miR-146a facilitates osteoarthritis by regulating cartilage homeostasis via targeting Camk2d and Ppp3r2
Citation: Cell Death and Disease
miR-146a facilitates osteoarthritis by regulating cartilage homeostasis via targeting Camk2d and Ppp3r2
Osteoarthritis (OA), characterized by insufficient extracellular matrix synthesis and cartilage degeneration, is known as an incurable disease because its pathogenesis is poorly elucidated. Thus far, limited information is available regarding the pathophysiological role of microRNAs (miRNAs) in OA. In this study, we investigated the specific function of miR-146a in OA pathophysiology using mouse OA models. We found that the articular cartilage degeneration of miR-146a knockout (KO) mice was alleviated compared with that of the wild-type (WT) mice in spontaneous and instability-induced OA models. We demonstrate that miR-146a aggravated pro-inflammatory cytokines induced suppressing the expression of cartilage matrix-associated genes. We further identified calcium/calmodulin-dependent protein kinase II delta (Camk2d) and protein phosphatase 3, regulatory subunit B, beta isoform (Ppp3r2, also known as calcineurin B, type II) were essential targets of miR-146a in regulating cartilage homeostasis. Moreover, we found that surgical-induced OA mice treated with a miR-146a inhibitor significantly alleviated the destruction of articular cartilage via targeting Camk2d and Ppp3r2. These results suggested that miR-146a has a crucial role in maintaining cartilage homeostasis. MiR-146a inhibition in chondrocytes can be a potential therapeutic strategy to ameliorate OA. Cell Death and Disease (2017) 8, e2734; doi:10.1038/cddis.2017.146; published online 6 April 2017
Osteoarthritis (OA) is the most prevalent musculoskeletal
disease in the elderly, and it is predicted to affect 60 million
people in the United States by 2020.1,2 However, effective
disease-modifying therapies for OA are unavailable because
of the limited understanding of the disease pathogenesis; as
such, joint replacement remains the preferred treatment for
patients with advanced OA.3
OA is primarily characterized by degradation of the articular
cartilage, as well as subchondral bone sclerosis, and
osteophyte formation.4,5 It is suggested that OA is caused by
the disruption of cartilage homeostatic balance between
anabolic and catabolic signals.6,7 Various risk factors, such
as abnormal mechanics, aging and inflammation, have been
identified to contribute to cartilage destruction.8?10 A better
understanding of the underlying molecular mechanisms of
deranged cartilage homeostasis may help to develop new
treatments for OA.
Recently, the function of microRNAs (miRNAs) in cartilage
homeostasis and OA disease received much attention.
miRNAs are a class of non-coding small RNAs, which regulate
gene expression through mRNA cleavage or translational
repression.11 Mice with chondrocyte-specific deletion of dicer,
which is required for miRNA biogenesis, exhibit severe
skeletal growth defects.12 This result suggests that miRNAs
have a critical role in skeletal development. Several miRNAs
have been identified involved in the regulation of cartilage
homeostasis. In IL-1?-stimulated OA chondrocytes, 42 miRNA
were downregulated, 2 miRNA (miR-491 and miR-146a) were
upregulated.13 MiR-140 was shown greatly reduced in human
OA cartilage and is suppressed by IL-1? treatment.14
Importantly, A disintegrin and metallopeptidase with
thrombospondin type 1 motif 5 (Adamts5) was identified as a direct
target of miR-140, and miR-140 knockout (KO) mice develop
more severe OA-like changes compared with wild-type (WT)
mice.15 Matrix metallopeptidase 13 (MMP13) was identified as
a direct target of miR-27b, which is a downregulated miRNA in
response to IL-1 stimulation.13 It seems that downregulation of
these miRNAs is responsible for the cartilage degradation
under the effects of IL-1. MiR-146a is intensely expressed in
cartilage in early OA, and it is strongly upregulated by IL-1?
stimulation in cultured normal human articular cartilage
chondrocytes.16 It has been suggested that miR-146a
functions as a negative feedback regulator of pro-inflammatory
signaling pathways by targeting TNF receptor-associated
factor 6 (TRAF6) and interleukin-1 receptor-associated kinase
1 (IRAK1) in the THP-1 human monocytic cell line.17 However,
the exact role of miR-146a in the pathogenesis of OA remains
unknown. Other recent findings suggest that miR-146a may
contribute to OA pathogenesis by impairing the TGF-?
signaling pathway via targeting Smad4 and increasing
apoptosis.18,19 Further in vivo functional analysis is necessary
to support this hypothesis. In this study, we sought to
determine the roles of miR-146a on cartilage homeostasis
and OA development. MiR-146a KO mice and genetic
background-matched WT mice were used to develop knee
OA with or without induced knee instability. The molecular
targets of miR-146a and potential pathway involved in the
pathogenesis of OA were further investigated.
