Association between circulating miRNAs and spinal involvement in patients with axial spondyloarthritis
Association between circulating miRNAs and spinal involvement in patients with axial spondyloarthritis
KlaÂ ra PrajzlerovaÂ 0 1
KristyÂ na GrobelnaÂ 0 1
MarkeÂ ta HusÏ aÂ kovaÂ 0 1
SÏ aÂ rka ForejtovaÂ 0 1
Astrid JuÈ ngel 1
Steffen Gay 1
JiřÂõ VencovskyÂ 0 1
Karel Pavelka 0 1
Ladislav SÏenolt 0 1
MaÂ ria FilkovaÂ 0 1
0 Institute of Rheumatology and Department of Rheumatology, 1st Faculty of Medicine, Charles University , Prague , Czech Republic , 2 Center of Experimental Rheumatology, University Hospital Zurich , Zurich , Switzerland
1 Editor: Yun Zheng, Kunming University of Science and Technology , CHINA
Dysregulation of miRNAs and their target genes contributes to the pathophysiology of autoimmune diseases. Circulating miRNAs may serve as diagnostic/prognostic biomarkers. We aimed to investigate the association between circulating miRNAs, disease activity and spinal involvement in patients with axial spondyloarthritis (AxSpA).
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was financially supported by a
project of the Ministry of Health of the Czech
Republic for conceptual research development
number 023728 and grant project 17-33127A.
There is no financial support or other benefits from
commercial sources for the work reported on in
Total RNA was isolated from the plasma of patients with non-radiographic (nr)AxSpA,
patients with ankylosing spondylitis (AS) and healthy controls (HC) via phenol-chloroform
extraction. A total of 760 miRNAs were analysed with TaqMan® Low Density Arrays, and
the expression of 21 miRNAs was assessed using single assays.
Comprehensive analysis demonstrated the differential expression of miRNAs among
patients with progressive spinal disease. Of the 21 miRNAs selected according to their
expression patterns, the levels of miR-625-3p were significantly different between nr-AxSpA
patients and HCs. We found no correlation between miRNA levels and Bath Ankylosing
Spondylitis Disease Activity Index (BASDAI) in nr-AxSpA patients. Selected miRNAs, such
as miR-29a-3p, miR-146a-5p or miR-222-3p with an established role in extracellular matrix
formation and inflammation were associated with spinal changes and/or disease activity
assessed by BASDAI in AS patients, including miR-625-3p reflecting disease activity in AS
with spinal involvement.
Our data indicate that circulating miRNAs play a role in the pathogenesis of AxSpA and are also suggestive of their potential as biomarkers of disease progression. We hypothesize
Competing interests: The authors have declared
that no competing interests exist.
that differential systemic levels of miRNA expression reflect miRNA dysregulation at sites of
spinal inflammation or bone formation where these molecules contribute to the development
of pathophysiological features typical of AxSpA.
Axial spondyloarthritis (AxSpA) is a chronic inflammatory disease that mainly affects the
axial skeleton, sacroiliac joints and entheseal spinal structures. It encompasses patients with
ankylosing spondylitis (AS) with radiographic sacroiliitis and syndesmophytes, as well as
patients with early or abortive forms of spondyloarthritis (SpA) characterized by the presence
of sacroiliac inflammation detected by magnetic resonance imaging (MRI) or the presence of
HLA-B27 in combination with features characteristic of SpA [
]. AS is an inflammatory
disease characterized by new bone formation. Mononuclear cells and osteoclasts initiate local
osteitis, which leads to cartilage erosion and bone destruction, as well as osteoblast
differentiation and subsequent syndesmophyte formation [
Inflammation develops several years before structural damage becomes visible on plain
radiographs. Although patients may have longstanding symptoms, the diagnosis of AS based
on the modified New York criteria delays early treatment, as radiographic sacroiliitis
represents a late sign of disease. Therefore, the Assessment of SpondyloArthritis International
Society (ASAS) developed new classification criteria for the diagnosis of AxSpA that takes
nonradiographic (nr-AxSpA) findings into account [
]. Shorter disease duration, younger age,
elevated baseline C-reactive protein (CRP) levels and active inflammatory changes involving the
sacroiliac joint are associated with better responses to anti-TNF therapy in patients with
]. Therefore, early diagnosis, disease monitoring and therapeutic response prediction
are very important.
