as a genetic
factor common to aniridia and glucose intolerance. Diabetes. 2002;
51(1):224 –230.
Wang Y, Liu J, Liu C, Naji A, Stoffers DA. MicroRNA-7 regulates
the mTOR pathway and proliferation in adult pancreatic ␤-cells.
Diabetes. 2013;62(3):887– 895.
Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal
integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol.
2011;12(1):21–35.
Bernal-Mizrachi E, Wen W, Stahlhut S, Welling CM, Permutt MA.
Islet ␤ cell expression of constitutively active Akt1/PKB␣ induces
striking hypertrophy, hyperplasia, and hyperinsulinemia. J Clin Invest. 2001;108(11):1631–1638.
Hamada S, Hara K, Hamada T, et al. Upregulation of the mammalian target of rapamycin complex 1 pathway by Ras homolog enriched in brain in pancreatic ␤-cells leads to increased ␤-cell mass
and prevention of hyperglycemia. Diabetes. 2009;58(6):1321–
1332.
Latreille M, Hausser J, Stutzer I, et al. MicroRNA-7a regulates
pancreatic ␤ cell function. J Clin Invest. 2014;124(6):2722–2735.
Plaisance V, Abderrahmani A, Perret-Menoud V, Jacquemin P, Lemaigre F, Regazzi R. MicroRNA-9 controls the expression of
Granuphilin/Slp4 and the secretory response of insulin-producing
cells. J Biol Chem. 2006;281(37):26932–26942.
Fukuda M. Rab27 and its effectors in secretory granule exocytosis:
a novel docking machinery composed of a Rab27.effector complex.
Biochem Soc Trans. 2006;34(Pt 5):691– 695.
Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T. Granuphilin
molecularly docks insulin granules to the fusion machinery. J Cell
Biol. 2005;171(1):99 –109.
El Ouaamari A, Baroukh N, Martens GA, Lebrun P, Pipeleers D,
van Obberghen E. miR-375 targets 3⬘-phosphoinositide-dependent
protein kinase-1 and regulates glucose-induced biological responses
in pancreatic ␤-cells. Diabetes. 2008;57(10):2708 –2717.
Ramachandran D, Roy U, Garg S, Ghosh S, Pathak S, KolthurSeetharam U. Sirt1 and mir-9 expression is regulated during glucose-stimulated insulin secretion in pancreatic ␤-islets. FEBS J.
2011;278(7):1167–1174.
Saunders LR, Sharma AD, Tawney J, et al. miRNAs regulate SIRT1
1932
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
Özcan
␤-Cell MicroRNAs
expression during mouse embryonic stem cell differentiation and in
adult mouse tissues. Aging (Albany NY). 2010;2(7):415– 431.
Moynihan KA, Grimm AA, Plueger MM, et al. Increased dosage of
mammalian Sir2 in pancreatic ␤ cells enhances glucose-stimulated
insulin secretion in mice. Cell Metab. 2005;2(2):105–117.
Joglekar MV, Parekh VS, Mehta S, Bhonde RR, Hardikar AA.
MicroRNA profiling of developing and regenerating pancreas reveal post-transcriptional regulation of neurogenin 3. Dev Biol.
2007;311(2):603– 612.
Lee CS, De Leon DD, Kaestner KH, Stoffers DA. Regeneration of
pancreatic islets after partial pancreatectomy in mice does not involve the reactivation of neurogenin-3. Diabetes. 2006;55(2):269 –
272.
Gradwohl G, Dierich A, LeMeur M, Guillemot F. neurogenin3 is
required for the development of the four endocrine cell lineages of
the pancreas. Proc Natl Acad Sci USA. 2000;97(4):1607–1611.
Roggli E, Britan A, Gattesco S, et al. Involvement of microRNAs in
the cytotoxic effects exerted by proinflammatory cytokines on pancreatic ␤-cells. Diabetes. 2010;59(4):978 –986.
Bravo-Egana V, Rosero S, Klein D, et al. Inflammation-mediated
regulation of MicroRNA expression in transplanted pancreatic islets. J Transplant. 2012;2012:723614.
Eizirik DL, Colli ML, Ortis F. The role of inflammation in insulitis
and ␤-cell loss in type 1 diabetes. Nat Rev Endocrinol. 2009;5(4):
219 –226.
Regazzi R, Sadoul K, Meda P, Kelly RB, Halban PA, Wollheim CB.
Mutational analysis of VAMP domains implicated in Ca2⫹-induced insulin exocytosis. EMBO J. 1996;15(24):6951– 6959.
Wheeler MB, Sheu L, Ghai M, et al. Characterization of SNARE
protein expression in ␤ cell lines and pancreatic islets. Endocrinology. 1996;137(4):1340 –1348.
Yaekura K, Julyan R, Wicksteed BL, et al. Insulin secretory deficiency and glucose intolerance in Rab3A null mice. J Biol Chem.
