Introduction
Ankylosing spondylitis (AS) is a chronic
inflammatory autoimmune disease involving the sacroiliac, axial and
hip joints that is caused by genetic, environmental and immune
disorders (1). The course of this
disease progresses from the inflammatory stage of bone marrow edema
to the chronic stage of ectopic ossification and joint fusion
(2). The incidence of AS in the
Chinese population is ~0.2% and tends to occur in young and
middle-aged men, which brings a burden to the family and society
(3,4). There are few cases of surgical
treatment in clinical application due to high perioperative risk,
cost, difficulty in operation and poor prognosis (5). Considering the adverse effects, such
as increased risk of cardiovascular disease, non-steroidal
anti-inflammatory drugs and anti-tumor necrosis factor-α therapy
are not approved for long-term treatment of AS (6,7).
Prevention of the ossification of ligament fibroblasts is one of
the primary goals in treating AS (8,9).
However, the underlying mechanism is not yet been fully
understood.
MicroRNAs (miRNAs or miRs) are non-coding small RNAs
comprising endogenous 21-23 nucleotides involved in regulation of
eukaryotic gene expression at the post-transcriptional level by
binding to the 3'-untranslated region (UTR) of target gene mRNA
(10). Evidence has revealed
abnormal expression of numerous miRNAs in AS, some of which may be
used as molecular markers for diagnosis and prognosis of AS
(11,12). Expression profile analysis of
patients with AS showed 9 differentially expressed circulating
miRNAs, among which miR-150-5p, located on chromosome 19q13.33, was
significantly decreased in patients with AS compared with healthy
controls (13). It was
hypothesized that decreased miR-150-5p expression may be associated
with the development of AS.
Materials and methods
Patients and tissue collection
The collection of clinical specimens involved was
approved by the Ethics Committee of Tianjin First Central Hospital
(approval no. TJIRB2015-198) and written informed consent was
obtained from all subjects. Patients and controls were recruited
from Tianjin First Central Hospital (Tianjin, China). A total of 20
patients with AS involving both hips who received joint replacement
between November 2015 to December 2017, including 17 males and 3
females with an average age of 32.8 years, were included as the
experimental group. The diagnostic criteria for patients with AS
complied with the 1984 modified New York criteria (14). A total of 20 patients with femoral
neck fracture who underwent open surgery or total hip arthroplasty
without AS or rheumatoid arthritis history (15 males and 5 females;
average age, 32.3 years) were selected as the control group between
November 2015 and December 2017. The joint capsules were acquired
during the surgery. Clinical characteristics of all subjects are
listed in Table I.
 | Table IClinical characteristics and
parameters of patients with ankylosing spondylitis and
controls. |
Table I
Clinical characteristics and
parameters of patients with ankylosing spondylitis and
controls.
| Parameter | Patient (n=20) | Control (n=20) |
|---|
| Age, years | 32.80±12.96 | 32.30±10.61 |
| Male/female | 17.00/3.00 | 15.00/5.00 |
| BASFI | 2.08±3.10 | - |
| BASDAI | 3.90±2.03 | - |
| BASMI | 2.80±1.05 | - |
| ASDAS-CRP | 2.85±1.11 | - |
| Disease duration,
years | 14.30±8.50 | - |
| WBC,
103/mm3 | 8.12±1.83 | - |
| Hb, g/dl | 12.93±2.68 | - |
| HLA-B27, n (%) | 18.00 (90.00) | - |
| CRP, nmol/l | 101.12±110.78 | 14.08±17.14 |
| ERS, mm/h | 16.90±18.30 | 6.10±5.20 |
Cell isolation and culture
Fibroblasts were obtained from ligament tissue of
joint capsules derived from patients with AS using tissue explant
adherent method. Briefly, 1 mm3 ligament pieces were
washed three times with PBS and transferred into a
25-cm2 tissue culture flask containing Dulbecco's
modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.)
supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher
Scientific, Inc.). The medium was refreshed every 3 days. After 7
days culture in a humidified atmosphere containing 5%
CO2 at 37˚C, the adherent cells were digested with 0.25%
trypsin at 37˚C for 2 min. Following centrifugation at 1,000 x g
for 5 min at room temperature, the single cell suspension was
sub-cultured in a ratio of 1:3 and fibroblasts at the third passage
were used for subsequent experiments. For induction of osteogenic
differentiation, cells were cultured at 37˚C in DMEM supplemented
with 50 bone morphogenic protein 2 (BMP-2) and 10 ng/ml
transforming growth factor-β1 (TGF-β1; both R&D Systems, Inc.)
for 14 days (15).