Several biomarkers have been tested regarding their usefulness in diagnosing disease,
monitoring disease activity and predicting therapeutic responsiveness but have thus far not been
implemented in clinical practice [
]. HLA-B27 remains the best genetic biomarker for
diagnosing AxSpA, and CRP remains the best circulating marker for assessing disease activity and
predicting treatment responsiveness and structural progression [
MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional
regulators of gene expression. Altered miRNA expression and target gene dysregulation have
been shown to contribute to the pathophysiology of many autoimmune diseases, including
rheumatic diseases [
]. Although the (patho) physiological roles of circulating miRNAs remain
largely unknown, cell-free circulating miRNAs appear to be promising disease biomarkers [
While rheumatoid arthritis (RA) has been extensively investigated, comprehensive studies
regarding miRNAs in patients with AxSpA are lacking.
The aim of the present study was to identify circulating miRNAs in patients with AxSpA
and to investigate their association with disease characteristics, including spinal disease
Material and methods
This study included 20 patients with nr-AxSpA, 24 AS patients with isolated sacroiliitis
without spinal involvement (AS stage I), 24 patients with AS with spinal involvement (presence of
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syndesmophytes, AS stages II-V), including 7 patients with a bamboo spine, and 29 healthy
controls (HC). Radiographic staging was performed as previously described [
]. All patients
fulfilled the 2011 ASAS classification criteria for the diagnosis of AxSpA [
]. Disease activity
was assessed using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) [
CRP. The clinical characteristics of the patients and HCs are shown in Table 1. Patients were
recruited from the outpatient clinic of the Institute of Rheumatology, Prague in 2013±2014.
Written informed consent was obtained from all participants prior to enrolment, and the
study was approved by the local Ethics committee at the Institute of Rheumatology in Prague.
Samples and RNA isolation
Whole blood samples collected to EDTA tubes were obtained from all participants and plasma
was separated by centrifugation within 4 hours of collection ensuring constant pre-analytical
condition for all samples. All plasma samples were stored at -80ÊC and experienced no
freezethaw cycles before use. Total RNA was extracted from plasma samples using
phenol-chloroform extraction, as previously described [
]. Briefly, 500 μl of plasma was homogenized with
500 μl of Trizol LS reagent (Thermo Fisher Scientific, Waltham, MA, USA) and then
centrifuged at 12,000 × g for 10 minutes at 4ÊC. Three cycles of acid phenol-chloroform (Thermo
Fisher Scientific) extraction were performed. RNA was precipitated by adding 5μg of
RNasefree glycogen (Roche Diagnostics, Mannheim, Germany) and 100% isopropanol and then
incubated for 10 minutes at room temperature before being centrifuged at 12,000 × g for 10
minutes at 4ÊC. The pellet was washed with 75% ethanol, and RNA was dissolved in
RNasefree water. Three synthesized C. elegans miRNAs, cel-miR-39, cel-miR-54 and cel-miR-238
(Integrated DNA Technologies, Coralville, IA, USA), 25 fmol each, were spiked into plasma
samples after denaturation and served as internal calibrators, as previously described [
RNA sample quality control was initially performed using Agilent 2100 Bioanalyser with
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Agilent Small RNA kit (Agilent, CA, USA) as RNA isolation quality measure and then using a
NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific) in remaining samples.
First, twenty non-pooled individual samples (5 samples from each group) were analysed using
a TaqMan1 Low Density Array (Thermo Fisher Scientific). Complementary DNA was
obtained by reverse transcription using a TaqMan1 MicroRNA Reverse Transcription Kit
with Megaplex RT Primers with equal RNA input. cDNA was preamplified using 2x
TaqMan1 PreAmp Master Mix and Megaplex™ PreAmp Primers (all Thermo Fisher Scientific) on
a PCR thermocycler (Bio-Rad Laboratories, CA, USA). The expression of 760 miRNAs was
measured using Human Pool A+B TaqMan1 Low Density Array platforms for microRNAs
on a 7900RT-PCR thermocycler (Thermo Fisher Scientific). All steps were performed
according to the manufacturer's instructions. Data were analysed with RQ Manager Software (Life
Technologies). The dCt method was used for relative quantification as follows: dCt = Ct(array
average)-Ct(miRNA of interest), followed by x-fold change calculations.