MiR-146a KO in mice suppresses spontaneous OA. At
ages of 3, 6 and 9 months, WT and miR-146a KO mice had
intact articular cartilage surfaces and vigorous proteoglycan
staining in knee joint (Figure 1a). At 12 months of age, the WT
mice showed roughened articular surface and
glycosaminoglycan loss in femoral condyles and tibial plateaus. In
contrast, osteoarthritic changes were less severe in the
age-matched miR-146a KO mice, as evident by a higher
safranin O staining (Figure 1a). Osteoarthritis Research
Society International (OARSI) scores were markedly
decreased in miR-146a KO mice compared with that of WT
mice, indicating alleviated cartilage degeneration in
miR-146a KO mice (Figure 1b). The 12-month-old
miR-146a KO mice exhibited reduced osteophyte formation
relative to the WT mice (Figures 1c and d). Moreover, the
percentages of MMP13 and type X collagen
(Col10a1)positive chondrocytes were markedly lower in 12-month-old
KO mice than the age-matched WT mice (Figures 1e-g). The
expression of TRAF6 and IRAK1, two well-established
targets of miR-146a, did not differ in articular chondrocytes
at 12 months between WT and KO mice (Figures 1h-j),
suggesting that inflammation may not be under the regulation
of miR-146a in spontaneous OA. The absence of miR-146a
expression in miR-146a KO mice was confirmed in articular
cartilage by qPCR (Figure 1k). These results indicated that
deficiency in miR-146a alleviated cartilage degeneration in
spontaneous OA model.
MiR-146a KO in mice alleviates knee
destabilizationinduced OA . We used three different knees
destabilizationinduced OA animal models, namely, destabilization of the
medial meniscus (DMM), anterior cruciate ligament
transection (ACLT), and medial collateral ligament+ partial medial
meniscectomy (PMM), to further evaluate whether the loss of
miR-146a affected OA progression. WT mice showed severe
proteoglycan loss and articular cartilage degeneration in
knee joints at 4 weeks post DMM surgery, but the
osteoarthritic changes were less severe in miR-146a KO
mice knee joints (Figure 2a). The OARSI scores were not
significantly different in sham surgery groups in both KO and
WT mice, but markedly decreased OARSI scores in
miR-146a KO mice with knee destabilization were observed
as compared with the WT mice (Figure 2b). Consistent with
this finding, immunostaining demonstrated that the protein
expression of type X collagen and Mmp13 was significantly
reduced in the miR-146a KO mice compared with that in the
WT mice. This result indicated the cartilage degradation may
be suppressed (Figures 2c-e). Similar results were observed
in ACLT and PMM models (Supplementary Figure 1). These
results collectively indicated that the deficiency of miR-146a
in mice inhibits the degeneration of articular cartilage during
secondary OA development.
MiR-146a exacerbates pro-inflammatory factors induced
suppressing the expression of cartilage
matrixassociated genes. We speculated that OA inflammatory
microenvironment may be responsible for the miR-146a
upregulation. We found that miR-146a was significantly
upregulated in the mouse articular cartilage in DMM group
as compared with the sham-operated group at 4 weeks post
operation (Figure 3a), and higher levels of TNF-? and IL-1?
were also found in DMM groups (Figure 3b). In order to test
the effects of pro-inflammatory cytokines on the expression of
miR-146a, mouse articular chondrocytes were isolated from
the hips and knees of 7-day-old C57BL/6 mice and confirmed
by type II collagen expression using immunofluorescence
(Figure 3c). We found that IL-1?, IL-17 and TNF-?
upregulated the expression of miR-146a in mouse primary
chondrocytes, whereas the other inflammatory cytokines had no
effect on miR-146a expression (Figure 3d). Ectopic
expression of miR-146a via infection of primary mouse articular
chondrocytes with Lenti-mimic 146a reduced the protein
levels of type II collagen (Col2a1) and sex determining region
Y (SRY)-box 9 (Sox9) in the absence or presence of
pro-inflammatory factors (Figure 3e). In contrast, knocking
down miR-146a with Lenti-inhibitor 146a markedly
upregulated the protein expression of Col2a1 and Sox9, and partially
rescued the inhibition of both anabolic genes by
proinflammatory cytokines (Figure 3f). In the mice with knee
destabilization, the expression levels of Sox9 and Col2a1
were higher in miR-146a KO mice as compared with WT mice
(Figures 3g and h). These results collectively showed that
miR-146a, which is induced by pro-inflammatory cytokines,
exacerbates suppression of cartilage anabolism in the
pathogenesis of OA.
