Circulating microRNA-144-5p is associated with depressive disorders
Wang et al. Clinical Epigenetics
Circulating microRNA-144-5p is associated with depressive disorders
Xiao Wang 0 2
Kristina Sundquist 0 1 2
Anna Hedelius 0 2
Karolina Palmér 0 2
Ashfaque A. Memon 0 2
Jan Sundquist 0 1 2
0 Center for Primary Health Care Research, Lund University/Region Skåne , Malmö , Sweden
1 Stanford Prevention Research Center, Stanford University School of Medicine , Stanford, CA , USA
2 Center for Primary Health Care Research, Lund University/Region Skåne , Malmö , Sweden
Background: Depressive/anxiety disorders are the most common types of mental illnesses in the world. The present study was the first to explore the association between plasma microRNAs (miRNAs) and depression/anxiety in primary care patients. Results: In total, 169 patients (aged 20-64 years) from 16 primary health centers were enrolled in the present study. The healthy controls were consisted of 52 individuals. We first performed miRNA screening of plasma samples from 11 patients using a Serum/Plasma Focus microRNA Panel comprising 179 miRNA primer sets. Six miRNAs were differentially expressed and were then validated by quantitative real-time (qRT)-PCR in the entire study cohort. The mean plasma miR-144-5p level in the depression/anxiety patients increased significantly compared to baseline (p < 0.0001) after the 8-week follow-up. No significant associations were found between the differentially expressed miRNAs and a change in the Montgomery-Åsberg Depression Rating Scale (MADRS-S) score after the follow-up. In linear regression analysis, the plasma miR-144-5p expression level was inversely related to the depression score (MADRS-S) (β = −0.02, p < 0.01), after adjustment for sex and age, at baseline. In addition, plasma miR-144-5p levels at baseline in the depression/anxiety patients were significantly lower compared with the healthy controls (p < 0.001). Conclusions: Our findings show that plasma miR-144-5p levels are associated with depressive symptoms. Although confirmatory analyses are required, plasma miRNA-144-5p is a potential peripheral biomarker for pathologic processes related to depression.
miR-144-5p; Depression; Severity
Depression and anxiety are common mental disorders.
The World Health Organization (WHO) has predicted
that depression will become the second leading
contributor to disease burden by the year 2020 [
]. In different
European studies, approximately 15 to 32 % of patients
who visited primary health care centers showed symptoms
of depression, anxiety, and stress-related disorders [
These in primary health care common psychiatric
disorders are associated with poor quality of life and reduced
life expectancy, placing a large economic burden on
society. Several studies have explored the underlying
pathophysiological and etiological mechanisms [
Current evidence suggests that the onset and progression
of depression appear to be influenced by multiple factors,
including neurotransmitter systems [
], stress [
and genetic susceptibility [
]. However, the
molecular pathophysiology underlying depression and anxiety
is still not fully understood. In recent years, the
emergence of small noncoding RNAs as regulators of
gene expression have gained much attention in various
disease pathophysiologies. MicroRNAs are some of the
most studied and well characterized among small
noncoding RNAs. [
MicroRNAs (miRNAs) are a class of small
(21-23nucleotide), noncoding, single-stranded RNAs that
inhibit gene expression by promoting messenger-RNA
(mRNA) degradation or inhibiting translation [
They influence a variety of physiological cell processes
during development and tissue homeostasis by
regulating the expression of around 90 % of all human
]. MicroRNAs are highly expressed in
neurons, where they regulate processes of brain
development, including neurogenesis, neuronal proliferation,
metabolism, and apoptosis [
]. Aberrant expression or
dysregulation of miRNA processing has been linked to
many neurological and psychiatric diseases. Studies in
postmortem brains from patients with Alzheimer’s
disease, schizophrenia, bipolar disorder, and major
depressive disorders showed alterations in several
]. Recently, numerous miRNAs have
been detected in several body fluids, including serum,
plasma, and cerebrospinal fluid (CSF) . The profile
of circulating miRNAs changes significantly under
pathological conditions compared to healthy