All miRNAs exhibiting a minimum 1.5 mean fold difference in expression between at least
2 groups according to across-group comparisons (HC vs. nr-AxSpA vs. AS) were taken
forward for pathway analysis and literature search as explained below. In total, 21 miRNAs were
selected for further validation using single assays. Total RNA from the remaining non-pooled
samples was reverse-transcribed using TaqMan Real Time miRNA specific primers (including
primers for cel-miR-39, cel-miR-54 and cel-miR-238) and then amplified by real-time PCR
with TaqMan probes and TaqMan Universal PCR Master Mix on a 7900RT-PCR thermocycler
(Thermo Fisher Scientific). Data were analysed with RQ Manager Software (Thermo Fisher
Scientific). The dCt method was used for relative quantification as follows: dCt = Ct(spike-in
average)-Ct(miRNA of interest); therefore, higher dCt values represent higher expression
levels of particular miRNAs.
Data are expressed as the mean±SD. One-way ANOVA with post-hoc comparison for multiple
comparisons or unpaired T test (with Welch's correction in case of homogeneity assumption
violations) for comparisons between 2 groups were used where applicable. Pearson's
correlation coefficient was used to correlate any two variables. P values less than 0.05 were considered
statistically significant. All analyses and graphs were performed and generated, respectively,
using GraphPad Prism 5.02 (GraphPad Software, La Jolla, CA).
First, DIANA-mirPath tool was used to analyze clustering of miRNAs and pathways. In the
next step, an online search (PubMed, performed in March 2016) of the functions of miRNAs
was performed and 21 miRNAs with a hypothesized role in the pathogenesis of AxSpA were
Comprehensive analysis of circulating miRNAs
A comprehensive screening of 760 miRNAs was performed using TaqMan Low Density
Arrays, as described above. Only miRNAs expressed in all 5 samples were taken forward for
the analysis. Overall, 162 miRNAs were detected in HCs, 154 miRNAs were detected in
patients with nr-AxSpA, 110 miRNAs were detected in patients with sacroiliitis, and 110
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miRNAs were detected in AS patients with spinal involvement (AS II-V). Of those miRNAs,
92 were detected in all tested samples, 10 were detected in HCs, nr-AxSpA patients and AS
patients with sacroiliitis and 25 were shared by HC and nr-AxSpA patients (S1 Fig). We found
no miRNA unique to AxSpA.
Using the approach described in the Methods, miRNAs exhibiting a minimum 1.5 mean
fold difference in expression between at least 2 groups according to across-group comparisons
(HC vs. nr-AxSpA vs. AS with sacroiliitis and with spinal involvement) were considered for
further analysis (S1 Table).
DIANA mir-Path cluster analysis and literature review enabled selection of 21 miRNAs for
further validation (Fig 1, S2 Table).
Differential expression of circulating miRNAs between HC and patients with AxSpA
As mentioned above, 21 selected miRNAs were analysed using single assays to confirm their
differential expression (S3 Table).
The expression was compared between HC and patients with AxSpA as follows:
Significantly lower expression (from 1.6 to 3.9 times) of 14 miRNAs most of which are
involved in osteoblast differentiation or the Wnt signalling pathway, were noted in all patients
with AxSpA irrespective of patient radiographic findings compared to HC (Table 2, extended
results shown in S4 Table).
As the group of AxSpA is heterogeneous, we next compared patients with AxSpA according
radiographic damage with HC (Table 2, S4 Table, Fig 2):
In patients with nr-AxSpA, only miR-625-3p appeared significantly different and exhibited
2.3 times lower expression levels than in HC. Eighteen miRNAs exhibited 2.1 to 5.6 times
lower expression levels in radiographic disease irrespective of spinal involvement than in HC,
and 14 miRNAs were 2.0±3.9 times lower in AS patients than in patients with
non-radiographic disease (Table 2, S4 Table).
These results indicate that some differences exist in the levels of circulating miRNAs
between HC and patients with non-radiographic disease, while more differences exist at
radiographic stage reflecting bony changes in patients with more advanced disease.