MiR-146a targets multiple genes to inhibit cartilage
anabolism. To explore the molecular mechanisms how
miR-146a regulates cartilage homeostasis, we combined
target prediction tools such as Miranda, TargetScan and
microarray gene expression analyses to search for potential
targets of miR-146a. Twelve genes were validated based on
the seed region of miR-146a in 3?-untranslated region (UTR)
of candidate gene, which is evolutionarily conserved in
mammals (Figure 4a). QPCR analysis showed that the
expression of the majority of these candidate genes can be
suppressed by miR-146a gain-of-function in mouse
chondrocytes (Figure 4b). We constructed luciferase reporters
containing WT 3?-UTR sequences of candidate genes, which
include the miR-146a binding site for the evaluation of direct
targeting by miR-146a. The results showed that miR-146a
markedly repressed the reporter activity of the 3?-UTR of the
genes encoding Tgif1, Bag1, Camta1, Sox5, Sema3g,
Camk2d, Ppp3r2 and Stim2 (Figure 4c). As the expression
of Tgif1, Camk2d and Ppp3r2 in cartilage were considerably
higher in KO mice as compared with WT mice (both with knee
destabilization) (Figures 4e-g), we further investigated if
these genes are regulated by direct binding of miR-146a.
Mutation of the 3?-UTR of these three genes abolished the
regulation by miR-146a (Figure 4d and Supplementary
Figure 2A). Moreover, overexpression of Tgif1, Camk2d and
Ppp3r2 in mouse chondrocytes significantly increased the
protein levels of Sox9 and Col2a1 (Figure 4h), which was
consistent with the results of inhibition of miR-146a in mouse
chondrocytes. Moreover, we found that overexpression of
Tgif1 markedly upregulated Ppp3r2 levels (Supplementary
Figures 2B-D). Knock-down of these three genes by specific
siRNA downregulated the expression of Sox9 and Col2a1
(Figure 4i and Supplementary Figure 2E). This result further
confirmed that miR-146a could inhibit the expression of
cartilage matrix-related genes by targeting Tgif1, Camk2d
Regulation of miR-146a level in cartilage has therapeutic
effect on OA. To investigate whether regulation of miR-146a
in OA had a therapeutic effect on OA disease, we performed
intra-articular (IA) injection of DMM-operated mice with
lentiviruses encoding miR-146a, miR-146a antagonist
sequence or corresponding control lentivirus weekly for
3 weeks 1 week after surgery (Figure 5a). The delivery of
miRNA to chondrocytes in vitro was confirmed by analyzing
miR-146a expression in mouse chondrocytes infected with
lentiviruses encoding miR-146a, whereas in vivo delivery was
confirmed by immunostaining of GFP after IA injection of
lentivirus integrated with GFP gene (Supplementary Figures
3A and B). We found that DMM mice treated with miR-146a
(Lenti-mimic 146) had significantly aggravated the extent of
articular cartilage degeneration as compared with the control
virus. Conversely, administration of miR-146a inhibitor
(Lenti-inhibitor 146a) markedly alleviated articular cartilage
degeneration caused by DMM surgery. The therapeutic effect
of miR-146a on OA progression was also reflected in OARSI
scores (Figures 5b and c). Moreover, the protein expression
of Col10a1 was significantly increased by miR-146a
overexpression and decreased by miR-146a inhibition (Figures 5d
and e), but differences in Mmp13 expression were not
significant (Supplementary Figure 3C).