conditions. Circulating miRNAs can be secreted from cells
into the blood in different ways—enclosed in exosomes
or associated with proteins [
]. They are resistant
to nuclease digestion, and can be measured
reproducibly, which makes them attractive as potential
Over the past several years, circulating miRNAs, as
potential biomarkers, have been well documented in
many diseases, including cancer, diabetes, and
psychiatric diseases [
]. Recently, two studies have
examined circulating miRNA expression in the blood in
patients with major depressive disorder [
found some dysregulated miRNAs. However, these
studies only had small numbers of patients with a focus
on major depressive disorders. A systematic analysis of
plasma miRNAs in depression and anxiety has so far not
The present study builds on a previously published
randomized controlled trial by our group, where we included
patients with depression/anxiety from 16 primary health
care centers [
] in Sweden in order to compare the
effects of mindfulness treatment with treatment as usual on
depressive/anxiety symptoms. In the present study, we
hypothesized that patients with depression/anxiety would
experience a significant change in plasma miRNAs after
an 8-week treatment and that there would be an
association between plasma miRNA levels and symptoms of
depression/anxiety at baseline. Using a broad miRNA
screening panel, we identified a group of miRNAs whose
expression differed between baseline and follow-up using
plasma samples from 11 patients who showed a decline
in the Montgomery-Åsberg Depression Rating Scale
(MADRS-S) total score of at least 50 % between
baseline and follow-up. The first aim was to examine whether
the miRNAs that were identified as differentially expressed
after treatment were associated with a response to
treatment. The second aim was to examine the association
between these differentially expressed miRNAs and
symptoms of depression/anxiety at baseline.
The clinical characteristics of the patients are shown in
Table 1. The mean age in the whole group was 42 years
and more of the participants were women. Most
antidepressants prescribed at primary health care centers in
Sweden are selective serotonin reuptake inhibitor
(SSRIs) (e.g., citalopram, fluoxetine, and sertraline, data
not shown). The median scores at baseline indicated
mild to moderate symptoms of depression and/or
anxiety. After treatment, the median scores decreased,
indicating none to mild symptoms. A total of 39 % of
the patients had a decrease in MADRS-S score of at
least 50 % after the 8-week treatment compared to
baseline (data not shown in tables).
miRNAs screening data
To increase the chances of identifying miRNAs that are
associated with treatment response, we performed initial
screening of plasma from 11 patients with at least a
IQR interquartile range, SD standard deviation
a16 patients had missing data for antidepressants, and 24 had missing data
50 % decline in MADRS-S total score at follow-up. Six
candidate miRNAs with a >1.5 fold differential
expressions between baseline and follow-up were selected
(Additional file 1: Table S1): miR-144-5p, miR-92b-3p,
miR-885-5p, miR-30a-5p, miR-29a-5p, and miR-29b-2-5p.
These six miRNAs were then measured in the entire
cohort. Five of them were detectable in all samples, but
miR-885 was not suitable for further analysis as more than
20 % of patients had Ct values of >35.
The selected five miRNAs were validated in all of the
169 patients at baseline and follow-up. Additional file 2:
Figure S1 shows that after treatment, the mean plasma
miR-144-5p level and miR-30a-5p in the
depression/anxiety patients increased significantly compared to
baseline, (p < 0.0001 and p = 0.007, respectively). Moreover,
to assess the potential association between candidates’
miRNAs and treatment response, we performed linear
regression analysis between miRNAs changes (ΔΔCt)
and MADRS score change in the patients. However, no
significant associations were found between the changes
of those selected miRNAs and MADRS-S score change
after treatment (Table 2).
Associations of miRNAs with symptoms of depression/ anxiety (cross-sectional study)
Unadjusted linear regression revealed that plasma
miR144-5p expression level at baseline was inversely related
to MADRS-S score at baseline (β = −0.02, p < 0.01)
(Table 3). Adjustment for sex and age did not affect
these values. The lower the plasma miR-144-5p
expression levels were the higher was the depression score.
