1. 570
COMPREHENSIVE EXPRESSION ANALYSIS OF CANDIDATE MIRNAS IN
OSTEOARTHRITIC JOINT TISSUES FROM A MOUSE MODEL OF POST-
TRAUMATIC OA
L.H. Kung y, L. Rowley y, V. Ravi z, K. Bell y, C.B. Little z, J.F. Bateman y.
y
Murdoch Childrens Res. Inst., Melbourne, Australia; z
Raymond Purves
Bone and Joint Res. Lab., Univ. of Sydney, Sydney, Australia
Purpose: Osteoarthritis (OA) is a degenerative joint disease charac-
terized by the progressive breakdown of articular cartilage. While car-
tilage degradation remains the hallmark of OA, it is clear that all joint
tissues contribute to the pathological process, however, the molecular
mechanisms that drive these degenerative events remain poorly
understood. This highlights the critical need to perform parallel
molecular studies on articular cartilage and other OA-affected joint
tissues, such as the synovium (SYN) and underlying subchondral bone
(SCB). Intriguingly, an increasing number of miRNAs are being identified
as novel regulators of OA disease initiation and progression, making
them exciting candidates for therapeutic targets and diagnostic bio-
markers. The purpose of this study is to investigate previously pub-
lished candidate miRNAs, known to be dysregulated in human OA, and
determine their role in extra-cartilaginous OA joint tissues.
Methods: OA was induced in 10e12 week old male wild type mice by
bilateral surgical destabilization of the medial meniscus (DMM). RNA
from SCB from DMM and sham-operated mice was isolated by laser
microdissection at 1 and 6 weeks post-surgery. RNA was also extracted
from the SYN of the same mice. miRNA expression profiling of SCB and
SYN was performed using Agilent miRNA microarrays. Histological
measurements examining the severity of OA in the contralateral joint
(including: SCB sclerosis; osteophyte size and maturity; anterior and
posterior synovitis - panus presence and bone erosion, sub-synovial
inflammatory cell infiltration, synoviocyte hyperplasia, and exudate)
were scored by one observer blinded to surgical intervention.
Results: There was no difference in SCB sclerosis between surgeries at 1
week but DMM > sham at 6 weeks (p ¼ 0.003), and DMM-6wk > DMM-
1wk (p ¼ 0.04). There was no osteophyte development at 1 week but at
6 weeks they had formed in DMM only, being larger (p ¼ 0.002) and
more mature (p ¼ 0.001) than sham. Anterior and posterior synovitis
decreased with post-operative time after sham and DMM (1wk > 6wk,
p < 0.01 for all comparisons). There was no difference between sur-
geries in the individual synovitis parameters in the anterior aspect of
the joint at either time, other than more panus in DMM at 1 week
(p ¼ 0.049). However, more severe joint inflammation in DMM com-
pared with sham was evident by significantly higher synovitis scores in
the posterior region of the joint (distant from the surgical incision) at
both 1 and 6 weeks (p ¼ 0.01 and 0.03, respectively). miRNA expression
analysis revealed 584 miRNAs to be differentially expressed between
SYN and SCB samples (adj.p.value < 0.05). Moreover, 384 and 164
miRNAs were dysregulated between 1 and 6 week time points
(adj.p.value < 0.05) in SYN and SCB, respectively. However, there were
no changes in miRNA expression between DMM and sham mice at both
1 and 6 weeks post-surgery in either the SCB or SYN. Additionally,
promising candidate miRNAs previously identified in human OA carti-
lage (for example, miR-140, miR-483, miR-16 and miR-25) were not
dysregulated in our data set.
Conclusions: We demonstrated typical OA pathology in both SCB
(sclerosis, osteophytosis) and SYN (synovial hyperplasia, sub-synovial
inflammatory cell infiltration) that differed significantly with post-
operative time and between DMM and sham surgeries. Dynamic
changes in miRNA expression were observed between joint tissues (SYN
v SCB) and time points (1 week v 6 weeks post-surgery), consistent with
the temporal changes in pathology severity. However, in contrast to
previously published data, we saw no associations with miRNAs and OA
(i.e. DMM v sham) in either SYN or SCB joint tissues. Our data dem-
onstrates that miRNAs in SYN and SCB of OA joints are unlikely to be
pathological contributors to post-traumatic OA disease progression and
our future studies are now focused on miRNAs in articular cartilage.