Cell transfection
miRNA-150-5p mimics and negative controls (miR-NC)
were designed and synthesized by Shanghai GenePharma Co., Ltd. as
follows: miR-150-5p mimics forward, 5'-UCUCCCAACCCUUGUACCAGUG-3'
and reverse, 5'-CACUGGUACAAGGGUUGGGAGA-3' and miR-NC forward,
5'-UCACAACCUCCUAGAAAGAGUAGA-3' and reverse,
5'-UCUACUCUUUCUAGGAGGUUGUGA-3'. Plasmid for vitamin D receptor
(VDR) overexpression was constructed by inserting the amplified
complementary DNA (cDNA) into pcDNA 3.1 vector (Invitrogen; Thermo
Fisher Scientific, Inc.). Fibroblasts were incubated at 37˚C
overnight in antibiotics-free DMEM medium until 70% confluence, and
transfection was performed with 50 nM oligonucleotides or 2 µg
vectors using Lipofectamine® 2000 (Invitrogen; Thermo
Fisher Scientific, Inc.) according to the manufacturer's
instructions for 4-6 h at 37˚C before changing to DMEM medium.
Following 48 h transfection, fibroblasts were stimulated at 37˚C
with 50 BMP-2 and 10 ng/ml TGF-β1 for 14 days before further
analysis.
Bioinformatics prediction
Bioinformatics algorithm from starBase v2.0
(starbase.sysu.edu.cn/starbase2/targetSite.php)
was used to predict the target genes of miR-150-5p.
Luciferase reporter assay
Fibroblasts were co-transfected with either
miR-150-5p mimics (forward, 5'-UCUCCCAACCCUUGUACCAGUG-3' and
reverse, 5'-CACUGGUACAAGGGUUGGGAGA-3') or mimic NC (forward,
5'-UCACAACCUCCUAGAAAGAGUAGA-3' and reverse,
5'-UCUACUCUUUCUAGGAGGUUGUGA-3') and wild-type or mutant psiCHECK-2
dual luciferase vector (Promega Corporation) comprising 3'UTR of
VDR. Following 48 h transfection using Lipofectamine®
2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C,
luciferase assay was performed using the Dual Luciferase Reporter
Assay System (Promega Corporation). Firefly luciferase activity was
normalized to Renilla luciferase activity.
RNA isolation and reverse
transcription-quantitative (RT-q)PCR
Total RNA was extracted from joint capsules and
fibroblasts using TRIzol® (Invitrogen; Thermo Fisher
Scientific, Inc.). cDNA was synthesized using Mix-X miRNA
First-Strand Synthesis and PrimeScript™ RT reagent kits (all from
Takara Bio, Inc.). Reverse transcription was performed at 37˚C for
15 min followed by 85˚C for 5 sec. The obtained cDNA was subjected
to PCR amplification using the following thermocycling conditions:
Denaturation at 92˚C for 10 sec, annealing at 55˚C for 20 sec and
extension at 68˚C for 20 sec (40 cycles). The mRNA expression
levels of miR-150-5p, VDR, collagen type I (Col I), osteopontin
(OPN) and runt-related transcription factor 2 (Runx2) were
quantified using SYBR Green PCR kit (Takara Bio, Inc.) and U6 or
GAPDH was used as the internal reference. The primer sequences were
as follows: miR-150-5p forward,
5'-ACACTCCAGCTGGGTCTCCCAACCCTTGTACCA-3' and reverse,
5'-CTCAACTGGTGTCGTGGA-3'; Col I forward, 5'-CCTGGATGCCATCAAAGTCT-3'
and reverse, 5'-ACTGCAACTGGAATCCATCG-3'; OPN forward,
5'-GCTATCACCTCGGCCGTTGGGG-3' and reverse,
5'-CATTGCCTCCTCCCTCCCGGTG-3'; Runx2 forward,
5'-CTCCCTGAACTCTGCACCAA-3' and reverse,
5'-GTTCTGAAGCACCTGAAATGCG-3'; GAPDH forward,
5'-CCATGGAGAAGGCTGGGG-3' and reverse, 5'-CAAAGTTGTCATGGATGACC-3'
and U6 forward, 5'-GCTTCGGCAGCACATATACTAAAAT-3' and reverse,
5'-CGCTTCACGAATTTGCGTGTCAT-3'. The relative quantification of mRNA
expression was calculated via the 2-ΔΔCq method as
previously described (16).