Effects of spinal involvement on circulating miRNA expression
Next, we evaluated the differences in circulating miRNA levels between patients with
nrAxSpA and definite radiographic disease in patients with isolated sacroiliitis and with spinal
involvement (classified as AS II-V) as follows (Fig 2):
nr-AxSpA vs. AS: The vast majority of miRNAs (miR-19a-3p, miR-24-3p, miR-27a-3p,
miR-29a-3p, miR-106a-5p, miR-140-3p, miR-146a-5p, miR-146b-5p, miR-151a-3p,
miR-2213p, miR-223-3p, miR-374a-5p) exhibited 2.0±5.2 times lower expression levels in both AS
groups than in the nr-AxSpA patients. As most of them are associated with bone remodelling,
these data indicate that an inverse association exists between radiographic bone formation and
circulating miRNA levels.
Sacroiliitis vs. AS II-V: However, there were no significant differences in the levels of
abovementioned 12 miRNAs between sacroiliitis and AS II-V groups. MiR-99b-5p,
miR-6253p and miR-885-5p exhibited significantly lower expression (2.3 times, 2.2 times, 3.3 times,
respectively) in AS patients with spinal involvement (AS II-V) than in those with sacroiliitis.
Interestingly, we found no data regarding the roles of miR-625-3p and miR-885-5p in bone
formation or inflammation (S2 Table).
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Fig 1. Significance cluster analysis of selected miRNAs using DIANA mirPath tool showing the involvement of miRNAs in
different signalling and pathogenic pathways. Although the involvement in certain pathways of several miRNAs overlapped, the
function of few miRNAs was unknown and required manual search for their function.
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Treatment response Hypothesized role in
NSAID vs. DMARDs. vs. AxSpA
- ** bone formation
*** *** bone formation
*** ** bone formation
* ** bone formation,
Abbreviations: HC, healthy controls; nr-AxSpA, non-radiographic axial spondyloarthritis; AS, ankylosing spondylitis; AS II-V, ankylosing spondylitis with
spinal involvement; DMARDs, disease modifying antirheumatic drugs; NSAID, non-steroidal anti-inflammatory drugs; T, T test; -, not significant;
Statistical significance was calculated using ANOVA unless stated otherwise.
Moreover, patients with a bamboo spine exhibited 1.6±8.0 times significantly lower
expression levels of some of the miRNAs mentioned above (miR-19a-3p, miR-24-3p, miR-27a-3p,
miR-99b-5p, miR-106a-5p, miR-140-3p, miR-145-5p, miR-146a-5p, miR-146b-5p,
miR-2223p, miR-223-3p, miR-374a-5p, miR-375), including miR-625-3p and miR-885-5p than AS
patients with less severe spinal damage (Fig 2).
Association between circulating miRNAs and disease activity
We next aimed to analyse the associations between circulating miRNA levels and disease
activity parameters (Fig 3).
In patients with nr-AxSpA, no significant associations between miRNA levels and BASDAI
were noted. CRP was positively correlated with the levels of miR-99b-5p, miR-140-3p,
miR145-5p and miR-374a-5p expression (Fig 3A).
In all AS patients, BASDAI positively correlated with the levels of miR-99b-5p,
miR-133a3p, miR-625-3p and miR-885-5p, while CRP was correlated with the levels of miR-146a-5p,
miR-151a-3p, miR-181a-5p and miR-221-3p (Fig 3B).
When further sub-analysis was performed, we noted no correlations between miRNAs and
CRP or BASDAI in patients with sacroiliitis. However, in AS patients with spinal involvement
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Fig 2. Differences in the levels of circulating miRNAs between healthy controls, patients with
nonradiographic AxSpA (nr-AxSpA) and patients with ankylosing spondylitis (AS) with isolated
sacroiliitis and spinal involvement (AS II-V). Black symbols indicate patients with bamboo spine. *p<0.05,
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Fig 3. Correlation between circulating miRNAs, CRP and BASDAI in nr-AxSpA (A) and AS patients (B).
(AS II-V), we noted a positive correlation between BASDAI and mi-29a-3p, miR-222-3p,
miR375, miR-625-3p and miR-885-5p levels and a positive correlation between CRP and
miR-29a3p, miR-133a-3p, miR-151a-3p and miR-409-3p levels.
