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Increased expression of miR-187 in human islets from individuals with type 2 diabetes is
associated with reduced glucose-stimulated insulin secretion
J. M. Locke1, G. da Silva Xavier2, H.R. Dawe3, G. A. Rutter2 and L. W. Harries1
1
Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter,
UK
2
Section of Cell Biology, Department of Medicine, Imperial College London, London, UK
3
College of Life and Environmental Sciences, University of Exeter, Exeter, UK
Corresponding author:Lorna W. Harries
University of Exeter Medical School
Barrack Road
Exeter
EX2 5DW
L.W.Harries@exeter.ac.uk
Tel: 01392 406749
Fax: 01392 406767
Word count (abstract) = 246
Word count (main text) = 3176
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Abstract
Aims/hypothesis Type 2 diabetes is characterised by progressive beta cell dysfunction, with
changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs)
are post-transcriptional regulators of gene expression and therefore alterations in miRNA
levels may be involved in the deterioration of beta cell function.
Methods Global TaqMan arrays and individual TaqMan assays were used to measure islet
miRNA expression in discovery (n=20) and replication (n=20) cohorts from individuals with
and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin
secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1
cells. Identification of miRNA targets was assessed using luciferase assays and by measuring
mRNA levels.
Results In the discovery and replication cohorts miR-187 expression was found to be
significantly increased in islets from individuals with type 2 diabetes compared to matched
controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin
secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation
paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187
markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the
gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of
insulin secretion, was identified as a direct target of miR-187 and displayed reduced
expression in islets from individuals with type 2 diabetes.
Conclusions/interpretation Our findings suggest a role for miR-187 in the blunting of insulin
secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of
type 2 diabetes.
Keywords Glucose-stimulated insulin secretion, HIPK3, Islets, MicroRNA, Type 2 diabetes,
Abbreviations
GSIS
glucose-stimulated insulin secretion
HIPK3
Homeodomain-interacting protein kinase-3
miRNA microRNA
sn
small nuclear
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Introduction
In type 2 diabetes the inability of beta cells to compensate for reduced insulin sensitivity is
associated with specific changes in gene expression, which may conceivably play a causal
role in beta cell dysfunction. MicroRNAs (miRNAs) are a class of small non-coding RNAs
that regulate gene expression by binding to target mRNAs, resulting in mRNA decay and/or
translational repression. There is growing evidence that changes in the expression of
miRNAs can affect beta cell function (reviewed by Guay et al [1]). Recent evidence has
suggested that changes in miRNA expression may either precede diabetes onset, and be
associated with positive effects on beta cell function, or occur on manifestation of the disease
and impact negatively on beta cell function [2, 3]. Indeed, a comparison of the islet
miRNome of the Goto-Kakizaki (GK) rat model of type 2 diabetes, with Wistar controls, has
revealed a set of up-regulated miRNAs enriched in targets within pathways known to be
involved in disease causation [4]. In the present study we hypothesised that a similar strategy,
measuring the expression of miRNAs in human islets from individuals with and without type
2 diabetes, might identify specific miRNAs with a causal role in human beta cell dysfunction.
Methods
Human islet tissue For global miRNA profiling snap-frozen human islets were received
from three centres (National Disease Research Interchange, Philadelphia, PA, USA; AMS
Biotechnology, Abingdon, UK; and ProCell Biotech, Newport Beach, CA, USA). All islets
were taken with approval from appointed ethics committees. For replication all islets came
from ProCell Biotech. Islet purity and viability were determined by dithizone and fluorescein
diacetate/propidium iodide staining, respectively [5, 6]. Islets from ProCell Biotech were
provided with information regarding glucose-stimulated insulin secretion (GSIS), determined
as follows. After isolation and overnight culture islets were incubated sequentially in media
containing low (2.8mM) and high (28mM) glucose for 1 h. The amount of insulin present in
each supernatant was measured by electrochemiluminescence immunoassay on a Cobas e601
analyzer (Roche, Indianapolis, IN, USA). GSIS was calculated as insulin secretion at 28
mmol l-1 / 2.8 mmol l-1 glucose.
Isolation and culture of primary rat islets and INS-1 cells The rat insulinoma cell line INS-1
was cultured in RPMI 1640 GlutaMAX (Life Technologies, Paisley, UK) media
supplemented with 10% FCS, 100 U/ml penicillin, 100µg/ml streptomycin and 100µmol/l βmercaptoethanol. Primary rat islets were isolated by collagenase digestion [7], hand-picked
and cultured, prior to transfection, for at least 24 h in RPMI 1640 medium containing 10
mmol/l glucose, 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin.
RNA extraction and real-time qPCR Total RNA, including miRNA, was extracted using the
miRVana miRNA isolation kit (Life Technologies). MiRNA reverse transcription reactions
were carried out using the miRNA reverse transcription kit (Life Technologies). For global
profiling human TaqMan arrays (version 2.0, cards A + B containing assays to 667 miRNAs,
Life Technologies) were used with a pre-amplification step included, following the
manufacturers’ instructions. Values for Ct were generated using automatic settings, ΔCt was
calculated using a global mean normalisation strategy [8], and ΔΔCt for each gene was
determined using a mean ΔCt value in control samples. Profiling of miR-187, miR-345, miR15b and U6 small nuclear (sn)RNA was conducted using inventoried TaqMan miRNA assays
(Life Technologies) with expression determined from three separate reverse transcription
reactions. For the measurement of rat Hipk3 and human HIPK3 expression, RNA was reverse
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transcribed using SuperScript III First-Strand Synthesis System (Life Technologies) with
oligo-dT priming. For rat Hipk3 expression RNA was, prior to reverse transcription, DNased
using the Turbo DNA-free kit (Life Technologies). TaqMan Gene Expression Assays (Life
Technologies) were used to measure expression of Hipk3 (Rn00582409_m1) and HIPK3
(Hs00178628_m1). Expression was normalised using GeNorm [9], with the following
housekeeping genes measured where appropriate; Ubc (Rn01789812_g1), Tbp
(Rn01455648_m1), Hprt1 (Rn01527840_m1), B2M (Hs00984230), GUSB (Hs00939627)
and RPLPO (Hs99999902_m1). Relative expression was calculated using the comparative Ct
method. All reactions were run on an ABI7900HT platform (Life Technologies).
INS-1 transfection and measurement of insulin secretion and beta-cell apoptosis Transient
transfection of INS-1 cells, at a density of ~2 x 106 cells, was conducted with miRVana
miRNA mimics (Life Technologies) and a Nucleofector Device (Lonza, Basel, Switzerland).
Negative Control miRVana miRNA mimic number 1 (Life Technologies), which has been
designed not to target any known human, mouse or rat gene, was used as a negative control.