Immunoblotting
Total protein was extracted from cultured
fibroblasts using RIPA lysis buffer (Pierce; Thermo Fisher
Scientific, Inc.). The protein quantification was performed using
bicinchoninic acid method (Thermo Fisher Scientific, Inc.). Equal
amounts of protein (40 µg) were electrophoresed via 10% SDS-PAGE
and electroblotted onto polyvinylidene fluoride membranes
(MilliporeSigma). After blocking in 5% skimmed milk for 1 h at room
temperature, the membranes were incubated with primary antibody
(1:1,000) overnight at 4˚C and horseradish peroxidase-conjugated
IgG secondary antibody (1:5,000) for 1 h at room temperature. The
primary antibodies against Col I (cat. no. ab6308), OPN (cat. no.
ab8448), Runx2 (cat. no. ab76956), VDR (cat. no. ab134826) and
GAPDH (cat. no. ab8245) and secondary antibody (cat. no. ab8245)
were all purchased from Abcam. Densitometric analysis was processed
using enhanced chemiluminescence reagent (Beckman Coulter, Inc.).
Protein bands were semi-quantified using Image J software (version
1.8.0; National Institutes of Health).
Determination of alkaline phosphatase
(ALP) activity
ALP activity was assessed using ALP assay kit (cat.
no. A059-2-1; Nanjing Jiancheng Bioengineering Institute) according
to the manufacturer's protocol. Fibroblasts in each group were
collected and seeded in 6-well plates at a density of
1x104 cells/well. A total of 50 µl p-nitrophenyl
phosphate (Sigma-Aldrich; Merck KGaA) was added to each well. The
reaction was stopped after 15 min at room temperature. The
absorbance at 520 nm was determined by a microplate reader.
Alizarin red staining
Fibroblasts in each group were fixed in 70% ethanol
for 1 h at 4˚C. After washing with distilled water three times, 40
mM alizarin red solution was added for 10 min at 37˚C. Cells were
rinsed with distilled water and washed with PBS. The stained cells
were observed under an inverted microscope (light microscope;
magnification, x100). Photomicrographs were obtained using a
charge-coupled device camera. Alizarin red was eluted with 10%
cetylpyridinium chloride and the optical density value was measured
at 510 nm to measure ossification.
Statistical analysis
Data are presented as the mean ± SD of three
independent experimental repeats. Comparisons between groups were
assessed by unpaired Student's t-test or one-way ANOVA followed by
Tukey's post hoc test using SPSS 19.0 (IBM Corp.). Spearman's
correlation analysis was used to determine the association between
miR-150-5p and VDR expression in AS joint capsules. P<0.05 was
considered to indicate a statistically significant difference.
Results
Downregulation of miR-150-5p in AS
joint capsules and fibroblasts
To determine whether miR-150-5p serves a key role in
the pathogenesis of AS, joint capsules from patients with AS and
non-AS controls with femoral neck fracture were used to analyze
expression levels of miR-150-5p via RT-qPCR. mRNA expression of
miR-150-5p was significantly lower in patients with AS compared
with controls (Fig. 1A).
miR-150-5p expression in AS patient-derived fibroblasts following
co-treatment with BMP-2 and TGF-β1 was detected. miR-150-5p
expression was significantly decreased in BMP-2 and TGF-β1-treated
AS fibroblasts compared with untreated cells (Fig. 1B).
Overexpression of miR-150-5p
suppresses osteogenic differentiation of AS fibroblasts
The association between miR-150-5p and osteoblast
differentiation was assessed by overexpressing miR-150-5p in AS
fibroblasts via transfection of miR-150-5p mimics. The
overexpression transfection efficiency was confirmed by RT-qPCR
(Fig. 2A). The mRNA and protein
expression levels of osteogenic genes, including Col I, OPN and
Runx2 were significantly elevated in BMP-2 and TGF-β1-induced AS
fibroblasts and miR-150-5p upregulation significantly reversed
these changes (Fig. 2B and
C). The osteogenic phenotype in AS
fibroblasts treated with BMP-2 and TGF-β1 was confirmed by
increased ALP activity, whereas miR-150-5p mimics resulted in
decreased ALP activity (Fig. 2D).
Additionally, mineralization visualized by alizarin red staining
showed more mineral deposition in AS patient-derived fibroblasts
following co-treatment with BMP-2 and TGF-β1 compared with control,
whereas miR-150-5p overexpression suppressed the mineralization of
fibroblasts (Fig. 2E).
 | Figure 2Overexpression of miR-150-5p
suppresses osteogenic differentiation of AS fibroblasts. (A)
Transfection efficiency. (B) mRNA expression of Col I, OPN and
Runx2 determined by reverse transcription-quantitative PCR. (C)
Protein levels of Col I, OPN and Runx2 determined by western
blotting. (D) ALP activity. (E) Alizarin red staining for
mineralization in BMP-2 and TGF-β1-induced AS fibroblasts
transfected with miR-150-5p mimics or miR-NC or untreated cells
(magnification, x100). Data are presented as the mean ± SD.