We then considered different HLA-B27 status, peripheral arthritis or extraarticular
manifestations on levels of miRNAs but no effect was found in any of these confounders. While no
significant differences were noted in miRNA levels between patients receiving non-steroidal
anti-inflammatory drugs (NSAID) or disease modifying antirheumatic drugs (DMARDs), the
patients receiving anti-TNF therapy exhibited significantly lower levels of all remaining
miRNAs than anti-TNF naïve patients (Table 2, S4 Table).
To our knowledge, this is the first study to perform comprehensive analyses of 760 circulating
miRNAs in patients with various stages of AxSpA. We observed differential expression of
some miRNAs in patients with more advanced spinal disease.
Circulating miRNAs have been shown to be unexpectedly stable, which makes them
accessible via body fluid sampling and potentially useful as biomarkers [
]. Some associations
between circulating miRNA levels and disease activity e.g., in early RA [
] or systemic lupus
], have previously been shown; however, data on circulating miRNAs in
AxSpA are lacking.
Of 760 screened miRNAs, 21 exhibiting differential expression among patients at various
disease stages were selected. We confirmed that 14 miRNAs (miR-19a-3p, miR-24-3p,
miR27a-3p, miR-29a-3p, miR-99b-5p, miR-133a-3p, miR-146a-5p, miR-146b-5p, miR-222-3p,
miR-223-3p, miR-374a-5p, miR-375, miR-409-3p and miR-625-3p) had lower expression
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levels in all AxSpA patients, irrespective of radiographic findings, than in HC. Most of them
were shown to be associated with osteoblast differentiation or the Wnt signalling pathway,
while others participate in cell differentiation and proliferation or inflammation. These data
support our hypothesis that dysregulation of circulating miRNA occurs in patients with
Interestingly, only miR-625-3p was significantly different in nr-AxSpA patients compared
to HC. Eighteen miRNAs were confirmed to have lower expression levels in AS patients than
in HCs. Of these, 14 exhibited lower expression levels in AS patients with radiographic disease
than in AS patients with non-radiographic disease. The levels of 9 miRNAs (miR-19a-3p,
miR24-3p, miR-27a-3p, miR-106a-5p, miR-140-3p, miR-146a-5p, miR-146b-5p, miR-223-3p,
miR-374a-5p) were lower in patients with a bamboo spine than in other patients with AS.
Interestingly, these 9 miRNAs that exhibited differential expression in conjunction with
progressive spinal damage, 6 miRNAs (miR-19a-3p, miR-24-3p, miR-27a-3p, miR-106a-5p,
miR223-3p, miR-374a-5p) play roles in bone formation by mediating Wnt signalling pathway
activity, and 3 (miR-140-3p, miR-146a-5p, miR-146b-5p) play roles in inflammation or cell
migration that may be related to the pathogenesis of AxSpA.
At present, it is not technically feasible to show the effect of native biofluid circulating
miRNA on target genes in tissues using functional experiments due to lack of data on their
specific source, trafficking and targeting mechanisms. Drawing firm conclusions on the role of
circulating miRNAs in the pathogenesis is therefore difficult and these are mostly inferred
from data published on intracellular miRNAs reviewed by literature and databases search.
Similarly, we inferred the potential of differentially expressed circulating miRNAs in the
pathogenesis of AxSpA based on published data on other diseases.
Many miRNAs shown here to be differentially expressed are involved in bone turnover.
MiR-19a was described as a negative regulator of the Wnt signalling pathway in endothelial
]. MiR-24 overexpression significantly inhibited osteogenic differentiation in
osteoblastic cells [
]. Runx2, a transcription factor essential for osteoblastogenesis, was shown to
negatively regulate the expression of miR-27a [
]. Another study showed that miR-27
promote osteoblast differentiation by modulating Wnt signalling . MiR-106a inhibits
osteogenesis in mesenchymal stem cells [
] and is a negative regulator of IL-8, the levels of which
are known to be elevated in AS patients [
]. MiR-223 affects bone metabolism, especially
osteoclast and osteoblast differentiation  and blocking miR-223 inhibits osteoclastogenesis
]. Moreover, miR-223 appears to be a biomarker of disease activity and treatment response
in RA [
]. MiR-374a has been shown to be an activator of the Wnt signalling pathway .