Like miR-144-5p, miR-29a-5p showed a significant
positive association with MADRS-S score (β = 0.02,
p = 0.04) in the unadjusted regression. After adjustment
for age and sex, the p value (0.06) no longer remained
significant (Table 3). We also corrected for multiple
testing according to Bonferroni. We adjusted the individual
p value for each of these five miRNAs and found that
the association between miR-144-5p and the MADRS-S at
baseline was still significant. The relationships between
miR-144-5p and the scores at baseline are also shown for
the other two scales (Additional file 1: Table S2a, b). The
overall pattern for the other two scales was similar to the
results for MADRS-S.
Comparison of miR-144-5p expression in depression/anxiety patients and healthy controls
Figure 1 shows the relative plasma miR-144-5p
expression levels in the healthy controls and the depression/
anxiety patients before and after the 8-week treatment.
The relative miR-144-5p expression levels at both
baseline and after treatment were significantly lower in the
depression/anxiety patients than in the healthy controls
(p < 0.001). In the depression/anxiety patients, the mean
plasma miR-144-5p level increased significantly after
treatment compared to baseline (p < 0.0001).
To our knowledge, this is the first study to investigate
the expression of miRNAs in primary health care
patients with depression/anxiety. Our major findings are
that plasma expression levels of miR-144-5p were
inversely associated with depression scores at baseline and
that plasma miR-144-5p levels in depressive patients
were significantly lower than in healthy controls. These
findings suggest that miRNA-144-5p may reflect the
pathologic processes of depression. Moreover, we found
that plasma levels of miRNA-144-5p were significantly
higher after treatment compared to baseline levels in
depression/anxiety patients. No significant associations
were found between the changes of those differentially
expressed miRNAs and MADRS-S score change after
treatment (response analysis).
The finding of an inverse correlation between plasma
miR-144-5p levels and the MADRS-S at baseline was
also present for the other two depression scores, i.e.,
Hospital Anxiety and Depression Scale (HADS)-D and
PHQ-9; however, the results for HADS-D and PHQ-9
did not reach statistical significance. We applied
correction for multiple testing according to Bonferroni, and
the lack of significance for HADS-D and PHQ-9 may be
due to the sample size in combination with the Bonferroni
correction. An advantage with the MADRS-S is that it is a
commonly used scale for depression [
], which is
sensitive to discriminate the severity of depression and it is
therefore preferred in studies where the intention is to
measure core depressive symptoms and states .
miR-144-5p (n = 160)
miR-29a-5p (n = 153)a
miR-29b-2-5p (n = 153)a
miR-30a-5p (n = 153)a
miR-92b-3p (n = 160)a
aInformation (clinical or qRT-PCR) is missing for some patients (Tables 2 and 3)
aInformation (clinical or qRT-PCR) is missing for some patients (Tables 2 and 3)
The presentation of depressive syndromes is
heterogeneous. Thus, identifying patients with depression using
only depression scores or patient self-report may lead to
a delayed or even wrong diagnosis [
]. It is thus
necessary to identify stable biomarkers to help with the
clinical diagnosis of depression and elucidate potential
biological mechanisms. A stable biomarker that is easily
assessed in peripheral blood might be an ideal and
objective measure to diagnose or evaluate the stage of
depression . Such biomarkers are still lacking.
Circulating miRNAs are stable and easily accessible, which may
enable their application in daily clinical practice. Studies
have reported that circulating miRNAs are released from
specific cells and transferred to recipient cells to exert
their function [
]. Furthermore, aberrant expression of
circulating miRNAs was demonstrated to be related to
tissue injury [
], which raises the possibility of
bloodbased miRNA profiles as fingerprints of diseases. Based on
the evidence described above, the notion of circulating
miRNAs as biomarkers for various diseases has been
Strengths of our study are that the present results were
obtained from an randomized controlled trial (RCT)
including a total of 169 patients, who are representative of
patients in primary health care. Furthermore, this is the
first study to provide evidence that a specific miRNA may
serve as a potential biomarker for the diagnosis of
depression. Presently, the role of circulating miR-144-5p in
depressive disorders remains unclear. However, there are
a few biological mechanisms that can explain our finding.
MiR-144 has broad expression and is enriched in brain,
as well as in normal and malignant hematopoietic cells
and tissues [
]. It is highly conserved and has multiple
predicted targets in both humans and rats [
studies have demonstrated that miR-144 is involved in the
response to mood stabilizer treatment [
], and aging diseases . Long-term
treatment with the mood stabilizers lithium and valproate
significantly up-regulates miR-144 expression in the rat
hippocampus, which is one of the brain regions involved
in mood regulation. Signaling pathways targeted by
miR144 include the protein kinase C (PKC), Wnt/β-catenin,
and PTEN pathways [
]. Some of them have been shown
to be involved in the development of depression [
In addition, miR-144 was reported to be selectively
upregulated in the aging brain of humans and was suggested
to have neuroprotective functions. miR-144 can inhibit
the expression of ataxin 1 (ATXN1) in human cells, and a
search of the Genetic Association Database shows that
ATXN1 is associated with mental disorders, such as
bipolar disorder, schizophrenia, and major depressive disorder
. It is known that stress is an important risk factor for
depression. In one study, circulating miR-144* level
changed with stress associated with the nationally administered
examination for academic promotion in healthy young
adults: miR-144* level was significantly increased after the
examination compared to before the examination [
However, there was no correlation between miR-144*
level and anxiety scores [
], which is consistent with
our findings (Additional file 1: Table S2b). We assume
that the increased circulating miR-144 level may reflect
changes in miR-144 expression in the brain, which may
in turn be associated with the progression of
depression. The psychopathological mechanisms of the effect
of miR-144 on depression remain to be elucidated. Our
results, however, is a complement to the recent findings
that decreased levels of miR-144* are associated with
several diseases [
Our study is the first RCT to explore the association
of circulating miRNA expression in depression. Despite
our promising results, there are several general
limitations to our experimental design that should be
considered when interpreting our results. First, we included
patients with depression as well as anxiety. Overlapping
symptoms could affect our findings. However,
overlapping symptoms are relatively common among these
conditions and our approach is in line with previous studies.
Second, in the initial screening analysis, we ran one well
instead of duplicates/triplicates for each sample.
However, when we setup technical replicates (duplicates or
triplicates) in our laboratory, the technical variation was
very small. Another limitation is that only six miRNAs
were selected from the initial miRNA screen and
validated in the whole cohort, suggesting that other
miRNAs that are potentially associated with depression
scores could have been missed in the current study.
However, these selected miRNAs were the only miRNAs
that were differentially expressed. In addition, this is the
first study to examine miRNAs’ potential association
with depression/anxiety, and there is so far no available
standard for selection of candidate miRNAs for
screening. In addition, the correlation between plasma
miR144-5p expression level and miR-144-5p in the brain is
unknown, as is the source of plasma miR-144-5p.
In summary, our findings show that plasma
miR-1445p levels are associated with depressive symptoms in a
primary health care setting. In further studies, plasma
miR-144-5p levels need to be measured in patients in
other clinical settings, including major depressive
disorders, to examine the validity of our findings. It will also
be of interest to examine whether the expression of
exosomal miR-144-5p is related to depression scores. In
addition, it would be informative to test the predictive
value of miR-144-5p in a larger cohort of patients with
Study subjects and sample collection
The study subjects were recruited from 16 primary
health care centers in a RCT of mindfulness group
therapy compared to treatment as usual. The RCT included
a group of patients with depression, anxiety or stress,
and adjustment disorder. A detailed description of the
study design is provided in our previous article [
overall aim of the RCT was to compare the effect of a
structured mindfulness-based group therapy program
with treatment as usual (mostly individual cognitive
behavioral therapy (CBT)) [
]. Briefly, patients were
recruited between 4 Jan 2012 and 22 March 2012 at the
16 primary health centers in urban and rural settings in
Skåne, in the southernmost part of Sweden. The
inclusion criteria were as follows: age 20–64 years and a score
of ≥10 on the Patient Health Questionnaire (PHQ)-9
or ≥7 on the HADS or a total score on the MADRS-S of
between 13 and 34 (mild to moderate depression). A