**P<0.01, ***P<0.001 vs. control;
#P<0.05, ##P<0.01,
###P<0.001 vs. miR-NC. miR, microRNA; AS, ankylosing
spondylitis; BMP, bone morphogenetic protein; TGF, transforming
growth factor; Col I, collagen type I; OPN, osteopontin; ALP,
alkaline phosphatase; NC, negative control. |
miR-150-5p decreases VDR expression by
targeting VDR 3'-UTR
Bioinformatics analysis (starBase) predicted VDR as
a candidate target gene of miR-150-5p (Fig. 3A). To verify whether VDR was a
direct target of miR-150-5p, luciferase activity assay was
performed. The relative luciferase activity of AS fibroblasts
co-transfected with miR-150-5p mimics in the presence of wild-type
VDR 3'-UTR was significantly decrease, while miR-150-5p
overexpression did not affect the relative luciferase activity of
mutant 3'-UTR-VDR (Fig. 3B). VDR
protein expression was significantly decreased in AS fibroblasts
transfected with miR-150-5p mimics (Fig. 3C). Increased expression of VDR mRNA
was observed in joint capsules of patients with AS (Fig. 3D), which was negatively correlated
with miR-150-5p mRNA expression (Fig.
3E). VDR protein expression in BMP-2 and TGF-β1-treated AS
fibroblasts was significantly higher compared with untreated cells
(Fig. 3F).
 | Figure 3miR-150-5p decreases VDR expression by
targeting VDR 3'-UTR. (A) Schematic diagram of the predicted
miR-150-5p binding sites to VDR. (B) Luciferase assay in AS
fibroblasts transfected with pmirGLO-VDR-WT or -MUT reporters and
miR-150-5p mimics or miR-NC. (C) Protein levels of VDR in AS
fibroblasts transfected with miR-150-5p mimics or miR-NC. (D) mRNA
expression of VDR in joint capsule tissues obtained from patients
with AS and non-AS controls (n=20/group) determined by reverse
transcription-quantitative PCR. (E) Spearman's correlation analysis
of miR-150-5p and VDR mRNA expression in AS joint capsules. (F) VDR
protein levels in BMP-2 and TGF-β1-treated AS fibroblasts or
untreated cells. Data are presented as the mean ± SD.
**P<0.01, ***P<0.001 vs. miR-NC or
control. miR, microRNA; AS, ankylosing spondylitis; BMP, bone
morphogenetic protein; TGF, transforming growth factor; VDR,
vitamin D receptor; UTR, untranslated region; WT, wild-type; MUT,
mutant; NC, negative control. |
miR-150-5p regulates osteogenic
differentiation in AS fibroblasts by downregulating VDR
To verify whether the role of miR-150-5p in
osteogenic differentiation was mediated by downregulating VDR
expression, rescue assay was performed using AS fibroblasts. High
transfection efficiency was confirmed by western blotting following
48 h transfection with VDR overexpression plasmid (Fig. 4A). Ectopic expression of VDR
antagonized inhibition of osteogenic differentiation potential
induced by miR-150-5p overexpression in AS fibroblasts treated with
BMP-2 and TGF-β1, as evidenced by the upregulation of Col I, OPN
and Runx2 (Fig. 4B and C) and ALP activity (Fig. 4D). Additionally, restoration of VDR
expression abrogated the inhibitory effects induced by miR-150-5p
overexpression in AS fibroblasts following co-treatment with BMP-2
and TGF-β1 (Fig. 4E).
 | Figure 4miR-150-5p regulates osteogenic
differentiation in AS fibroblasts by downregulating VDR. (A)
Transfection efficiency. (B) mRNA expression of Col I, OPN and
Runx2. (C) Protein levels of Col I, OPN and Runx2. (D) ALP
activity. (E) Alizarin red staining for mineralization in BMP-2 and
TGF-β1-treated AS fibroblasts transfected with miR-NC or miR-150-5p
mimics and with VDR (magnification, x100). Data are presented as
the mean ± SD. *P<0.05, **P<0.01,
***P<0.001 vs. Vector or miR-NC;
#P<0.05, ##P<0.01,
###P<0.001 vs. miR-150-5p. miR, microRNA; AS,
ankylosing spondylitis; BMP, bone morphogenetic protein; TGF,
transforming growth factor; VDR, vitamin D receptor; UTR,
untranslated region; Col I, collagen type I; OPN, osteopontin; ALP,
alkaline phosphatase; NC, negative control. |
Discussion
The present study aimed to investigate expression of
miR-150-5p and its molecular mechanism in progression of AS.
miR-150-5p expression was decreased in hip capsule tissue of
patients with AS and decreased the osteogenic differentiation
capability of AS fibroblasts. VDR was confirmed as a direct target
gene of miR-150-5p. miR-150-5p targeted VDR to exert
anti-osteogenic effects in AS fibroblasts (Fig. 5).