Camk2d and Ppp3r2 are functional targets of miR-146a
in vivo. In order to verify the involvement of Tgif1, calcium/
calmodulin-dependent protein kinase II delta (Camk2d), and
protein phosphatase 3, regulatory subunit B, beta isoform
(Ppp3r2) in the pathogenesis of OA, we detected their
distribution in mouse knee joints by immunostaining. We
found that Camk2d and Ppp3r2 were expressed abundantly at
articular cartilage and growth plate in 3-month-old WT mice,
but their expression levels were markedly decreased in the
articular cartilage after DMM operation (Supplementary
Figures 3D and E).The expression of Tgif1 is too low to be
detected. The expression levels of Camk2d and Ppp3r2 in
articular cartilage were markedly reduced in 12-month-old WT
mice compared with that of 3-month-old WT mice. This
agerelated downregulation of Camk2d and Ppp3r2 was
significantly alleviated in KO mice as compared with that in WT mice
(Figures 6a-c). We also observed that the expression of
Camk2d and Ppp3r2 in bone marrow cells of 3-month-old
miR-146a KO mice was much higher than that of age-matched
WT mice (Supplementary Figure 3F). The protein expression
of Camk2d and Ppp3r2 were downregulated by miR-146a
overexpression and upregulated by miR-146a inhibition
(Figures 6f-h), it indicated that they are the direct targets of
miR-146a in vivo, as their 3?-UTR sequences containing
miR-146a-binding site. As one of the regulatory subunit of
calcineurin, Ppp3r2 is required for the activation of nuclear
factor of activated T cells (NFAT) proteins,20 which were
expressed significantly higher at the hyaline cartilage and
bone marrow cells of miR-146a KO mice as compared with
age-matched WT mice. The ratio of nuclear NFAT-positive
chondrocytes/total NFAT-positive chondrocytes at cartilage
was also markedly higher in 12-month-old miR-146a KO mice
than age-matched WT mice (Figures 6a, d and e and
Supplementary Figure 3F and G). Additional data showed
that SMAD family member 4 (SMAD4), a well-known mediator
of TGF-? pathway, was scarcely distributed at the hyaline
cartilage of 12-month-old WT mice, but highly expressed in
miR-146a KO mice (Supplementary Figures 3H and I).
Collectively, these results suggest that TGF-? signaling and
calcineurin-mediated activation of NFAT pathway may be
involved in the miR-146a regulation of OA progression.
miRNAs may have an important role in the pathogenesis of
OA. For the miRNAs downregulated by pro-inflammatory
cytokines, previous reports suggest that induction of MMPs
and catabolic enzymes by IL-1? were regulated by
suppression of miR-140, for example, miR-140 targets Adamts5,
which mediate degeneration of articular cartilage.14 However,
the function of miRNAs, which are upregulated by
proinflammatory cytokines treatment in OA remains ambiguous.
It is reported that miR-146a, which is induced in response to
lipopolysaccharide (LPS) and pro-inflammatory mediator
stimulation,17 was highly expressed in human RA synovial
tissue and less highly expressed in OA tissue.21 miR-146a
may have the potential to be a novel targets in OA by negative
feedback regulation of inflammatory responses,22?25 or by
promoting chondrocytes autophagy.26 However, other studies
showed that miR-146 may contribute to OA pathogenesis by
promoting VEGF expression and impairing the TGF-?
signaling pathway through targeting of Smad4.18,19 In this study, we
found that miR-146a was upregulated in cartilage in the early
stage of OA induced by knee destabilization (including PMM,
ACLTand DMM models). We speculated that OA inflammatory
environment may be responsible for the miR-146a
upregulation. We found that the mRNA levels of IL-1? and TNF-? were
elevated in OA lesions. IL-1? induced highest levels of
miR-146a in chondrocytes than that of TNF-? and IL-17,
indicating that the upregulation of miR-146a in eroded
cartilage is correlated with inflammatory cytokines especially
IL-1? synthesized by resident chondrocytes. It should be
noted that mechanical pressure injury and pro-inflammatory
cytokines secreted by synovium or meniscus are potentially
involved in the induction of miR-146a in OA cartilage.19,27
MiR-146a has been considered as a negative regulator of
inflammation,28 and administration of miR-146a prevented joint
destruction in mice with collagen-induced arthritis.29 However,
we found that abolishing the expression of miR-146a in mice by
removal of miR-146a precursor sequence in mouse genome
did not exacerbate the destruction of articular cartilage. On the
contrary, KO of miR-146a in vivo significantly alleviated the
cartilage degeneration of OA caused by aging or imbalanced
mechanical loading, suggesting that miR-146a may serve a role
other than restriction of inflammation. We found that inhibiting
the endogenous expression of miR-146a in mouse primary
chondrocytes significantly enhanced the levels of Col2a1 and
Sox9, which are well-known markers of mature chondrocytes,
and partially rescued pro-inflammatory factors induced
inhibition of both anabolic genes. Pro-inflammatory cytokines
induced miR-146a strengthened the cytokines-mediated
suppression of anabolic genes, suggesting that miR-146a is
responsible for the disordered cartilage homeostasis. The role
of miR-146a in OA was further confirmed by our in vivo
therapeutic experiment in which treated DMM mice with
miR-146a or miR-146a inhibitor significantly aggravated or
alleviated the destruction of articular cartilage, respectively. Our
study provides the first insight that regulation of miR-146a level
in vivo has a therapeutic effect for OA.
We further elucidated the mechanism of miR-146a
suppresses cartilage anabolism. We identified the targets of
miR-146a, namely, Tgif1, Camk2d and Ppp3r2. CaMKII is a
widely distributed Ser/Thr protein kinase that is encoded by
four genes (?, ?, ? and ?).30 As soon as CaMKII was activated
by Ca2+/calmodulin, CaMKII phosphorylated transcription
factor cAMP response element (CRE)-binding protein
(CREB), thereby facilitating the activation of downstream
genes.31 In addition to activating CaMKII, Ca2+ also activated
the phosphatase activity of calcineurin to dephosphorylate
NFAT by binding to the 19-kDa regulatory subunit of
calcineurin (calcineurin B).32 The activated NFAT then
translocated to the nucleus and induced the expression of
NFAT target genes.33 There are two isoforms of calcineurin B
exist in mammals, calcineurin B, Type I and calcineurin B, type
II.34 The two subunit isoforms are indistinguishable in their
binding to bacterially expressed forms of mouse catalytic
subunits,35 and are required for the activation of all NFAT
proteins.20,36 Although the importance of CaMK?CREB, as
well as calcineurin?NFAT pathway, in osteoclasts and
osteoblasts has been well documented,37?40 the contribution of both
pathways in OA remains poorly elucidated. In this study, we
identified Camk2d and Ppp3r2 were direct targets of miR-146a
in the development of OA. Ectopic expression of Camk2d and
Ppp3r2 significantly enhanced the protein amounts of Col2a1
and Sox9. Moreover, the expression of Camk2d and Ppp3r2 in
normal cartilage is mainly located at the hyaline cartilage or
growth plate, seldom distributed at the calcified cartilage or OA
cartilage. This result suggests that Camk2d and Ppp3r2 may
be required to maintain the phenotype of mature
chondrocytes. Although the specific mechanism by which the Camk2d
and Ppp3r2 regulate the expression of Col2a1 or Sox9
remains unclear, the calcineurin?NFAT pathway may be
involved in the regulation of cartilage homeostasis. We found
that miR-146a KO mice expressed more activated Nfatc1 and
NFatc2 than WT mice in articular cartilage, especially in
hyaline cartilage zone. The calcineurin?NFAT pathway had a
critical role in promoting chondrogenesis through induction of
Sox9,41,42 or by activating bone morphogenetic protein
expression.43 Mice lacking Nfatc2, a member of NFAT
transcription factor family, displayed loss of Col2a1 and
aggrecan (Acan) with high expression of specific
matrixdegrading proteinases in adult articular chondrocytes, all of
which resemble human OA.44 Further study revealed that
Nfatc2 bound to the promoter regions of Acan, Col2a1,
Mmp13 and Tnfa genes in articular chondrocytes of aged
mice,45 suggested that large changes in Sox9 and Col2a1
levels caused by overexpression and inhibition of miR-146a in
chondrocytes in vitro may attribute to regulation of Nfatc2 by
miR-146a. Moreover, cartilage-specific ablation of NFATc1 in
NFATc2 KO mice led to aggressive OA, as well as upregulation
of Mmp13.46 It may explain why the KO mice have less OA and
lower expression of MMPs than the WT mice.
It should be noted that some limitations are present in this
study. For example, safer delivery of miRNA to articular
chondrocytes in vivo should be sought because we found that
a mouse died 1 week later after the last injection in the
therapeutic experiment. We speculate the undesirable
sideeffect of lentivirus is the likely cause. Moreover, the role of
Tgif1 in the pathogenesis of OA was partly ignored because
we failed to detect it in vivo. However, Tgif1 may have the same
effect as Camk2d and Ppp3r2, as overexpression of Tgif1 in
chondrocytes greatly enhanced the expression of Ppp3r2.
Further investigation should be performed to elaborate the role
of above-mentioned genes in OA.
Inflammatory mediators in joint space greatly reduced the
capacity of chondrocytes to repair damaged cartilage by
suppressing the cartilage anabolism-related genes. Here, we
identified miR-146a, an upregulated miRNA in response to
inflammation stimulation, act as an accomplice of
proinflammatory factors in promoting the progression of OA by
targeting Camk2d and Ppp3r2 (Figure 7). Our study provides
the first in vivo evidence that inhibiting the levels of miR-146
can alleviate cartilage degeneration in OA. MiR-146a may be a
potential therapeutic target for OA.
Figure 7 Schematic model of the role of miR-146a in the pathogenesis of OA.
MiR-146a is induced in chondrocytes in an NF-?B-dependent manner by
pro-inflammatory cytokines stimulation. Once activated, miR-146a disrupts cartilage
homeostasis via targeting Camk2d and Ppp3r2, which are associated with cartilage
anabolism, thereby inhibiting the activation of downstream NFAT pathway
Materials and Methods
Mice. MiR-146a heterozygous KOs on a C57BL/6 background were obtained from
the Jackson Laboratory (Bar Harbor, ME, USA). Upon arrival at our animal facility, the
miR-146a heterozygotes were intercrossed. The miR-146a homozygous KOs
(designated as miR-146a KO) and WT C57BL/6 mice were screened by genotyping
and mating as homozygote ? homozygote or WT ? WT. The WT and miR-146a KO
offspring were used in spontaneous and surgically induced OA models. The primer
sequences used for genotyping are as follows: forward primer 5?-ACCAGCA
GTCCTCT-TGATGC-3? and reverse primer 5?-GACGAGCTGCTTCAAGTTCC-3?.
C57BL/6J male mice were purchased from Shanghai Laboratory Animal Center
(Shanghai, China), Chinese Academy of Sciences (Shanghai, China) and used in OA
therapeutic experiment. All mice were maintained under pathogen-free conditions. All
animal experiments were approved by the Institutional Biomedical Research Ethics
Committee of the Shanghai Institutes for Biological Sciences (Chinese Academy of
Experimental OA. Surgical OA was induced in 12-week-old male miR-146a
KO and WT mice through DMM (n = 10?14 per group), ACLT (n = 6 per group) or
PMM (n = 6 per group) as previously described.47?49 Briefly, the mice were
anesthetized with chloral hydrate (400 mg/kg) intraperitoneally, and the anterior
cruciate ligament (for ACLT) or medial meniscotibial ligament (for DMM) was
transected in the right knee joint. PMM model was performed by resection of the
medial collateral ligament and removal of the cranial horn of the medial meniscus.
Sham operation was done on independent mice. Mice were killed 4 weeks (DMM
and PMM groups) or 8 weeks (ACLT group) after surgery as previously
described,48?50 and right knee joints were processed for histological and
biochemical analyses. For spontaneous OA experiments, male mice were killed
and right knee joints were harvested from 3-, 6-, 9- and 12-month-old miR-146a KO
and WT mice. For the therapeutic experiment, 3-month-old C57BL/6 male mice
receiving DMM surgery were assigned into four groups (n = 8?9 per group). One
week after surgery, mice were treated with IA injection of lentivirus-incorporated
miR-146a mimic and inhibitor, or corresponding control lentivirus (1 ? 109
plaqueforming units in a total volume of 5 ?l) once every week for 3 weeks. Mice were
killed 3 weeks after the first IA injection for histological analyses.
Histology and immunohistochemistry. Right knee joints were fixed in
4% paraformaldehyde for 24 h, decalcified in 12.5% EDTA (pH 7.4) for 2 weeks,
embedded in paraffin. In all, 5 ?m sagittal sections were cut from the whole medial
compartment of the joints and stained with safranin O and fast green. Cartilage
destruction in the medial tibial plateau of the joint was scored by two blinded
observers using the OARSI system.51 In short, five serial sections from each
individual were scored according to the formula: score = grade ? stage. The individual
score was calculated by averaging the scores of five sections. Osteophyte
development in the medial tibial plateau was determined by safranin O staining
and quantified as described previously.47 Immunohistochemistry was performed
according to the manufacturer?s instructions (Maixin Biotech. Co., Ltd, Fuzhou, China).