total of 135 patients met the inclusion criteria for
MADRS-S, 118 for HADS-A, 159 for HADS-D, and 111
for PHQ-9. The rationale for using multiple scales to
assess symptoms of depression and anxiety was that
different scales are used in clinical practices worldwide
]. Eligible patients had a clinical diagnosis of
depression, anxiety or stress, and adjustment disorder,
according to the International Classification of Diseases
(ICD)10 criteria. All clinical diagnoses were made by doctors
at the 16 primary health care centers. The exclusion
criteria were as follows: severe personality disorder, risk
of suicide, pregnancy, thyroid disease, current
psychotherapy of any kind, and participation in any other
psychiatric intervention study. The clinical characteristics
and miRNA distributions of the total study population are
presented in Table 1. In total, 169 patients (86 % females,
13 % males, 1 % gender not specified) aged 42 ± 11 years
(mean ± standard deviation) were enrolled in the present
study. All the patients were fluent in Swedish, and 90 %
were born in Sweden and/or had at least a high school
degree (not shown in table). Each patient filled in three
self-rated questionnaires (above mentioned PHQ-9,
HADS-A/HADS-D, and MADRS-S) at baseline and after
8 weeks of follow-up. The patients received
antidepressants and tranquilizers if deemed necessary. Blood
samples were collected at the same time as the assessment of
self-rated symptoms before and after treatment. The
control group was consisted of 52 healthy individuals (74 %
females, 26 % males) aged 47 ± 12 years. They were
recruited from all types of personnel groups among the
For patients, whole blood (6 mL) was collected from
each participant in EDTA tubes. Blood samples were
centrifuged at 2000 g for 10 min at 4 °C, and the plasma
was then aliquoted and stored at −80 °C before further
processing. For healthy controls, whole blood was
collected in buffered citrate tubes and centrifuged at 2000 g
for 20 min at room temperature. Blood samples were
processed and plasma frozen within 8 h of collection.
Sampling strategy and miRNA analysis
For the miRNA screening, 11 individuals (8 females and 3
males, age 41 ± 9. 2 years) with a minimum reduction in
MADRS-S score of 50 % from the initial evaluation were
selected. We selected those individuals who showed the
largest change in the magnitude of the depressive
symptoms. We assumed that a linear change in the magnitude
of the depressive symptoms would also be associated with
the magnitude of the depressive/anxiety symptoms at
We used MADRS-S score as our selection criteria
because it is a commonly used scale for depression [
]. The individuals in this initial screening analysis
were chosen to resemble the whole group as much as
possible, based on sex and age. Fifty microliter of total
RNA was isolated from 200 μl of plasma using the
Qiagen miRNeasy Mini Kit (Qiagen GmbH, Hilden,
Germany) according to the manufacturer’s protocol,
with minor modifications. miRNAs were reverse
transcribed using a Universal cDNA Synthesis kit (Exiqon,
Vedbaek, Denmark). The resulting reverse transcription
reaction product was stored at −20 °C before analysis. A
detailed description of the methodology is provided in a
previous article . miRNA expression was screened
using a Serum/Plasma Focus microRNA PCR Panel
(Exiqon) comprising 179 LNA™ microRNA primer sets
focusing on serum/plasma-relevant human miRNAs.
Quantitative real-time PCR (qPCR) was conducted using
a CFX384 Real-Time PCR Detection System (Bio-Rad).
Undetectable data were assigned a default threshold
cycle (Ct) value of 36. A mean of 172 miRNAs were
detected in the 11 samples. As there is no current
consensus as to an appropriate reference miRNA for the
normalization of plasma miRNAs in qPCR analysis, all
qPCR data were normalized to the Ct average of all
miRNA measurements for each sample [
Comparison of the 172 miRNAs in the 11 samples revealed six
miRNAs with >1.5 fold differential expression between
baseline and follow-up (Additional file 1: Table S1). We
used a >1.5 fold change between baseline and follow-up
as the cutoff, which is based on several previous
profiling studies confirming that a >1.5 fold change in miRNA
expression can have a significant impact on the biology
of the cell [
]. These six miRNAs were then
measured in duplicate by qPCR in the whole cohort
(n = 169). miR-451a and miR-23a-3p were used to test
hemolysis in plasma samples [
]. The Ct values were
normalized according to the ΔCt method with the
internal controls miR-425-5p and miR-186-5p. The
normalization stability of those two miRNAs was
confirmed with geNorm software [
]. Ct values were
normalized to miR-425-5p and miR-186-5p using the
following equation: ΔCt = CtmiR-425-5p&miR-186-5p
(geometric mean of two miRNAs) − CtmiR of interest. Relative
expression was calculated as 2ΔCt.