2003;278(11):9715–9721.
Ruan Q, Wang T, Kameswaran V, et al. The microRNA-21PDCD4 axis prevents type 1 diabetes by blocking pancreatic ␤ cell
death. Proc Natl Acad Sci USA. 2011;108(29):12030 –12035.
Vinciguerra M, Sgroi A, Veyrat-Durebex C, Rubbia-Brandt L,
Buhler LH, Foti M. Unsaturated fatty acids inhibit the expression of
tumor suppressor phosphatase and tensin homolog (PTEN) via microRNA-21 up-regulation in hepatocytes. Hepatology. 2009;49(4):
1176 –1184.
Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A,
Lund AH. Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol
Chem. 2008;283(2):1026 –1033.
Peyton M, Stellrecht CM, Naya FJ, Huang HP, Samora PJ, Tsai MJ.
␤3, a novel helix-loop-helix protein, can act as a negative regulator
of ␤2 and MyoD-responsive genes. Mol Cell Biol. 1996;16(2):626 –
633.
Iguchi H, Ikeda Y, Okamura M, et al. SOX6 attenuates glucosestimulated insulin secretion by repressing PDX1 transcriptional activity and is down-regulated in hyperinsulinemic obese mice. J Biol
Chem. 2005;280(45):37669 –37680.
Zhu Y, You W, Wang H, et al. MicroRNA-24/MODY gene regulatory pathway mediates pancreatic ␤-cell dysfunction. Diabetes.
2013;62(9):3194 –3206.
Vijayaraghavan J, Maggi EC, Crabtree JS. miR-24 regulates menin
in the endocrine pancreas. Am J Physiol Endocrinol Metab. 2014;
307(1):E84 –E92.
Karnik SK, Hughes CM, Gu X, et al. Menin regulates pancreatic
islet growth by promoting histone methylation and expression of
genes encoding p27Kip1 and p18INK4c. Proc Natl Acad Sci USA.
2005;102(41):14659 –14664.
Crabtree JS, Scacheri PC, Ward JM, et al. A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors. Proc Natl Acad Sci USA. 2001;98(3):1118 –1123.
Mol Endocrinol, December 2014, 28(12):1922–1933
66. Crabtree JS, Scacheri PC, Ward JM, et al. Of mice and MEN1:
Insulinomas in a conditional mouse knockout. Mol Cell Biol. 2003;
23(17):6075– 6085.
67. Karnik SK, Chen H, McLean GW, et al. Menin controls growth of
pancreatic ␤-cells in pregnant mice and promotes gestational diabetes mellitus. Science. 2007;318(5851):806 – 809.
68. Pullen TJ, da Silva Xavier G, Kelsey G, Rutter GA. miR-29a and
miR-29b contribute to pancreatic ␤-cell-specific silencing of monocarboxylate transporter 1 (Mct1). Mol Cell Biol. 2011;31(15):
3182–3194.
69. Bagge A, Clausen TR, Larsen S, et al. MicroRNA-29a is up-regulated in ␤-cells by glucose and decreases glucose-stimulated insulin
secretion. Biochem Biophys Res Commun. 2012;426(2):266 –272.
70. Ishihara H, Wang H, Drewes LR, Wollheim CB. Overexpression of
monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in ␤ cells. J Clin
Invest. 1999;104(11):1621–1629.
71. Zhao C, Wilson MC, Schuit F, Halestrap AP, Rutter GA. Expression and distribution of lactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas. Diabetes.
2001;50(2):361–366.
72. Roggli E, Gattesco S, Caille D, et al. Changes in microRNA expression contribute to pancreatic ␤-cell dysfunction in prediabetic NOD
mice. Diabetes. 2012;61(7):1742–1751.
73. Baran-Gale J, Fannin EE, Kurtz CL, Sethupathy P. ␤ Cell 5⬘-shifted
isomiRs are candidate regulatory hubs in type 2 diabetes. PLoS
One. 2013;8(9):e73240.
74. He A, Zhu L, Gupta N, Chang Y, Fang F. Overexpression of micro
ribonucleic acid 29, highly up-regulated in diabetic rats, leads to
insulin resistance in 3T3-L1 adipocytes. Mol Endocrinol. 2007;
21(11):2785–2794.
75. Herrera BM, Lockstone HE, Taylor JM, et al. MicroRNA-125a is
over-expressed in insulin target tissues in a spontaneous rat model
of type 2 diabetes. BMC Med Genomics. 2009;2:54.
76. Pandey AK, Verma G, Vig S, Srivastava S, Srivastava AK, Datta M.
miR-29a levels are elevated in the db/db mice liver and its overexpression leads to attenuation of insulin action on PEPCK gene expression in HepG2 cells. Mol Cell Endocrinol. 2011;332(1–2):125–
133.
77. Kurtz CL, Peck BC, Fannin EE, et al. MicroRNA-29 fine-tunes the
expression of key FOXA2-activated lipid metabolism genes and is
dysregulated in animal models of insulin resistance and diabetes.