Transfected cells were plated in 24-well poly-D-lysine coated plates at a density of ~3 x 105
cells/well. After 48 h, media were removed and cells washed once and then incubated for 2 h
in modified Krebs-Ringer medium (125mmol/l NaCl, 4.74mmol/l KCl, 1mmol/l CaCl2,
1.2mmol/l KH2PO4, 1.2mmol/l MgSO4, 5mmol/l NaHCO3, 25mmol/l Hepes, pH 7.4)
containing 0.1% BSA and 2.8mmol/l glucose. Cells were then subjected to either high
(28mM) or low (2.8mM) glucose treatment for 1 h before the supernatant fraction was
removed for insulin determination. Levels of insulin were measured by radioimmunoassay
(Linco Research Inc., St. Charles, MO, USA) and normalised to protein content as
determined by BCA assay (Pierce, Rockford, IL, USA). For the analysis of INS-1 apoptosis
48 h post-transfection the ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit
(Millipore, Billerica, MA, USA) was used according to the manufacturer’s instructions.
Samples were co-stained with DAPI, mounted in VectorShield (Vector Laboratories,
Peterborough, UK) and viewed using a Zeiss AxioObserver Z1 microscope (Carl Zeiss,
Oberkochen, Germany) equipped with a x40 1.3 numerical aperture (NA) oil objective and
controlled by AxioVision software (Carl Zeiss). As a positive control untransfected cells
were heat shocked at 56 0C for 3 min. The cells were allowed to recover at 37oC for 1 h
before the assay was performed. Heat shock induced ~40% cells to become apoptotic (data
not shown).
Primary rat islet transfection and measures of insulin secretion and beta cell apoptosis
Transfections were carried out using TransIT-TKO (Mirus Bio Corporation, Madison, WI,
USA) in the presence of 1 nmol/l miRVana miRNA mimic (Life Technologies) for 48 h prior
to assays. Insulin secretion was determined as previously described [10] with insulin content
assayed following acidified ethanol extraction. We note that while transfection of the intact
islet is likely to affect only the outermost layers of cells [11] it is from these that the majority
of stimulated insulin secretion is likely to be observed in vitro given the loss of islet
vasculature which occurs rapidly during culture [12]. For the analysis of apoptosis, islets
were fixed in 4% paraformaldehyde and stained using the DeadEnd fluorometric TUNEL
system (Promega, Madison, WI, USA), as per the manufacturer’s protocol for non-adherent
cells. Following nicked end labelling using the kit, islets were washed in PBS and then
incubated overnight at 4oC with guinea pig anti-insulin antibody (1:200; Dako, Glostrup,
Denmark) in PBS containing 0.1% Triton X-100 and BSA. Islets were then washed and
incubated in goat anti-guinea pig Alexa Fluor 568 (1:1,000; Life Technologies) in PBS for 1
h at room temperature. Subsequently, islets were washed twice in PBS and spotted on
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superfrost slides. The slides were left to set overnight in Vectashield HardSet Mounting
Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) at room temperature in the
dark. Islets were imaged using a Zeiss Axiovert-200 confocal microscope with an
Improvision/Nokigawa spinning disc, and running Volocity 5.0 (Improvision, Coventry, UK)
software.
Image
analysis
was
performed
using
ImageJ
v.1.43m
(http://rsbweb.nih.gov/ij/download.html).
Luciferase assay
The pMirTarget plasmids containing the 3’UTR of human HIPK3
(NM_001048200) downstream of firefly luciferase, and a mutant version differing only by a
C-to-G substitution (underlined) within the predicted miR-187 binding site
(UUCUAACUAGUGCAAGACACGU), were purchased (Origene, Rockville, MD, USA).
Luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega).
To account for differences in transfection efficiency firefly luciferase activity was normalised
to Renilla expression which originated from co-transfected pRL-SV40 (Promega).
Statistical analysis Except where stated otherwise, statistical differences were assessed using
two-tailed one-sample or two-sample t tests, with correction for multiple testing as indicated.
A p value <0.05 was considered significant. All data presented as mean ± SEM.
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Results
miR-187 expression is increased in islets from donors with type 2 diabetes The global
miRNA profile of islets from 20 donors (11 with type 2 diabetes, nine controls) was
determined using TaqMan arrays. Of the 667 miRNA assys on these arrays 255 amplified in
all samples (electronic supplementary material [ESM] Table 1) and the use of preamplification meant we decided to limit our analysis to these miRNAs (we found many
‘on/off’ changes in expression, most likely due to inconsistent amplification of very weakly
expressed miRNAs). Two miRNAs, miR-187 and miR-345, displayed higher and
statistically significant (after Benjamini-Hochberg false discovery rate correction) differential
expression between islets from donors with and without type 2 diabetes (Table 1).
We next sought to replicate our results in an independent cohort using individual
miRNA-specific TaqMan assays. We measured the expression of miR-345 and miR-187 in
another 20 islet samples (ten from individuals with type 2 diabetes, ten without diabetes) and
again found significantly higher miR-187 expression in islets from donors with type 2
diabetes vs healthy donors. The increase in miR-345 islet expression observed in individuals
with type 2 diabetes in the initial cohort did not replicate (Table 1). No significant differences
in age, sex, BMI, ethnicity, islet purity or islet viability between groups were identified in
either islet cohort. Also consistent with previous findings [13] and with a primary role for
reduced GSIS in disease pathogenesis, islets in the replication cohort from individuals with
type 2 diabetes displayed reduced glucose-stimulated insulin release when compared with
islets from individuals without diabetes (Table 2).
miR-187 expression is inversely correlated with GSIS Given the observed increase in miR187 expression in both islet cohorts we investigated whether this miRNA might play a role in
islet function or survival. In islets from 35 normoglycaemic donors we found a significant
inverse correlation between miR-187 expression and GSIS (Fig. 1a). No significant
correlation was found between levels of miR-15b and GSIS (data not shown), despite levels
being similarly normalised to U6 snRNA.
Overexpression of miR-187 in islets and beta cells reduces GSIS To determine whether
increased miR-187 expression might contribute directly to reduced GSIS, miR-187 mimics or
control sequences were transiently transfected into primary rat islets. Compared with mimiccontrol-transfected islets the introduction of miR-187 mimics sharply reduced insulin
secretion stimulated by high (20 mmol/l) glucose and to a far lesser, albeit significant degree,
on KCl-stimulation (Fig. 1b). There were no significant differences in insulin content
between miR-187 and mimic-control-transfected islets (Fig. 1c).
We next tested whether the above actions of miR-187 were likely to be through a cellautonomous effect of miR-187 on beta cells within the islet. Correspondingly, measured in
the rat pancreatic beta cell line, INS-1, near-physiological levels of miR-187 over-expression
(ten- to 20-fold increase in cells transfected with miR-187 compared with mimic control;
ESM Fig.1a) also resulted in a significant reduction in insulin secretion under high (28
mmol/l) but not low (2.8 mmol/l) glucose conditions, compared with cells transfected with
mimic control (ESM Fig. 1b).
Transfection of miR-187 mimic into primary rat islets and INS-1 cells resulted in no
significant difference in rates of apoptosis, as assessed by TUNEL assay, when compared
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with mimic-control-transfected cells (ESM Fig. 2), and no evident effects on cell viability as
judged through overall cellular morphology (data not shown).
HIPK3 is a direct target of miR-187 In order to identify putative miR-187 targets we used
miRWalk[14], a database that compiles results from multiple commonly used prediction
programs (TargetScan, miRanda, miRDB and RNA22). One of the putative targets identified
was the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known
regulator of insulin secretion [15]. Strikingly, the blunted insulin secretory responses at high
glucose concentrations in Hipk3-/- mice, or on small interfering (si)RNA-mediated
knockdown of HIPK3 in isolated mouse islets [15], are similar to the phenotype we observe
on miR-187 overexpression.
To explore the possibility that HIPK3 may be regulated by miR-187 we first
measured Hipk3 mRNA levels in INS-1 cells transfected with miR-187 mimic and mimic
control. The putative miR-187 binding site is conserved from human to rat so a decrease in
Hipk3 expression was expected. Indeed, as determined by real-time qPCR, Hipk3 mRNA
levels were significantly reduced in INS-1 cells transfected with miR-187 mimic compared
with mimic-control-transfected cells (Fig. 2a). Furthermore overexpression of miR-187 in
INS-1 cells significantly inhibited the expression of a construct in which the 3’UTR sequence
of human HIPK3 was fused downstream of luciferase cDNA. This inhibition was not seen
when the predicted miR-187 target sequence was mutated, indicating a direct interaction
between miR-187 and the 3’UTR of human HIPK3 (Fig. 2b). Subsequent measurement of
HIPK3 transcripts in the islets comprising the discovery and replication cohorts for the
miRNA profiling also revealed a reduction of HIPK3 mRNA expression in islets from
individuals with type 2 diabetes vs those without (Fig. 2c).
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Discussion
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By providing one possible mechanism through which miR-187 may act we show here
that HIPK3 is a target for this miRNA in the beta cell. Importantly, HIPK3 has recently been
shown to be required for the normal stimulation of insulin secretion by glucose. Thus, Hipk3/mice are glucose intolerant and show depressed islet levels of two key mediators of glucose
responsiveness: pancreatic duodenum homeobox-1 (PDX1) and phosphorylated glycogen
synthase kinase-3 beta (GSK3β) [15]. Although we observed only small decreases in
luciferase activity and Hipk3 transcript levels on miR-187 overexpression, this is consistent
with a role for miRNAs in fine-tuning gene expression [21, 22]. Additionally, given that in
mouse islets a modest 46% knockdown of HIPK3 reduces insulin secretion at 20 mmol/l to
Despite concerns regarding the effect of hyperglycaemia on transcript levels, differential
mRNA expression in human islets from donors with type 2 diabetes vs those without diabetes
can successfully identify genes with a causal role in beta cell dysfunction [16]. Using a
similar global profiling approach we have identified a specific miRNA, miR-187, with
reproducibly higher expression in islets from donors with type 2 diabetes. We have
consequently elucidated a role for miR-187 in regulating insulin secretion, possibly through a
direct targeting of HIPK3.
Many previous studies describing differential islet gene expression from individuals
with and without type 2 diabetes have been plagued by a failure to replicate. One of the
strengths of our study was the use of a second islet cohort, the prudence of such an approach
highlighted by differential miR-345 expression not replicating. A failure to match groups for
age, BMI, sex, ethnicity, islet purity and viability can also lead to spurious results. In the
present study there were no significant differences in such confounding factors, all of which
can influence mRNA expression [17, 18], and are likely to affect miRNA expression. There
may, of course, be other confounders we were unable to control for in the present study. For
example, changes in the cellular composition of the islets (i.e. alpha/beta cell ratios) cannot
be excluded, though this would seem to be unlikely. Future studies, involving even larger
islet cohorts, seem very likely to find further aberrantly expressed miRNAs in islets from
individuals with type 2 diabetes; however, our study provides proof-of-principle that miRNA
expression profiling in islets from individuals with and without diabetes can identify miRNAs
with a causal role in beta cell dysfunction.
Using direct functional assays in both primary rodent islets and a rat insulinomaderived cell line, we provide evidence here that increases in miR-187 affect glucose-, and to a
lesser extent, depolarisation-induced insulin secretion, without evident effects on cell
viability or apoptotic index. Importantly, the dramatic inhibition of GSIS elicited by miR187 was similar in extent to what we observed after transfection with a mimic of miR-375
(data not shown). MiR-375 has a well-established role in the control of insulin release [19]
and our results suggest that miR-187 may play an equally important role in this process after
its induction in the beta cell of individuals with type 2 diabetes. Given the much greater
impact of miR-187 over-expression on glucose- compared with KCl-stimulated secretion, it
would appear that miR-187 acts chiefly on events upstream of membrane depolarisation,
potentially impairing glucose metabolism or increases in free cytosolic Ca2+ [20]. Detailed
future studies will be needed to investigate these possibilities. Likewise, analysing the effects
of over-expressing miR-187 in primary human islets and in response to additional
physiological secretagogues (such as glucagon-like peptide-1, acetylcholine and amino acids)
also represent important future experiments.
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54% of that seen in control cells [15], it seems plausible that small decreases in Hipk3
expression may have a significant impact on GSIS. We emphasise that while the decrease in
Hipk3 mRNA levels in the presence of miR-187 may conceivably be compounded by a
decrease in translational efficiency, recent studies suggest that the actions of miRNAs are
largely (~84%) mediated by RNA degradation [23]. Nonetheless, studies are needed to
explore this possibility.
Aberrant miR-187 expression has, to our knowledge, not been reported in any
previous study examining miRNA expression in islets from animal models of type 2 diabetes,
nor in cell-line models using culture conditions that mimic those found in an individual with
diabetes. While evidence of deregulated miR-187 expression in these experiments might
strengthen and support our data it is perhaps not surprising, given the difficulties in modelling
polygenic diseases in controlled animal and cell culture systems, that no such result has been
reported previously. Indeed, the molecular mechanisms through which miR-187 expression is
increased in islets from donors with type 2 diabetes remain obscure.
It is interesting that two recent studies detailing the human islet miRNome in
individuals without diabetes report either low miR-187 expression [24] or a failure to detect
miR-187 in the majority of samples studied [25] (it is assumed that ΔCt values and
normalised RNA-Seq read counts can be used as appropriate proxies for absolute miRNA
levels). We also find that miR-187 is relatively weakly expressed in individuals without
diabetes. However, the five- to sevenfold increase in miR-187 expression in islets from
individuals with type 2 diabetes means its levels in this pathophysiological state are
comparable with, or greater than, levels of several other miRNAs with known roles in beta
cell function (miR-29b [26], miR-9 [27] and miR-96 [28]). Perhaps, akin to what has been
assumed to account for the relatively low steady-state expression of other regulatory genes
(such as transcription factors) [29], a low expression of miR-187 is needed to provide a builtin fail-safe mechanism, controlling its persistence and thus preventing aberrant beta cell
function.
We are aware of only one other publication comparing miRNA expression in human
islets from individuals with and without glucose intolerance. While using a far smaller
number of samples than the present study (n=9 islets from individuals without diabetes, n=6
from individuals with an HbA1c ≥ 6.1), and also examining the expression of only a small
number of miRNAs (not including miR-187), this earlier study provided some evidence for
abnormal miRNA expression in islets from individuals with type 2 diabetes [30]. The results
of our larger study highlight that aberrantly expressed miRNAs may be causally involved in
human islet dysfunction during type 2 diabetes. Future studies identifying further
dysregulated miRNAs may discover novel pathways involved in islet dysfunction that could
provide novel therapeutic targets for diabetes treatment.
Acknowledgements The authors are grateful to H. Welters (University of Exeter Medical
School, Exeter, UK) for help with the insulin secretion assays. The kind assistance of D.
Hodson and R. Mitchell (Section of Cell Biology, Department of Medicine, Imperial College
London, London, UK) with confocal imaging is also much appreciated.
Funding
This work was supported by the Wellcome Trust (project grant number
089845/Z/09/Z). G.A.R. is the recipient of Royal Society Wolfson Research and Wellcome
Trust Senior Investigator (WT098424AIA) Awards, and thanks the Medical Research
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Council (MRC) for Programme Grant MR/J0003042/1. GdSX and GAR. were supported by a
project grant from Diabetes UK (BDA 13/0004672) and HDR by MRC grant G1001644.
Duality of interest The authors declare that there is no duality of interest associated with this
manuscript.
Contribution statement JML designed and conducted experiments, analysed data and wrote
the manuscript. GdSX and HRD designed, conducted and analysed data from experiments.
GAR contributed to the study design, data analysis and writing the manuscript. LWH was
involved in the study design, writing the manuscript and managed the project. All authors
participated in data interpretation, revision of the article and approved the final version of the
manuscript.
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[15] Shojima N, Hara K, Fujita H, et al. (2012) Depletion of homeodomain-interacting
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453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
12
Diab-13-0685 (D-RSW-04)
470
471
472
473
Table 1 Results of miRNA expression profiling in human islets from individuals with and
without type 2 diabetes
Global TaqMan array profiling
Individual
profiling
miRNA
Relative
expressiona
Unadjusted
p value
Adjusted
p valueb
Relative
expressiona
Unadjusted
p value
miR-187
7.55
7 x 10-5
0.009
5.38
0.021
miR-345
1.92
2 x 10-5
0.006
0.64
0.112
0.002
0.115
ND
ND
miR-129-3p 3.32
474
475
476
477
478
479
480
481
assay
a
Calculated using the formula: mean expression in those with type 2 diabetes/mean
expression in controls
b
Adjusted using Benjamini-Hochberg false discovery rate
Statistical significance determined using two-sample t tests and Fisher’s exact test
ND, not determined.
Table 2 Clinical characteristics of donors in discovery and replication cohorts
Characteristic
482
483
484
485
486
TaqMan
Global TaqMan array profiling
Individual
profiling
T2D
islets
T2D
islets
Control
islets
p value
TaqMan
Control
islets
assay
p value
n
9
11
10
10
Sex (M/F)
7/2
5/6
0.20
3/7
5/5
0.65
Ethnicity
5/2/2
9/2/0
0.19
4/6/0
9/1/0
0.06
(Caucasian/African
American/Asian)
Age (years)
53 (8)
47 (9)
0.09
55 (9)
51 (6)
0.24
BMI
36 (14)
30 (6)
0.27
32 (4)
29 (5)
0.08
Islet purity (%)
81 (8)
82 (10)
0.94
89 (5)
90 (5)
0.51
Islet viability (%)
90 (6)
88 (8)
0.58
91 (2)
92 (2)
0.30
Cold
ischaemic 14 (7)
16 (5)
0.54
16 (6)
15 (6)
0.61
time (hours)
GSIS a
ND
ND
2.6 (1.3) 4.2 (1.4)
0.02
a
Calculated using formula: insulin secretion at 28 mmol/l glucose/insulin secretion at 2.8
mmol/l glucose
Where appropriate, data presented as mean (SD)
Statistical significance determined using two-sample t tests and Fisher’s exact test
ND, not determined; T2D, type 2 diabetes
13
Diab-13-0685 (D-RSW-04)
487
a
Insulin secretion (log2 fold of basal)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-2.5
488
489
-1.5 -0.5
0.5
1.5
Log2 relative miR-187
expression
b
Insulin secretion (% insulin content)
3.0
2.5
2.0
1.5
1.0
0.5
**
0.0
3mmol/l glucose
20mmol/l glucose
490
491
492
493
494
495
496
* 497
498
499
500
501
502
503
504
20mmol/l KCl505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
14
Diab-13-0685 (D-RSW-04)
520
521
c
30
Insulin content (ng/islet)
25
20
15
10
5
0
522
523
524
525
526
527
528
529
530
531
532
533
534
535
Control miR-187
Fig. 1 Increased miR-187 expression is associated with reduced GSIS. (a) In islets from 35
non-diabetic donors higher levels of miR-187 expression correlated with reduced GSIS
9calculated as amount of insulin secreted at 28 mmol l-1/amount of insulin secreted at 2.8
mmol l-1). miRNA expression was determined form three separate reverse transcriptions
using real-time PCR. Statistical significance was assessed by the Pearson correlation
coefficient test; r=-0.34, p=0.049. (b) Compared with mimic-control-transfected cells, the
introduction of miR-187 mimic into primary rat islets reduced insulin secretion under high
glucose (20 mmol/l) conditions. **p<0.01 and *p<0.05 vs cells transfected with mimic
control, n=3 independent experiments. Black, control; grey, miR-187. (c) No significant
difference in insulin content in primary rat islets transfected with control and miR-187
mimics, n=3 independent experiments. All data expressed as mean ± SEM
15
Diab-13-0685 (D-RSW-04)
536
a
Relative Hipk3 expression
1.2
1.0
*
0.8
0.6
0.4
0.2
0.0
Control miR-187
537
538
539
b
Relative luciferase expression
1.2
1.0
*
0.8
0.6
0.4
0.2
0.0
540
541
542
HIPK3
WT
HIPK3
MT
c
Relative HIPK3 expression
1.2
1.0
*
0.8
0.6
0.4
0.2
0.0
543
Control T2D
16
Diab-13-0685 (D-RSW-04)
544
545
546
547
548
549
550
551
552
553
554
555
556
557
Fig. 2 HIPK3 is a direct target of miR-187. (a) In INS-1 cells transfected with miR-187
mimic endogenous Hipk3 mRNA expression is reduced, *p<0.05 vs cells transfected with
negative control miRNA mimic. Statistical significance assessed by one-tailed one-sample t
test, n=8 independent experiments. (b) In INS-1 cells overexpression of miR-187 inhibited
luciferase expression form a construct containing the 3’UTR sequence of human HIPK3
(WT), but not expression from a construct containing the 3’UTR sequence of human HIPK3
where the putative miR-187 binding site has been mutated (MT). Statistical significance
assessed by one-sample t test; n=4 independent experiments. Blacnk, control; grey, miR-187.
(c) HIPK3 mRNA expression is reduced in islets from individuals with type 2 diabetes (T2D)
(n=17) compared with islets from matched controls (n=18). Statistical significance assessed
by two-sample t test. All data presented as mean ± SEM
17
Diab-13-0685 (D-RSW-04)
558
559
18
Diab-13-0685 (D-RSW-04)
560
ESM Table 1. Global miRNA profiling results of islets from individuals with and without type 2 diabetes
561
T2D
expression
mean (SD)
Control
expression
mean (SD)
miR-345
2.01 (0.42)
miR-187
adjusted562
p
value
unadjusted p
value
1.05 (0.33)
Relative
Expression
(T2D mean /
control
mean)
1.92
2 x 10-5
0.006
11.74 (9.17)
1.55 (1.68)
7.55
7 x 10-5
0.009
miR-129-3p
4.43 (2.24)
1.33 (0.89)
3.32
0.002
0.115
miR-28-3p
1.78 (0.47)
1.07 (0.37)
1.67
0.002
0.115
miR-29c
2.83 (0.94)
1.30 (1.04)
2.17
0.002
0.115
miR-95
2.29 (0.99)
1.31 (1.19)
1.75
0.014
0.392
miR-203
2.18 (1.07)
1.14 (0.52)
1.91
0.015
0.392
miR-210
2.42 (2.19)
1.16 (0.75)
2.10
0.016
0.392
miR-125a-5p
2.11 (1.11)
1.24 (0.98)
1.70
0.018
0.392
miR-200a
1.79 (0.48)
1.17 (0.68)
1.53
0.021
0.392
miR-31
5.85 (6.11)
1.54 (1.43)
3.80
0.022
0.392
miR-185
1.81 (0.48)
1.18 (0.62)
1.53
0.024
0.392
miR-885-5p
3.32 (2.09)
1.29 (1.10)
2.57
0.025
0.392
miR-374b
2.74 (1.37)
1.32 (0.81)
2.07
0.030
0.392
miR-372
30.37 (27.69)
6.21 (7.16)
4.89
0.040
0.392
miR-18a
9.53 (16.31)
2.12 (2.26)
4.50
0.040
0.392
miR-200b
2.29 (1.07)
1.20 (0.51)
1.90
0.042
0.392
miR-148b*
3.18 (1.68)
1.52 (1.07)
2.09
0.044
0.392
miR-9
3.52 (3.20)
1.34 (0.95)
2.64
0.049
0.392
let-7i*
6.00 (3.95)
2.27 (2.70)
2.64
0.050
0.392
miR-141
1.72 (0.57)
1.18 (0.65)
1.45
0.051
0.392
miR-204
1.97 (0.71)
1.21 (0.69)
1.62
0.052
0.392
miR-454*
4.70 (3.72)
1.86 (1.61)
2.53
0.052
0.392
miR-411
1.66 (0.84)
1.11 (0.57)
1.49
0.055
0.392
miR-517a
3.43 (2.69)
1.52 (1.52)
2.26
0.055
0.392
miR-30c
2.02 (0.81)
1.19 (0.62)
1.70
0.055
0.392
miR-26b*
2.61 (1.52)
1.43 (0.99)
1.82
0.055
0.392
miR-24
1.65 (0.64)
1.17 (0.80)
1.41
0.056
0.392
miR-148b
2.16 (0.87)
1.24 (0.58)
1.74
0.056
0.392
miR-27b
1.91 (1.20)
1.24 (0.90)
1.55
0.059
0.392
miR-331-3p
1.61 (0.51)
1.21 (0.86)
1.33
0.060
0.392
miR-320
1.80 (0.99)
1.22 (0.96)
1.47
0.061
0.392
miR-942
2.42 (1.79)
1.23 (0.78)
1.97
0.064
0.392
miR-17
2.10 (1.32)
1.15 (0.62)
1.82
0.065
0.392
563
19
Diab-13-0685 (D-RSW-04)
miR-132
2.31 (1.43)
1.31 (0.96)
1.77
0.065
0.392
miR-29c*
2.37 (1.14)
1.44 (1.05)
1.65
0.065
0.392
miR-34a
2.64 (1.81)
1.40(0.96)
1.89
0.065
0.392
miR-409-5p
2.66 (1.71)
1.58 (1.52)
1.68
0.066
0.392
miR-99b
1.77 (0.64)
1.24 (0.75)
1.43
0.066
0.392
miR-101
2.26 (1.30)
1.24 (0.65)
1.82
0.066
0.392
miR-28-5p
2.43 (1.92)
1.26 (0.74)
1.92
0.067
0.392
miR-374a
2.33 (1.55)
1.09 (0.39)
2.13
0.067
0.392
miR-886-3p
0.40 (0.33)
4.81 (8.90)
0.08
0.071
0.392
miR-186
2.16 (1.23)
1.25 (1.16)
1.72
0.075
0.392
miR-376a
2.12 (1.34)
1.37 (1.41)
1.55
0.077
0.392
miR-660
1.97 (0.79)
1.27 (0.70)
1.55
0.078
0.392
miR-103
2.16 (1.30)
1.34 (0.79)
1.61
0.078
0.392
miR-324-3p
1.63 (0.72)
1.15 (0.64)
1.41
0.080
0.392
miR-146a
1.66 (0.67)
1.19 (0.83)
1.40
0.080
0.392
miR-27a
2.28 (2.27)
1.17 (0.58)
1.95
0.082
0.392
miR-106a
2.19 (1.49)
1.15 (0.58)
1.91
0.083
0.392
miR-652
2.43 (1.17)
1.60 (1.22)
1.51
0.086
0.392
miR-24-1*
0.67 (0.31)
1.15 (0.53)
0.58
0.087
0.392
miR-34a*
2.38 (1.36)
1.24 (0.73)
1.92
0.090
0.392
miR-195
1.67 (0.68)
1.17 (0.63)
1.42
0.090
0.392
miR-551b*
2.36 (1.52)
1.49 (1.19)
1.59
0.092