The study was performed according to the principles of
the Declaration of Helsinki. It was reviewed and
approved by the Ethics Committee of Lund University,
prior to its commencement, on 5 October 2011
(application no. 2011/491). Written informed consent
was obtained from all participants.
For the miRNA screening analysis, we tested the difference
between baseline and follow-up using the Wilcoxon
signed-rank test due to the nature of data, repeated
measurements and nonnormality (Additional file 1: Table S1).
MADRS-S, HADS-D (depression), HADS-A (anxiety), and
PHQ-9 scores are presented as the median and
interquartile range (IQR); age is presented as the mean and standard
deviation (SD); and sex and antidepressant and tranquilizer
use are presented as numbers and percentages (Table 1).
The mean and 95 % confidence interval (CI) at baseline
and follow-up are shown for plasma miR-144-5p levels. A
paired t test was used to test the difference between
baseline and follow-up (Additional file 2: Figure S1). The
association between change in miRNA levels and treatment
response was tested using linear regression, adjusted for
MADRS-S at baseline (Table 2). Linear regression was also
used to examine the associations between miRNA levels at
baseline and MADRS-S, HADS-D, HADS-A, and PHQ-9
scores (Table 3 and Additional file 1: Table S2a-S2c). We
considered important potential confounders to be sex, age,
drug treatment (antidepressant and/or tranquilizer), body
mass index (BMI), and smoking. When adjusting the
models for these variables, the beta-coefficients did not
change much (at the most 5 %) and no p values were
significant for drug treatment, BMI, or smoking. Hence,
we kept in the final models only sex and age as adjusting
Linear regression analyses (adjusted for age and sex)
were used to test the difference of miR-144-5p levels
between healthy controls and patients at baseline and after
treatment (Figure 1). STATA version 12 (StataCorp LP)
was used for all statistical analyses.
Additional file 1: Supplementary Tables. Table S1. six candidate
miRNAs with >1.5 fold differential expression between baseline and
follow-up and changes in more than half of the samples (n≥6) were
selected for further validation. Table S2a and Table S2b. The relationships
between miR-144-5p and two depression scores at baseline, HADS-D
(β=-0.02, p=0.02) and PHQ-9 (β=-0.02, p=0.06) were also significant or
borderline significant, whereas the association with HADS-A score
(β=-0.01, p=0.37) was non-significant (Table S2c).
Additional file 2: Figure S1. Supplementary Figure. Plasma 5 miRNAs
levels (determined by the 2ΔCt method)changed after treatment. Data
are shown as the mean and 95 % CI. P calculated with paired t-test
ATXN1: ataxin 1; BMI: body mass index; CBT: cognitive behavioral therapy;
CI: confidence interval; HADS: Hospital Anxiety and Depression Scale;
ICD-10: International Classification of Diseases 10; IQR: interquartile range;
MADRS-S: Montgomery-Åsberg Depression Rating Scale; miRNAs: microRNAs;
PHQ-9: Patient Health Questionnaire; PKC: protein kinase C;
qPCR: quantitative real-time; RCT: randomized controlled trial; SD: standard
deviation; SSRIs: selective serotonin reuptake inhibitor.
The authors declare that they have no conflict of interests.
XW conducted the molecular studies, designed and performed the
experiments and data analyses, and drafted the manuscript. AH and AAM
participated in the experimental design and running and helped draft the
manuscript. KP participated in data analyses and helped draft the
manuscript. JS and KS conceived the study, supervised the work, and were
responsible for manuscript edits. All authors read and approved the final
version of the manuscript.
This project was supported by the Swedish Research Council and FORTE to
Jan Sundquist, the Swedish Research Council to KS (K2012-70X-15428-08-3),
as well as the ALF funding from the Region Skåne awarded to JS and KS. We
would like to thank science editor Stephen Gilliver for critical reading of the
manuscript as well as to Professor Björn Dahlbäck and Cecilia Frej for their
support with this manuscript.
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