571
MECHANO-SENSITIVITY OF MICRORNAS IN ARTICULAR CARTILAGE
P.Z. Stadnik y, J. Tarn z, A. Skelton x, T. Stone k, V.C. Duance y, D. Young z,
E.J. Blain y. y
Pathophysiology and Repair Div., Sch. of BioSci.s, Cardiff Univ.,
Cardiff, United Kingdom; z
Inst. of Cellular Med., Newcastle Univ.,
Newcastle, United Kingdom; x
Musculoskeletal Res. Group Bioinformatics
Support Unit, Newcastle Univ., Newcastle, United Kingdom; k
Inst. for
Translation, Innovation, Methodology and Engagement, Cardiff Univ. Sch.
of Med., Cardiff Univ., Cardiff, United Kingdom
Purpose: microRNAs (miRs) are small non-coding molecules that
negatively control the expression of their target genes at the post-
transcriptional level. The role of miRs in articular cartilage is an
emerging field. miR-140 is known to be involved in cartilage develop-
ment and homeostasis, and several other miRs are also differentially
regulated in healthy and osteoarthritic (OA) cartilage. One of the major
risk factors for OA is abnormal mechanical load. A few studies have been
conducted to date, with miR-221, -222, -146a and -365 being found to
be mechano-responsive in chondrocytes. The aim of this project is to (i)
examine the mechano-sensitivity of miRs in articular cartilage sub-
jected to normal (turnover genotype) and high (degradative genotype)
loads and (ii) study the correlation between mechano-regulated miRs
and mechano-sensitive matrix molecules involved in OA development.
Methods: Full-depth articular cartilage explants were collected from
the metacarpophalangeal joint of immature bovine calves. Explants
were stabilised in culture for 72 hours prior to loading. Using the BOSE
ElectroForce 3200® cartilage explants were either left unloaded or
subjected to a load of 2.5MPa (normal) or 7MPa (high) (1Hz, 15
minutes), and analysed 24 hours post-cessation of load. Extracted RNA
samples from each loading regime (n¼6) were pooled to create repre-
sentative samples, and three independent repeats performed (N¼3) to
assess expression levels of mechano-sensitive miRs, measured using
Next Generation Sequencing (The Genome Analysis Centre, Norwich,
UK), and mRNAs using Affymetrix GeneChip® Bovine 1.0 ST arrays
(Central Biotechnology Services, Cardiff, UK). Correlation of mechan-
ically regulated miRs with changes in matrix molecule mRNA levels is
currently being performed to identify miR targets for validation.
Results: Expression levels of specific miRs that are known to play
important roles in cartilage homeostasis altered in response to the
magnitude of compressive load. The known mechano-responsive
miRs miR-221 and miR-222 were significantly increased (3.4-fold;
padj<0.001 and 7.4-fold; padj<0.001, respectively) in response to a
high (7MPa) load in comparison to unloaded explants. Furthermore,
both miR-221 and-222 expression increased with increasing magni-
tude of load i.e. response to 7MPa compared to 2.5MPa load (2.6-fold;
padj<0.001 and 4.1-fold; padj<0.001); only miR-222 was elevated
significantly in response to the 2.5MPa load (1.8-fold; padj<0.01).
Interestingly, miR-21 and miR-27a that control essential genes in
cartilage homeostasis and are, respectively, either up- or down-
regulated in OA cartilage, are upregulated in response to 7MPa load
compared to unloaded (2.67-fold; padj<0.001 and 3.02-fold;
padj<0.001 respectively) or compared to the 2.5MPa load (1.72-fold;
padj<0.05 and 2.02-fold; padj<0.001 respectively). Correlation
between changes in expression of miRs and matrix molecules is cur-
rently being performed.
Conclusions: Our results confirm the reported mechano-regulation of
miR-221 and -222, and demonstrate the novel mechano-regulation of
miR-21 and -27a, miRs known to be involved in OA. Our data demon-
strates that mechanical load does regulate cartilage miR expression
which is likely to mediate downstream effects that may lead to alter-
ations in the mRNA level of genes responsible for tissue homeostasis
and cartilage degradation.
572
IDENTIFICATION OF SYNOVIAL FLUID MIRNA SIGNATURE IN KNEE
OSTEOARTHRITIS: DIFFERENTIATING EARLY AND LATE KNEE
OSTEOARTHRITIS
Y.-H. Li y, G. Tavallaee y, T. Tokar z, K. Sundararajan x, A. Sharma y,
R. Gandhi k, I. Jurisica z, M. Kapoor k. y
Toronto Western Res. Inst., Toronto,
ON, Canada; z
Princess Margaret Cancer Ctr./ Univ. of Toronto, Toronto, ON,
Canada; x
Toronto Western Hosp., Toronto, ON, Canada; k
Toronto Western
Hosp./Univ. of Toronto, Toronto, ON, Canada
Purpose: This study was aimed to identify circulating microRNA
(miRNA) signatures in knee synovial fluid (SF) from early-stage and
late-stage knee osteoarthritis (OA) patients.
Methods: miRNAs were screened by miRNA PCR-arrays and further
validated by qPCR in SF from patients with early-stage (Kellgren Law-
erence Score-I and -II) and late-stage OA (score: III and IV). Cartilage and
synovial explants from OA patients were cultured to study the source
and release of identified miRNAs. Computational approach was utilized
to predict genes/pathways modulated by validated miRNAs.
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534 S347
Downloaded from ClinicalKey.com.au at University of Melbourne May 05, 2016.
For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved.