osteoarthritis ( oa ) is a degenerative joint disease characterized by  articular cartilage degradation , subchondral bone thickening , and osteophyte formation.1 , 2 cartilage cellularity is reduced in oa through chondrocyte death , in which chondrocytes are stimulated by cytokines and growth factors to undergo catabolic and abnormal differentiation , leading to extracellular matrix ( ecm ) degradation.3 , 4 , 5 , 6 in oa , the medial compartment of the articular cartilage is the most susceptible to degeneration , whereas the lateral compartment remains relatively unaffected.7 , 8 differences in mechanical factors underlie the disparity in disease susceptibility between the medial and lateral compartments ; the damaged cartilage regions are usually subjected to mechanical loading , whereas the intact regions are not .
the chondrocytes in the damaged cartilage are particularly susceptible to mechanical stress , and the tensile properties of the damaged cartilage are lost because of the destructed collagen network.9 , 10 circular rnas ( circrnas ) are a large class of noncoding rnas that exist ubiquitously in the cytoplasm of eukaryotic cells;11 , 12 these endogenous rnas are characterized by stable structure and high tissue - specific expression . compared with linear rnas , circrnas can remarkably undergo non - canonical splicing without a free 3 or 5  end.14 , 15 recent reports show that circrnas function as microrna ( mirna ) sponges that naturally sequester and competitively suppress mirna activity .
the involvement of circrnas is demonstrated in the development of several types of diseases , such as atherosclerosis and nervous system disorders.17 , 18 , 19 however , the role of circrnas in cartilage and their overall contribution to oa pathogenesis are still unknown .
the present study identified a small number of up- or downregulated circrnas in different cartilage regions .
we specifically identified a new circrna , called mechanical stress - related circrna ( circrna - msr ) , involved in mechanical stress .
we demonstrate that circrna - msr controls tumor necrosis factor alpha ( tnf- ) expression and promotes the degradation of ecm .
the cartilage was assessed by histologic examination , and the histologic scores were graded according to a modified mankin scale .
the  score of < 4 points was considered intact cartilage and a score of > 6 represented damaged cartilage .
hierarchical clustering revealed the circrna expression in the cartilage samples ( figure  1a ) .
the scatter and volcano plots showed varied circrna expressions between the damaged and intact cartilage samples ( figures 1b and 1c ) .
we identified 104 differentially expressed circrnas in the damaged cartilage compared to the intact cartilage .
of these circrnas , 44 were upregulated and 60 were downregulated in the  damaged tissue samples ( table s1 ) . to validate the circrna microarray results , we performed qpcr to analyze the changes in  expression among the differentially expressed circrnas .
the data confirmed that circrna_000598 , circrna_103387 , circrna_101975 , and circrna_100226 ( circrnas - msr ) were overexpressed in the damaged region compared with the intact region of the cartilage in oa ( figure  2a ) .
a  total of 89 circrnas and 61 mrnas were noted in the damaged cartilage network , whereas 94 circrnas and 36 mrnas were in the intact cartilage network . in these co - expression networks ,
circrnas and mrnas were connected by a string , indicating a tight correlation between these genes and a potential regulatory relationship ( figures s1 and s2 ) .
furthermore , we constructed another network with circrnas , mrnas , and the common binding mirnas .
the differentially expressed circrnas were annotated in  detail with the mirna interaction information ( table s1 ) .
we selected cartilage - specific mrnas according to gene ontology ( go )  and pathway analyses ( figures 2b and 2c ) .
we constructed a network of circrnas - mirnas - mrnas , with a total of 45 circrnas , 42 mrnas , and 42 mirnas , by merging the commonly targeted mirnas of circrnas and mrnas ( figure  3 ) .
this indicated the tight  correlation and regulation relationship of these genes in the network .
we assumed that circrna - msr was a special cartilage circrna ( hsa_circ_100226 , i d circ_0005567 in circbase ; http://circbase.org ) , with its gene located at chr1:51868106 - 51874004 and the symbol of the associated gene being eps15 .
the circrna - msr was chosen because it is one of the circrnas indicated to be associated with mechanical stress and inflammatory response according to the bioinformatics analysis mentioned above . to investigate the effect of mechanical stress on chondrocytes , the cells were exposed to pre - optimized magnitudes of cyclic tensile strain ( cts ) using flexcell-5000 ( figure  4a ) .
the expression levels of col2a1 and aggrecan were significantly downregulated , whereas the expression levels of tnf- and il-1 were significantly upregulated by cts for 24  hr ( figure  4b ) .
moreover , we found that circrna - msr expression paralleled tnf- expression under mechanical stress ( figure  4c ) .
therefore , we speculated the co - regulated expression of circrna - msr and tnf- under mechanical stress in  vitro conditions . to analyze the effects of circrna - msr on tnf- expression in chondrocyte ecm degradation , we examined the effect of circrna - msr knockdown on damaged chondrocytes .
the small interfering rna ( sirna ) used in this experiment was specific for circrna - msr .
as a consequence of this inhibition , the mrna expression for tnf- decreased , whereas the ecm expression significantly rose by si - msr treatment ( figure  5a ) .
the immunofluorescence results also showed that ecm proteins were significantly elevated by the si - msr treatment ( figures 5b and 5c ) .
circrnas have been reported to function as mirna sponges that naturally sequester and competitively suppress mirna activity .
we assumed that circrna - msr functions as a decoy to regulate tnf- expression through the same mechanism . according to the  informatics analysis
, there were five mirna - binding sites for circrna - msr , and they were mir-138 , mir-145 , mir-24 , mir-620 , and mir-875 ( figure  6a ) .
the circrna - msr 3 utr sequence matched these mirnas , and the tnf- 3 utr matched mir-875 ( figure  6b ) .
thus , we identified a common mirna ( mir-875 ) for circrna - msr and tnf- targets .
oa is a multifactorial disease characterized by progressive inflammation , pain , and cartilage destruction in load - bearing surfaces of the knee joints . among various pathogenic factors ,
aberrant mechanical stimulation results in a physiological imbalance between mechanical stress on the joint and the joint s ability to withstand such stress .
chondrocytes are directly exposed to compression force during cartilage loading ; thus , the ecm of chondrocytes tends to stretch the cells during cartilage compression .
many studies on oa focused on the epigenetic regulation of its pathogenesis and potential targets for therapy , including mirnas and long noncoding rnas ( lncrnas ) . however , the occurrence of circrnas in cartilage remains largely unknown .
this study identified a number of aberrantly expressed circrnas in damaged regions compared with the intact regions of oa .
we found that circrna - msr is vital under mechanical stress in loss - of - function experiments and that it regulates the tnf- in the process of ecm degradation in chondrocytes .
recent studies showed that numerous exonic transcripts can form circrnas through non - linear reverse splicing or gene rearrangement .
the two properties of circrnas are the most important : first , they are highly conserved sequences ; second , they show a high degree  of stability in mammalian cells . compared with other noncoding rnas , such as mirnas and lncrnas ,
these properties provide circrnas with the potential to be used as ideal biomarkers and potential therapy targets .
we assumed that circrna - msr functioned as a decoy to regulate tnf- expression through the same mechanism .
we found that circrna - msr harbors mirna - binding sites , including mir-138 , mir-145 , mir-24 , mir-620 , and  mir-875 .
in addition , mir-875 can bind to the 3 utr of tnf-. however , this study focused on mir-875 as  the only mirna that can target both circrna - msr and tnf-. this study demonstrated that circrna - msr regulated tnf- expression and participated in the chondrocyte ecm degradation process .
we confirmed that the silencing of circrna - msr by sirna can suppress tnf- expression and increase ecm formation .
thus , circrna - msr can be used as a potential target and specific sirnas can be used as therapeutic agents in oa therapy .
the most attractive aspect of this therapeutic is the ability to target the gene(s ) , which may not be possible with small molecules or protein - based drugs .
this opens up a whole new therapeutic approach for the treatment of oa by targeting genes that are causally involved in the pathological process .
although this approach is promising , several challenges have been identified , including the lack of stability against extracellular and intracellular degradation by nucleases , poor uptake and low potency at target sites of sirnas , and off - target effects .
collectively , our data indicate that 104 circrnas were either over- or underexpressed in oa .
the observed changes were suggested to have biologic effects and that circrnas are key regulators of gene expression .
we confirmed that circrna - msr is the decoy for tnf-. the mechanism needs to be confirmed with further specific studies .
deciphering the precise molecular mechanisms of circrna function in oa is critical to understanding oa pathogenesis and exploring new potential therapeutic targets .
oa cartilage was isolated from the knee joints of 30 patients undergoing total knee arthroplasty .
joint tissue was immediately shock - frozen in liquid nitrogen , and the articular cartilage was isolated within 24  hr from the condyles and tibia plateaus with the use of a surgical blade .
the study was approved by the human ethics committee of the peking university third hospital ( china ) .
the cartilage was removed and fixed in 4% paraformaldehyde in pbs solution ( ph 7.4 ) for 48  hr at 4c , and then it was demineralized in 15% edta ( ph 7.2 ) in pbs for 2  weeks .
the cartilage specimens were dehydrated in a graded series of alcohol and xylene , and then they were embedded in paraffin and cut serially into 5  m sagittal sections . the sections were stained with toluidine blue , safranin - o , and h&e as per routine protocol .
scores of < 4 and > 6 points indicated intact and damaged cartilage , respectively .
joint tissue was immediately shock - frozen in liquid nitrogen , and then the articular cartilage was isolated from the condyles and tibia plateaus using a surgical blade within 24  hr .
afterward , the samples were homogenized in trizol reagent ( invitrogen ) , and the total rna in each sample was quantified using a nanodrop nd-1000 . sample preparation and microarray hybridization
the total rna from each sample  was amplified and transcribed into fluorescent crna utilizing random primers according to the arraystar super rna labeling protocol .
the labeled crnas were hybridized onto the arraystar human circrna array ( 8   15 k ) .