0.392
miR-365
6.11 (6.72)
3.20 (3.88)
1.91
0.093
0.392
miR-192
1.46 (0.44)
1.13 (0.57)
1.29
0.095
0.392
miR-29a
1.61 (0.71)
1.19 (0.86)
1.36
0.095
0.392
miR-501-5p
1.60 (0.70)
1.13 (0.53)
1.41
0.096
0.392
miR-532-3p
1.52 (0.51)
1.09 (0.40)
1.39
0.096
0.392
miR-452
2.35 (2.04)
1.26 (0.94)
1.87
0.097
0.392
miR-140-3p
1.83 (1.19)
1.13 (0.67)
1.63
0.097
0.392
miR-455-5p
1.93 (0.98)
1.21 (0.62)
1.59
0.103
0.405
miR-155
3.52 (2.70)
1.46 (1.31)
2.41
0.104
0.405
miR-149
1.47 (0.47)
1.15 (0.60)
1.28
0.105
0.405
miR-222
1.86 (1.01)
1.35 (1.22)
1.38
0.107
0.405
miR-138
2.44 (2.27)
1.25 (0.88)
1.95
0.108
0.406
miR-410
2.15 (1.48)
1.43 (1.24)
1.50
0.110
0.406
miR-183
1.72 (0.78)
1.24 (0.79)
1.39
0.116
0.418
miR-340
2.13 (1.14)
1.27 (0.66)
1.67
0.116
0.418
miR-592
2.82 (1.69)
1.44 (0.72)
1.95
0.120
0.423
miR-500
1.76 (0.93)
1.09 (0.47)
1.61
0.121
0.423
miR-590-5p
1.70 (0.76)
1.26 (0.88)
1.35
0.127
0.439
miR-598
1.75 (0.92)
1.14 (0.52)
1.54
0.135
0.456
miR-339-3p
5.31 (7.23)
1.70 (1.74)
3.12
0.137
0.456
miR-532-5p
1.60 (0.72)
1.09 (0.40)
1.47
0.139
0.456
miR-642
1.56 (0.44)
1.29 (0.94)
1.21
0.140
0.456
20
Diab-13-0685 (D-RSW-04)
miR-27b*
2.08 (1.14)
1.36 (0.78)
1.53
0.146
0.470
miR-551b
2.24 (1.04)
1.70 (1.16)
1.32
0.157
0.480
miR-744
1.49 (0.45)
1.30 (1.11)
1.15
0.161
0.480
miR-425*
2.32 (1.29)
1.58 (1.48)
1.47
0.163
0.480
miR-362-5p
2.18 (1.39)
1.22 (0.52)
1.78
0.164
0.480
miR-330-3p
1.69 (0.84)
1.27 (0.89)
1.33
0.165
0.480
miR-15b
1.73 (1.07)
1.23 (0.71)
1.41
0.166
0.480
miR-183*
1.61 (0.60)
1.38 (1.14)
1.16
0.167
0.480
miR-7
2.46 (1.44)
1.96 (1.89)
1.26
0.168
0.480
miR-26a
1.73 (0.87)
1.15 (0.50)
1.51
0.168
0.480
miR-487b
1.88 (0.95)
1.21 (0.75)
1.55
0.169
0.480
miR-19b-1*
1.99 (1.22)
1.25 (0.72)
1.59
0.171
0.480
miR-106b
1.79 (0.98)
1.34 (0.88)
1.34
0.171
0.480
miR-361-5p
1.43 (0.45)
1.17 (0.62)
1.22
0.174
0.483
miR-25
1.89 (1.52)
1.22 (0.73)
1.55
0.188
0.516
miR-378
2.69 (2.17)
1.82 (1.69)
1.48
0.190
0.516
miR-190b
3.81 (2.81)
1.82 (1.85)
2.09
0.200
0.532
miR-379
3.16 (0.88)
2.51 (2.19)
1.26
0.202
0.532
miR-212
2.27 (1.59)
1.49 (1.50)
1.53
0.202
0.532
miR-224
4.45 (6.50)
1.48 (1.30)
3.02
0.205
0.532
miR-335
1.48 (0.51)
1.15 (0.54)
1.28
0.212
0.540
miR-136*
1.75 (1.06)
1.42 (1.00)
1.23
0.214
0.540
miR-16
1.83 (1.21)
1.29 (1.26)
1.41
0.215
0.540
miR-636
0.85 (0.36)
1.09 (0.52)
0.78
0.216
0.540
miR-889
1.70 (0.90)
1.28 (0.84)
1.32
0.218
0.540
miR-128
1.52 (0.52)
1.24 (0.77)
1.23
0.221
0.540
miR-26b
1.62 (0.71)
1.20 (0.63)
1.34
0.224
0.540
miR-655
1.73 (0.86)
1.28 (0.75)
1.34
0.228
0.540
miR-223
0.83 (0.59)
1.22 (0.83)
0.68
0.229
0.540
miR-20a
2.98 (3.17)
1.27 (0.75)
2.34
0.230
0.540
miR-376c
1.50 (0.53)
1.26 (1.01)
1.20
0.231
0.540
miR-221
2.08 (1.56)
1.30 (0.79)
1.59
0.233
0.540
miR-502-3p
1.47 (0.52)
1.24 (0.74)
1.18
0.241
0.546
miR-21*
0.84 (0.76)
1.59 (1.57)
0.53
0.242
0.546
miR-375
1.50 (0.64)
1.19 (0.73)
1.26
0.244
0.546
miR-301a
1.80 (0.89)
1.27 (0.68)
1.41
0.246
0.546
miR-135b
4.72 (6.13)
1.36 (1.15)
3.47
0.246
0.546
miR-151-3p
1.57 (0.63)
1.53 (1.52)
1.03
0.254
0.558
miR-99a*
1.42 (0.46)
1.23 (0.72)
1.15
0.256
0.559
miR-193a-5p
1.92 (1.32)
1.22 (0.70)
1.57
0.261
0.559
miR-33a*
1.62 (0.52)
1.30 (0.68)
1.25
0.261
0.559
miR-323-3p
1.71 (0.98)
1.21 (0.83)
1.41
0.263
0.559
miR-126*
4.33 (4.38)
2.17 (2.42)
1.99
0.271
0.560
let-7e
1.82 (1.00)
1.38 (0.93)
1.32
0.271
0.560
21
Diab-13-0685 (D-RSW-04)
miR-133a
2.39 (3.15)
1.79 (2.28)
1.33
0.272
0.560
miR-130a
2.14 (2.36)
1.25 (0.80)
1.70
0.272
0.560
miR-32
1.88 (1.20)
1.24 (0.53)
1.52
0.281
0.566
miR-135a
2.10 (1.42)
1.40 (0.95)
1.50
0.286
0.566
miR-941
1.45 (0.65)
1.20 (0.64)
1.21
0.289
0.566
miR-550
1.61 (0.83)
1.25 (0.76)
1.29
0.290
0.566
miR-137
2.12 (1.30)
1.97 (1.77)
1.08
0.293
0.566
miR-491-5p
1.66 (0.94)
1.11 (0.47)
1.49
0.294
0.566
miR-708
1.57 (0.52)
1.62 (1.58)
0.97
0.299
0.566
miR-429
2.01 (1.43)
1.22 (0.59)
1.65
0.301
0.566
miR-30e*
1.66 (1.16)
1.10 (0.51)
1.52
0.302
0.566
miR-92a
1.73 (1.10)
1.22 (0.73)
1.41
0.302
0.566
miR-26a-1*
2.33 (1.97)
1.54 (1.37)
1.51
0.302
0.566
miR-22*
2.16 (1.47)
1.63 (1.53)
1.33
0.303
0.566
miR-380*
0.84 (0.73)
1.44 (1.06)
0.58
0.306
0.566
miR-125b
1.50 (0.65)
1.27 (0.78)
1.19
0.309
0.566
miR-200c
1.33 (0.48)
1.09 (0.51)
1.22
0.314
0.566
miR-1
2.04 (1.49)
1.36 (1.09)
1.51
0.315
0.566
miR-181a*
5.02 (6.25)
4.68 (6.60)
1.07
0.315
0.566
miR-342-3p
1.93 (1.86)
1.35 (1.25)
1.43
0.315
0.566
miR-139-5p
1.85 (1.49)
1.23 (0.90)
1.51
0.327
0.583
miR-92a-1*
1.49 (1.68)
1.54 (1.56)
0.97
0.333
0.586
miR-191
1.98 (2.37)
1.18 (0.71)
1.68
0.333
0.586
miR-769-5p
1.78 (1.03)
1.38 (1.07)
1.29
0.337
0.588
miR-369-5p
1.52 (0.82)
1.20 (0.59)
1.27
0.340
0.588
miR-19a
1.99 (1.32)
1.28 (0.75)
1.55
0.343
0.588
miR-324-5p
1.66 (1.07)
1.27 (0.78)
1.31
0.344
0.588
miR-505*
2.73 (2.63)
1.43 (0.96)
1.91
0.347
0.590
miR-331-5p
1.54 (1.13)
1.11 (0.63)
1.39
0.358
0.601
miR-7-2*
1.56 (0.65)
1.63 (1.58)
0.96
0.359
0.601
miR-301b
2.12 (1.67)
1.54 (0.73)
1.38
0.364
0.601
miR-21
5.02 (10.18)
1.61 (1.38)
3.11
0.366
0.601
miR-362-3p
1.93 (2.05)
1.27 (0.62)
1.51
0.367
0.601
miR-886-5p
1.05 (1.37)
2.00 (2.34)
0.53
0.370
0.601
miR-483-5p
2.81 (2.79)
1.88 (2.46)
1.50
0.370
0.601
let-7c
1.56 (0.82)
1.21 (0.66)
1.29
0.375
0.605
miR-181a
2.07 (2.20)
1.28 (0.89)
1.62
0.381
0.608
miR-194
1.28 (0.40)
1.19 (0.76)
1.07
0.382
0.608
miR-425
1.29 (0.48)
1.12 (0.57)
1.15
0.385
0.611
miR-628-5p
1.83 (1.45)
1.56 (1.88)
1.17
0.406
0.636
miR-192*
1.50 (0.80)
1.19 (0.58)
1.27
0.406
0.636
miR-7-1*
1.81 (1.19)
1.46 (1.26)
1.24
0.415
0.638
miR-184
1.57 (0.94)
1.41 (1.15)
1.11
0.415
0.638
miR-96
2.11 (1.58)
1.37 (1.30)
1.55
0.421
0.638
22
Diab-13-0685 (D-RSW-04)
miR-20b
2.03 (1.93)
1.12 (0.43)
1.82
0.424
0.638
miR-23b
1.42 (0.97)
1.62 (0.83)
0.88
0.427
0.638
miR-143
1.30 (0.54)
1.17 (0.67)
1.11
0.431
0.638
miR-93*
1.42 (0.74)
1.57 (1.89)
0.91
0.433
0.638
miR-489
2.36 (3.21)
2.29 (3.14)
1.03
0.434
0.638
miR-181c
1.42 (0.69)
1.26 (0.88)
1.13
0.438
0.638
miR-19b
1.74 (1.32)
1.17 (0.54)
1.48
0.446
0.638
miR-625
1.34 (0.66)
1.18 (0.52)
1.13
0.446
0.638
miR-29b
1.47 (0.93)
1.41 (1.06)
1.04
0.446
0.638
miR-10b*
0.92 (0.44)
1.35 (1.17)
0.68
0.450
0.638
miR-454
1.73 (1.25)
1.68 (2.44)
1.03
0.451
0.638
miR-29b-2*
1.44 (0.62)
1.41 (0.93)
1.03
0.451
0.638
miR-296-5p
0.91 (0.47)
1.45 (1.25)
0.63
0.452
0.638
miR-543
1.66 (1.04)
1.38 (0.99)
1.21
0.454
0.638
miR-10b
4.12 (2.88)
3.71 (2.34)
1.11
0.454
0.638
miR-381
0.98 (0.76)
1.26 (0.79)
0.78
0.455
0.638
let-7a
1.40 (0.60)
1.24 (0.81)
1.13
0.462
0.642
miR-432
2.25 (2.27)
2.17 (3.88)
1.04
0.464
0.642
miR-493
1.73 (1.35)
1.41 (1.26)
1.22
0.483
0.663
miR-216a
1.12 (0.95)
1.20 (0.75)
0.93
0.484
0.663
miR-93
1.61 (1.12)
1.34 (1.21)
1.21
0.488
0.663
miR-127-3p
1.59 (0.90)
1.32 (0.89)
1.20
0.489
0.663
miR-30d
1.89 (1.12)
1.57 (1.51)
1.21
0.497
0.671
miR-328