In recent years, researchers have proposed aberrant
expression of miRNAs in AS serves role in regulating osteoblast
differentiation (17,18). For example, miR-124 increases
osteoblast differentiation of AS fibroblasts via glycogen synthase
kinase-3β-mediated Wnt/β-catenin pathway signaling (19). Moreover, miR-17-5p improves
osteogenic differentiation and heterotopic ossification in AS via
downregulation of ANKH (20).
Overexpression of miR-96 enhances osteoblast growth and
differentiation as well as bone formation in AS via activation of
Wnt signaling pathway by targeting sclerostin (21). The present study demonstrated that
miR-150-5p was downregulated in AS patients-derived hip joint
tissue and ligament fibroblasts. The involvement of miR-150-5p has
been reported in several types of disease, especially immune
disease. For example, Chen et al (18) showed that exosomal miR-150-5p
serves a therapeutic role in rheumatoid arthritis (RA) by
decreasing migration and invasion of fibroblast-like synoviocytes,
synoviocyte hyperplasia and angiogenesis. Qiu et al
(19) reported that miR-150-5p
inhibits apoptosis of RA synovial fibroblasts and promotes
proliferation by downregulating suppressor of cytokine signaling 1.
During osteoarthritis (OA) progression, overexpression of
miR-150-5p suppresses proliferation and induces apoptosis and
extracellular matrix degradation of OA chondrocytes (22,23).
To the best of our knowledge, the present study is the first to
demonstrate decreased miR-150-5p expression in AS fibroblasts
treated with BMP-2 and TGF-β1. BMP-2 is a widely studied growth
factor that enhances bone tissue regeneration (24,25).
TGF-β1 serves a key role in regulating cell proliferation and
differentiation and promoting osteogenesis (26,27).
Therefore, the combination of BMP-2 with TGF-β1 is hypothesized to
promote bone induction and regeneration and to exhibit a
synergistic effect on induction of osteogenic differentiation
(28-30).
In vitro study of BMP-2 and TGF-β1-treated AS fibroblasts
confirmed that miR-150-5p overexpression suppressed osteogenic
potential, as shown by decreased expression of osteogenic
markers.
VDR is a nuclear transcription factor that regulates
bone metabolism and the inflammatory process by binding with
specific ligands (31,32), such as steroid hormone
1alpha,25(OH)2-vitamin D3 (1,25(OH)2-D3) (33,34).
VDR gene polymorphsim confers increased risk of AS in Chinese Han
population (35,36). Zhang et al (33) recently demonstrated that long
non-coding RNA H19 initiates IL-17A/IL-23 inflammatory pathway in
AS pathogenesis by upregulating VDR by targeting miR-675-5p and
miR-22-5p. The inhibitory effect of miR-351 in targeting VDR in
osteogenic differentiation of bone marrow mesenchymal stem cells
has been demonstrated (37). Here,
VDR was highly expressed in AS fibroblasts and miR-150-5p inhibited
expression of VDR by binding to its 3'-UTR. VDR overexpression
abolished the anti-osteogenic role of miR-150-5p in AS fibroblasts.
However, certain limitations should be considered. Other mechanisms
underlying miR-150-5p-associated pathways that contribute to AS
pathogenesis in different types of cell need further validation.
Furthermore, additional animal models are required to confirm the
role of miR-150-5p in AS. Lack of controls treated with BMP-2- and
TGF-β1-alone is as a limitation of the present study and should be
investigated in future.
In summary, the present study demonstrated that
miR-150-5p directly targeted VDR and prevented osteogenic
differentiation of AS fibroblasts, indicating a potential novel
therapeutic option for AS.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by Tianjin Science and
Technology Commission (grant no. 2012KZ026).
Availability of data and materials
The datasets used and/or analyzed during the current
study are available from the corresponding author on reasonable
request.
Authors' contributions
YL performed experiments and wrote the manuscript.
WFQ performed experiments and analyzed the data. YQS designed the
study and revised the manuscript. All authors have read and
approved the manuscript. YL and YQS confirm the authenticity of all
the raw data.
Ethics approval and consent to
participate
The present study was approved by the ethics
committee of Tianjin First Central Hospital (approval no.
TJIRB2015-198). All subjects provided written informed consent.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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