Sections were deparaffinized and rehydrated. Antigen retrieval was performed by
digesting with pepsin, trypsin or hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) at
37 ?C for several minutes. Sections were blocked with 5% goat serum, 0.1% tween-20
in phosphate-buffered saline (PBS) for 30 min at room temperature, and then
incubated with following primary antibodies to Mmp13 (1:200, ab39012; Abcam,
Cambridge, UK), type X collagen (1:1000, ab58632, Abcam), GFP (1:1000, ab290,
Abcam), Camk2d (1:100, 20667-1-AP; ProteinTech Group, Chicago, IL, USA), Ppp3r2
(1:100, 14005-1-AP, ProteinTech Group), Nfatc1 (1:50, ab175134, Abcam), Nfatc2
(1:100, 22023-1-AP, ProteinTech Group) and isotype control antibody (ab27478,
Abcam) overnight at 4 ?C. Streptavidin-horse radish peroxidase (HRP) detection
system (Maixin Biotech. Co., Ltd) was used to detect antigen and was visualized with
2, 2?-diaminobenzidine tetrahydrochloride. Sections were then counterstained with
hematoxylin, and sealed with mounting medium. Images were obtained with ZEN lite
2011 software and Axio imager A2 microscope (Carl Zeiss, Oberkochen, Germany).
Cell culture. HEK293T cells were purchased from ATCC (Manassas, VA, USA),
and mouse primary articular chondrocytes were isolated from femoral heads,
femoral condyles and tibial plateaus of mice postnatal 1 week as previously
described.52 Chondrocytes were maintained as a monolayer in Dulbecco?s
modified Eagle?s medium (DMEM) supplemented with 10% FBS (Gibco, Thermo
Fisher Scientific, Waltham, MA, USA) and 1% penicillin?streptomycin (Hyclone, GE
Healthcare Life Sciences, Logan, UT, USA) and identified by type II collagen
fluorescence immunoassay. Chondrocytes at passage 1?2 (P1?P2) were used in all
Immunofluorescence. Chondrocytes at P1 were seeded in a 24-well culture
plate at 2 ? 104 cells per well. At 70?80% confluence, the cells were rinsed twice
with PBS, fixed in 4% paraformaldehyde for 10 min at room temperature. After three
washes in PBS, the cells were blocked with 5% bovine serum albumin (BSA), 0.3%
tween-20 in PBS for an hour, and incubated with anti-type II collagen antibody
(1 : 200, BS1071; Bioworld Technology, St. Louis Park, MN, USA) diluted in PBS
overnight at 4 ?C. After three washes in PBS, the cells were incubated with the
fluorescein isothiocyanate-labeled secondary antibody diluted 1:1000 in PBS for 2 h
at room temperature. After three washes in PBS, images were photographed
protected from light using a microscope equipped with a mercury lamp.
Lentiviruses, plasmids and siRNAs. We purchased lentiviruses that
expressed a mature sequence of murine miR-146a (Lenti-mimic 146a) or an
antagonist sequence (Lenti-inhibitor 146a) and negative control viruses
(GenePharma Co., Ltd, Shanghai, China) to effectively reinforce or silence miR-146a
expression in vitro and in vivo. Chondrocytes were seeded on the 12-well plate
1 day before transduction and then infected with viruses for 24 h. Then, cells were
processed for further analyses after 72 h. We also purchased vector-based
miR-146a precursor and inhibitor clones (GeneCopoeia, Rockville, MD, USA). For
gene expression experiments, full-length mRNA encoding mouse TGFB-induced
factor homeobox 1 (Tgif1, gene ID 21815), mouse Camk2d (gene ID 108058) and
mousePpp3r2 (gene ID 19059) were cloned into a pEGFP-N1 vector (Clontech,
Takara Bio USA, Inc., Mountain View, CA, USA). siRNAs targeting mouse Tgif1,
Camk2d and Ppp3r2 are obtained from GenePharma Co., Ltd. Scrambled siRNA
was used as a control. Chondrocytes were transfected with either the expression
plasmid or siRNA using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific,
Waltham, MA, USA). Cells were collected 48 h after transfection for further
analyses. Experiments were performed in duplicates and repeated at least three
times independently. The sequences of siRNAs and primers that were used for
gene cloning can be found in Supplementary Table 1.