In summary, we believe that lower systemic levels of miRNAs may reflect their low local
expression levels. Our data show a trend towards lower levels according the extent of spine
involvement with the lowest levels in patients with most advanced disease. We hypothesize
that lower systemic levels of miRNAs negatively correlate with new bone formation promoted
by induction of osteoblastogenesis and to lesser extend also by local inflammation or osteitis
initiated by osteoclast infiltration. However, the data regarding miR-27a and miR-374a remain
controversial and do not entirely support our theory.
In addition, several miRNAs modulate inflammatory process. IL-1β suppressed miR-140
expression and induced ADAMTS5, a member of the extracellular protease enzyme family.
Conversely, transfection of chondrocytes with miR-140 downregulated IL-1β-induced
ADAMTS5 expression [
]. In keeping with these findings, miR-140-3p was shown to
ameliorate autoimmune arthritis [
]. Administration of miR-146a prevents joint
destruction in arthritic mice presenting miR-146a as a negative regulator of inflammation [
These data suggest that miR-140-3p and miR-146a dysregulation may be associated with the
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proinflammatory state characteristic of AxSpA. Also, miR-146a polymorphisms have been
suggested to be a potential pathogenic factor for AS [
Here we propose circulating miRNAs as markers of disease activity. In nr-AxSpA patients,
miR-140-3p, miR-223-3p, miR-99b-5p and miR-145-5p levels correlated with CRP. Both
miR99b and miR-145 were shown to be associated with osteoclast/osteoblast differentiation [
]. However, no associations with BASDAI were observed in these patients or those with
Furthermore, in AS patients with spinal involvement, we observed correlations between
miR-29a-3p and both BASDAI and CRP, as well as associations between miR-222-3p,
miR625-3p and miR-885-5p and BASDAI. The data on miR-29a levels in AS are rather
inconsistent. While miR-29a expression in peripheral blood mononuclear cells was lower in active AS
patients than in controls and decreased after anti-TNF therapy [
], another showed otherwise
]. TGF-β, an important stimulator of bone formation [
], inhibits miR-29a expression [
]. In addition, miR-29a has been described as a negative regulator of Wnt signalling and
production of extracellular matrix [
]. We hypothesize that advanced-stage AS patients with
extensive bone formation have higher levels of TGF-β, which ultimately results in miR-29a
suppression and increased bone formation. MiR-222 has been predicted to exert inhibitory
effects on genes associated with osteogenic differentiation [
], and low levels of miR-222 may
result in increased MMP-13 expression in osteoarthritic cartilage [
]. As mentioned above,
miR-625-3p was significantly decreased in nr-AxSpA and may be associated with early disease.
There is no pathophysiologic explanation for this finding, nor is there an explanation for its
association with BASDAI in advanced AS patients. Similarly, data regarding the potential role
of miR-885-5p in AxSpA are lacking.
There are some limitations in our study. The selection of miRNAs for validation analysis
may appear biased. The initial mirPath screen of all miRNAs of 1.5 mean fold difference
obtained by TLDA provided us with a broad-spectrum unspecific data since such an online
tool is based on validated/predicted targets genes coming from different fields (mostly cancer)
that may be yet largely unknown in AxSpA. Due to practical reasons, it was not feasible to
validate all miRNAs fulfilling the abovementioned cut-off. Therefore, a manual review was
implemented to narrow down the list of miRNAs taken forward for further analysis and to enable
drawing hypotheses. Next, two different normalization methods were used in our study. Array
data were normalized to Ct average of all miRNAs as miRNAs of non-human origin are not
included on the array platform, while the normalization to the average of 3 spike-in controls
was performed in single-assay analysis. At present, there is no consensus on normalization of
cell-free miRNAs. As the normalization to endogenous cell-free miRNAs may be conditioned
by their altered expression due to (patho)physiological condition of each individual, the use of
spike-in controls of non-human origin appears an acceptable alternative. Moreover, this
approach reflects potential errors during the workflow, although these were minimized. We
appreciate that the differential expression pattern based on TLDA analysis was not always
reflected in single assay analysis. More studies involving larger patient cohorts are needed to
confirm these data, as only a small proportion of the circulating mirnome has been analysed,
and the functions of many miRNAs remain unknown. Moreover, due to nature of circulating
miRNAs direct functional experiments are not feasible and the involvement of miRNAs in the
pathogenesis of AS was hypothesized based on published data.
We have shown for the first time the differential expression levels of several circulating
miRNAs in radiographic AxSpA patients compared to non-radiographic disease patients and HC.