Diabetes. 2014;63(9):3141–3148.
78. Kong L, Zhu J, Han W, et al. Significance of serum microRNAs in
pre-diabetes and newly diagnosed type 2 diabetes: a clinical study.
Acta Diabetol. 2011;48(1):61– 69.
79. Ozcan S. MiR-30 family and EMT in human fetal pancreatic islets.
Islets. 2009;1(3):283–285.
80. Tang X, Muniappan L, Tang G, Ozcan S. Identification of glucoseregulated miRNAs from pancreatic ␤ cells reveals a role for miR30d in insulin transcription. RNA. 2009;15(2):287–293.
81. Zhao X, Mohan R, Ozcan S, Tang X. MicroRNA-30d induces
insulin transcription factor MafA and insulin production by targeting mitogen-activated protein 4 kinase 4 (MAP4K4) in pancreatic
␤-cells. J Biol Chem. 2012;287(37):31155–31164.
82. Liao X, Xue H, Wang YC, et al. Matched miRNA and mRNA
signatures from an hESC-based in vitro model of pancreatic differentiation reveal novel regulatory interactions. J Cell Sci. 2013;
126(Pt 17):3848 –3861.
83. Kim JW, You YH, Jung S, et al. miRNA-30a–5p-mediated silencing
of Beta2/NeuroD expression is an important initial event of glucotoxicity-induced ␤ cell dysfunction in rodent models. Diabetologia.
2013;56(4):847– 855.
84. Wijesekara N, Zhang LH, Kang MH, Abraham T, Bhattacharjee A,
Warnock GL, Verchere CB, Hayden MR. miR-33a modulates
ABCA1 expression, cholesterol accumulation, and insulin secretion
in pancreatic islets. Diabetes. 2012;61(3):653– 658.
doi: 10.1210/me.2014-1306
85. Kang MH, Zhang LH, Wijesekara N, et al. Regulation of ABCA1
protein expression and function in hepatic and pancreatic islet cells
by miR-145. Arterioscler Thromb Vasc Biol. 2013;33(12):2724 –
2732.
86. Brunham LR, Kruit JK, Pape TD, et al. ␤-Cell ABCA1 influences
insulin secretion, glucose homeostasis and response to thiazolidinedione treatment. Nat Med. 2007;13(3):340 –347.
87. Baroukh N, Ravier MA, Loder MK, et al. MicroRNA-124a regulates Foxa2 expression and intracellular signaling in pancreatic
␤-cell lines. J Biol Chem. 2007;282(27):19575–19588.
88. Joglekar MV, Parekh VS, Hardikar AA. New pancreas from old:
microregulators of pancreas regeneration. Trends Endocrinol
Metab. 2007;18(10):393– 400.
89. Lovis P, Gattesco S, Regazzi R. Regulation of the expression of
components of the exocytotic machinery of insulin-secreting cells
by microRNAs. Biol Chem. 2008;389(3):305–312.
90. Jing G, Westwell-Roper C, Chen J, Xu G, Verchere CB, Shalev A.
Thioredoxin-interacting protein promotes islet amyloid polypeptide expression through miR-124a and FoxA2. J Biol Chem. 2014;
289(17):11807–11815.
91. Klein D, Misawa R, Bravo-Egana V, et al. MicroRNA expression in
mend.endojournals.org
92.
93.
94.
95.
96.
97.
98.
1933
␣ and ␤ cells of human pancreatic islets. PLoS One. 2013;8(1):
e55064.
Gregory PA, Bert AG, Paterson EL, et al. The miR-200 family and
miR-205 regulate epithelial to mesenchymal transition by targeting
ZEB1 and SIP1. Nat Cell Biol. 2008;10(5):593– 601.
Poy MN, Spranger M, Stoffel M. microRNAs and the regulation of
glucose and lipid metabolism. Diabetes Obes Metab. 2007;9(suppl
2):67–73.
Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk
RH. Targeted inhibition of miRNA maturation with morpholinos
reveals a role for miR-375 in pancreatic islet development. PLoS
Biol. 2007;5(8):e203.
Poy MN, Hausser J, Trajkovski M, et al. miR-375 maintains normal pancreatic ␣- and ␤-cell mass. Proc Natl Acad Sci USA. 2009;
106(14):5813–5818.
Wei R, Yang J, Liu GQ, et al. Dynamic expression of microRNAs
during the differentiation of human embryonic stem cells into insulin-producing cells. Gene. 2013;518(2):246 –255.
Erener S, Mojibian M, Fox JK, Denroche HC, Kieffer TJ. Circulating miR-375 as a biomarker of ␤-cell death and diabetes in mice.
Endocrinology. 2013;154(2):603– 608.
van Rooij E, Kauppinen S. Development of microRNA therapeutics
is coming of age. EMBO Mol Med. 2014;6(7):851– 864.