1.57 (0.92)
1.39 (1.01)
1.13
0.500
0.671
miR-495
1.49 (0.76)
1.26 (0.81)
1.18
0.508
0.678
miR-140-5p
1.51 (0.82)
1.11 (0.51)
1.36
0.520
0.691
miR-27a*
1.77 (1.01)
1.64 (1.76)
1.08
0.523
0.691
miR-200a*
1.36 (0.83)
1.22 (0.88)
1.12
0.532
0.698
miR-30b
1.46 (0.92)
1.17 (0.49)
1.24
0.534
0.698
miR-181a-2*
1.79 (1.42)
1.41 (0.93)
1.26
0.538
0.700
miR-340*
1.50 (0.71)
1.62 (1.33)
0.93
0.542
0.701
miR-138-1*
1.52 (2.13)
2.25 (2.85)
0.68
0.546
0.704
miR-10a
1.37 (0.77)
1.27 (0.97)
1.08
0.549
0.704
miR-338-3p
1.14 (0.25)
1.13 (0.51)
1.01
0.553
0.706
miR-423-5p
1.31 (0.73)
1.06 (0.34)
1.24
0.558
0.708
miR-382
1.25 (0.53)
1.17 (0.63)
1.07
0.565
0.711
miR-455-3p
1.41 (0.73)
1.30 (0.82)
1.08
0.568
0.711
miR-485-3p
1.39 (0.64)
1.23 (0.75)
1.13
0.569
0.711
miR-494
1.20 (0.38)
1.25 (0.84)
0.96
0.594
0.737
miR-484
1.33 (0.73)
1.37 (1.21)
0.97
0.597
0.737
miR-100
1.45 (0.98)
1.21 (0.59)
1.20
0.598
0.737
miR-625*
1.24 (1.32)
1.20 (0.76)
1.03
0.604
0.739
miR-770-5p
1.46 (2.29)
1.59 (1.61)
0.92
0.606
0.739
miR-125b-1*
1.21 (0.46)
1.27 (0.89)
0.95
0.618
0.751
23
Diab-13-0685 (D-RSW-04)
miR-758
1.34 (0.68)
1.20 (0.66)
1.12
0.633
0.765
miR-888
1.74 (1.52)
1.24 (0.81)
1.41
0.641
0.771
miR-424
1.26 (0.62)
1.32 (0.97)
0.95
0.651
0.779
miR-629*
1.51 (1.35)
1.08 (0.49)
1.40
0.655
0.781
miR-99a
1.40 (0.96)
1.21 (0.69)
1.16
0.664
0.783
miR-126
1.04 (0.62)
1.26 (0.99)
0.83
0.666
0.783
let-7b
1.11 (0.76)
1.21 (1.03)
0.92
0.666
0.783
miR-199a-3p
1.56 (1.68)
1.12 (0.42)
1.39
0.670
0.784
miR-409-3p
1.32 (0.53)
1.53 (1.36)
0.87
0.678
0.789
miR-145*
1.51 (1.10)
1.36 (0.92)
1.12
0.682
0.789
miR-99b*
4.83 (11.72)
2.25 (2.88)
2.14
0.685
0.789
miR-146b-5p
1.16 (1.04)
1.13 (0.61)
1.03
0.687
0.789
miR-20a*
1.80 (1.87)
1.56 (1.02)
1.15
0.696
0.794
miR-30a*
1.52 (1.12)
1.11 (0.49)
1.37
0.700
0.794
miR-218
1.18 (0.47)
1.20 (0.71)
0.98
0.701
0.794
miR-539
1.69 (1.52)
1.37 (1.15)
1.23
0.709
0.798
miR-891a
1.50 (1.26)
1.21 (0.74)
1.24
0.710
0.798
miR-222*
1.20 (1.15)
1.76 (2.41)
0.68
0.720
0.805
miR-152
0.97 (0.26)
1.12 (0.46)
0.87
0.752
0.828
miR-15a*
1.26 (0.67)
1.45 (0.93)
0.87
0.752
0.828
miR-30a
1.62 (1.40)
1.45 (1.27)
1.12
0.753
0.828
miR-574-3p
1.28 (1.13)
1.45 (1.34)
0.89
0.753
0.828
miR-197
1.31 (0.75)
1.36 (1.45)
0.96
0.774
0.847
miR-150
1.19 (0.97)
1.68 (2.12)
0.71
0.783
0.853
miR-628-3p
1.19 (0.52)
1.32 (1.09)
0.90
0.794
0.862
miR-216b
2.01 (1.82)
1.30 (1.07)
1.54
0.810
0.875
miR-335*
1.12 (0.71)
1.25 (0.75)
0.90
0.825
0.888
miR-125a-3p
2.48 (1.78)
1.72 (1.21)
1.44
0.833
0.893
miR-145
1.07 (0.57)
1.18 (0.75)
0.91
0.842
0.899
miR-339-5p
1.26 (0.56)
1.08 (0.43)
1.17
0.862
0.913
miR-541
1.72 (1.54)
1.32 (0.95)
1.31
0.863
0.913
miR-214
1.13 (0.76)
1.14 (0.60)
1.00
0.866
0.913
miR-337-5p
1.12 (0.40)
1.46 (1.28)
0.76
0.874
0.914
miR-130b
1.06 (0.48)
1.20 (0.72)
0.88
0.875
0.914
miR-509-3p
1.10 (0.52)
1.40 (1.04)
0.78
0.888
0.921
let-7g
1.13 (0.45)
1.11 (0.45)
1.03
0.891
0.921
miR-370
1.17 (0.63)
1.18 (0.74)
0.99
0.892
0.921
miR-148a
1.19 (0.85)
1.51 (1.26)
0.79
0.906
0.932
miR-671-3p
1.28 (0.86)
1.11 (0.49)
1.15
0.923
0.944
miR-433
1.16 (0.57)
1.37 (1.14)
0.84
0.926
0.944
miR-214*
1.39 (1.32)
1.21 (0.57)
1.15
0.944
0.959
miR-193b
1.26 (0.90)
1.08 (0.48)
1.16
0.970
0.982
miR-193a-3p
1.36 (1.31)
1.42 (1.15)
0.96
0.987
0.995
miR-182
1.14 (0.63)
1.34 (1.22)
0.85
0.992
0.996
24
Diab-13-0685 (D-RSW-04)
let-7d
1.19 (0.80)
1.11 (0.56)
1.07
0.998
0.998
564
25