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Moreover, the levels of these miRNAs appear to reflect progressive spinal involvement.
Interestingly, while some of these miRNAs play roles in bone formation and associated signalling
pathways, others play roles in inflammation. It can be hypothesized that differential systemic
miRNA expression levels reflect their dysregulation at sites of spinal inflammation or bone
formation where they contribute to the development of pathophysiological features characteristic
of AS. However, only miR625-3p, whose role in AS is unknown, exhibited significantly
different expression levels in nr-AxSpA patients compared to HC and, interestingly, correlated with
disease activity in AS. Our data support the role of circulating miRNAs in the pathogenesis of
AxSpA and their potential as biomarkers of disease progression. Additional studies involving
larger patient cohorts are needed to confirm these data, as only a small proportion of the
circulating mirnome has been analysed, and the functions of many miRNAs remain unknown.
S1 Fig. The expression of miRNAs in healthy controls (HC) and patients with
non-radiographic axial spondyloarthritis (nr-AxSpA), sacroiliitis and ankylosing spondylitis with
spinal involvement (AS II-V) using TaqMan Low Density Array and their shared
expression among groups.
S1 Table. Original Taq Man Low Density Array (TLDA) data obtained in 5 healthy
controls (HC), and 5 patients with non-radiographic axial spondyloarthritis (nr-AxSpA), 5
patients with sacroiliitis and 5 patients with ankylosing spondylitis with spinal
involvement (AS II-V). The statistical analysis was performed as described in Methods paragraph.
S2 Table. Functions of the 21 miRNAs selected for further analysis based on a literature
search as explained in Methods paragraph.
S3 Table. Data obtained from single assay analysis of 21 miRNAs. The data are provided as
mean±SD calculated from data obtained from 29 healthy controls (HC), 20 patients with
nonradiographic axial spondyloarthritis (nr-AxSpA), 24 patients with sacroiliitis and 24 patients
with ankylosing spondylitis with spinal involvement (AS II-V). The statistical analysis was
performed as described in Methods paragraph. Abbreviations: HC, healthy controls; nr-AxSpA,
non-radiographic axial spondyloarthritis; AS, ankylosing spondylitis.
S4 Table. Extended summary of expression and function of selected miRNAs as markers of
disease activity and hypothesized role in AxSpA. Abbreviations: HC, healthy controls;
nrAxSpA, non-radiographic axial spondyloarthritis; AS, ankylosing spondylitis; AS II-V,
ankylosing spondylitis with spinal involvement; DMARDs, disease modifying antirheumaticdrugs;
NSAID, non-steroidal anti-inflammatory drugs; T, T test; -, not significant. Statistical
significance was calculated using ANOVA unless stated otherwise.
Conceptualization: KlaÂra PrajzlerovaÂ, Astrid JuÈngel, Steffen Gay, JiřÂõ VencovskyÂ, Karel
Pavelka, Ladislav SÏenolt, MaÂria FilkovaÂ.
Data curation: KlaÂra PrajzlerovaÂ, KristyÂna GrobelnaÂ, MarkeÂta HuÏsaÂkovaÂ, SÏaÂrka ForejtovaÂ.
12 / 15
Formal analysis: KlaÂra PrajzlerovaÂ, KristyÂna GrobelnaÂ.
Investigation: KlaÂra PrajzlerovaÂ, KristyÂna GrobelnaÂ, MarkeÂta HuÏsaÂkovaÂ, SÏaÂrka ForejtovaÂ,
Methodology: KlaÂra PrajzlerovaÂ, Astrid JuÈngel, Karel Pavelka, Ladislav SÏenolt, MaÂria FilkovaÂ.
Supervision: Steffen Gay, JiřÂõ VencovskyÂ, Karel Pavelka, Ladislav SÏenolt, MaÂria FilkovaÂ.
Writing ± original draft: KlaÂra PrajzlerovaÂ, MaÂria FilkovaÂ.
Writing ± review & editing: KlaÂra PrajzlerovaÂ, KristyÂna GrobelnaÂ, MarkeÂta HuÏsaÂkovaÂ, SÏaÂrka
ForejtovaÂ, Astrid JuÈngel, Steffen Gay, JiřÂõ VencovskyÂ, Karel Pavelka, Ladislav SÏenolt, MaÂria
13 / 15
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