qPCR. Mouse cartilage was cut with a surgical blade from the medial tibial
plateau and medial femoral condyle; avoid the contamination from the brown
subchondral bone. Cartilage was rinsed with PBS, transferred to 1 ml TRIzol
reagent (Invitrogen), and then homogenized with a power homogenizer at highest
speed on ice. Total RNA was extracted from homogenized cartilage samples or
primary cultured chondrocytes with TRIzol reagent and treated with DNase I
(Sigma-Aldrich) to remove genomic DNA. For mRNA expression, RNA was reverse
transcribed into complementary DNA using a PrimeScript RT Master Mix Kit (Takara
Bio Inc., Dalian, China) and then analyzed using real-time PCR (SYBR Green). All
mRNA expression was normalized by housekeeping gene
glyceladehyde-3phosphate dehydrogenase (GAPDH). qPCR primers are summarized in
Supplementary Table 2. Mature miR-146a were assayed with specific Taqman
kits from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, USA) and
normalized by U6 snRNA.
Western blot analysis. Cells were lysed in lysis buffer (1 ? PBS, 0.1% SDS,
0.5% sodium deoxycholate, 1% NP-40, 5 mM EDTA, 1 mM PMSF and protease
inhibitor cocktail), subjected to SDS-PAGE, and transferred to a nitrocellulose
membrane. The membranes were blocked with 5% BSA and probed with antibodies
against COL2A1 (1:1000, BS1071; Bioworld Technology), SOX9 (1:1000, ab26414,
Abcam) or GAPDH (1:5000, G9545, Sigma-Aldrich).
3?-UTR cloning and luciferase assay. The 3?-UTRs of candidate genes
containing the predicted miR-146a target sequences were PCR amplified and then
inserted between XhoI and NotI site downstream from the Renilla luciferase gene in a
psiCHECK-2 vector (Promega, Sunnyvale, CA, USA). Binding site mutations were
performed using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent
Technologies, Inc., Santa Clara, CA, USA). Primers that used for 3?-UTRs cloning are
summarized in Supplementary Table 3. For luciferase assay, 293T cells were seeded
at 3000 cells per well in a 96-well plate. The cells in each well were transfected with a
mixture of 20 ng of luciferase constructs and 80 ng of miR-146a precursor mimic or
control mimic plasmid after 24 h. At 48 h after transfection, cells were lysed, and
luciferase activity was measured on a luminometer using dual-luciferase reporter
assay system (Promega) according to the manufacturer?s instructions. Luciferase
activity was normalized by firefly luciferase activity. Experiments were performed in
triplicate and repeated at least three times independently.
mRNA microarray. Total RNA was isolated from mouse chondrocytes
transfected with miR-146a inhibitor or control inhibitor using TRIzol reagent. RNA
integrity was assessed using standard denaturing agarose gel electrophoresis.
Whole Mouse Genome Oligo Microarray (4x44K, Agilent Technologies) platform
was used for microarray analysis. Sample preparation and microarray hybridization
were performed based on the manufacturer?s standard protocols. Agilent Feature
Extraction software (version 220.127.116.11, Agilent Technologies, Inc., Santa Clara, CA,
USA) was used to analyze acquired array images. Quantile normalization and
subsequent data processing were performed using the GeneSpring GX v11.5.1
software package (Agilent Technologies).
Statistical analyses. All statistical analyses were performed with SPSS 19.0
software (SPSS Inc., IBM Corporation, Armonk, NY, USA). Data are presented as
mean ? S.D. Statistical differences between two groups were determined by
two-tailed Student?s t-test or a Mann?Whitney test. Po0.05 was considered
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. We thank Dr Ning Zhang (Chinese University of Hong
Kong) for providing technical guidance in OA models. This work was supported by
grants from National Natural Science Foundation of China (no. 81190133, 81401844
and 81572123), Science and Technology Commission of Shanghai (no.
14431900900, 14140903700 and 15411951100), Shanghai Municipal Commission
of Health and Family Planning (no. 2013ZYJB0501) and Shanghai Municipal
Education Commission-Gaofeng Clinical Medicine Grant Support (20161314).
Xudong Zhang and Xiaoling Zhang designed the experiments, analyzed the data and
prepared the manuscript. Xudong Zhang and CW performed most of the
experiments; JZ helped with mouse experiments. JX and YG were responsible for
study conduct and data collection. LD and YH provided technical support. Dr S-CF
helped with carefully revising the manuscript. KD and Xiaoling Zhang supervised the
study and revised the manuscript.
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