we selected specific mrnas to construct the network according to our previous microarray data and the enrichment analyses of go .
co - expression networks were constructed according to the normalized signal intensities of circrnas and mrnas in the original microarray data .
pearson s correlation analysis was applied to measure the significance of the correlation of the expressions between each gene pair .
when the expression levels of two genes were similar above a preselected threshold in the pearson analysis , they were considered to exhibit a co - expression relationship and would be connected .
each gene corresponded to a node , and two genes were connected by a string , indicating a tight correlation .
the degree of correlation determined gene importance in the  network.30 , 31 an mrna - mirna - circrna network was constructed according to the common target mirnas of the circrnas and mrnas .
the interactions of circrnas and mrnas with mirnas were predicted with the arraystar mirna target prediction software based on targetscan and miranda.32 , 33 donor chondrocytes were isolated as previously described .
chondrocytes ( 5   105/well ) were at passage 2 and grown on pronectin f - coated bioflex six - well culture plates ( flexcell international ) to 80% confluence .
our previous study selected the pre - optimized magnitudes of cts , so the chondrocytes were enforced at 10% elongation ( 0.5  hz ) for the indicated times.34 , 35 the sirnas targeting circrna - msr ( referred to as si - circmsr ) were designed and synthesized by ribobio .
chondrocytes were transfected with si - circmsr using lipofectamine 3000 ( invitrogen ) according to the manufacturer s protocol .
prior to  transfection , the culture medium was replaced with a medium without antibiotics , and the cells were cultured for 24  hr ( cyagen biosciences ) .
the rnai lipofectamine 3000 complex was prepared  by  mixing for 20  min , and the complex was then added to each cell .
total rna was isolated from cartilage tissues or monolayer - cultured primary chondrocytes using trizol reagent .
homogenized tissue samples were in 1  ml trizol reagent per 50100  mg , and the lysed cells were directly added to 1  ml trizol reagent in a 3.5-cm diameter dish . for mirna qpcr analysis , reverse transcription of specific mirnas was performed with the bulge - loop mirna primer set ( ribobio ) according to the manufacturer s instructions . for mrna analysis ,
the  mrna expression levels were reported relative to glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) , whereas mirna expression levels were reported relative to u6 .
the primers used in the present study are as follows : tnf- forward : 5-cctctctctaatcagccctctg-3 , reverse : 5-gaggacctgggagtagatgag-3;circrna - msr forward : 5-tccagtctgatccttttgttgg-3 , reverse : 5-ctgtttcttgctgtagacggct-3;hsa_circrna_103387 forward : 5-agtctttccaccttggctct-3 , reverse : 5-tggacagggtacttctcgttt-3;hsa_circrna_000598 forward : 5-gtcccttccctgtcactacct-3 , reverse : 5-tctgttgatgccgccttgg-3;hsa_circrna_101975 forward : 5-gcccaaaccagacctcactt-3 , reverse : 5-tccttctcgggctcctga-3 ; andgapdh forward : 5-gggaaactgtggcgtgat-3 , reverse : 5-gagtgggtgtcgctgttga-3. tnf- forward : 5-cctctctctaatcagccctctg-3 , reverse : 5-gaggacctgggagtagatgag-3 ; circrna - msr forward : 5-tccagtctgatccttttgttgg-3 , reverse : 5-ctgtttcttgctgtagacggct-3 ; hsa_circrna_103387 forward : 5-agtctttccaccttggctct-3 , reverse : 5-tggacagggtacttctcgttt-3 ; hsa_circrna_000598 forward : 5-gtcccttccctgtcactacct-3 , reverse : 5-tctgttgatgccgccttgg-3 ; hsa_circrna_101975 forward : 5-gcccaaaccagacctcactt-3 , reverse : 5-tccttctcgggctcctga-3 ; and gapdh forward : 5-gggaaactgtggcgtgat-3 , reverse : 5-gagtgggtgtcgctgttga-3. the cultured cells were rinsed in pbs and fixed with 4% paraformaldehyde for 15  min at room temperature .
the cultured cells were incubated with anti - col2 ( 1:200 dilution ) and aggrecan ( 1:200 dilution ) at 4c overnight .
the cells were subsequently incubated for 1  hr with fluorescein isothiocyanate - conjugated affinipure goat anti - rabbit igg ( 1:100 dilution ) .
finally , the samples were incubated for 5  min with hoechst 33342 and observed with a confocal microscope ( fv  1000 olympus ix-81 ) .
the results are reported as the mean   sem ; p  < 0.05 was considered statistically significant .
 circular rnas ( circrnas ) are involved in the development of various diseases ; however , knowledge on circrnas in osteoarthritis ( oa ) is limited . 
 this study aims to identify circrna expression in different regions affected by oa and to explore the function of mechanical stress - related circrnas ( circrnas - msr ) in cartilage . 
 bioinformatics was employed to predict the interaction of circrnas and mrnas in the cartilage . 
 loss - of - function experiments for circrnas - msr were performed in  vitro . 
 a total of 104 circrnas were differentially expressed in damaged versus intact cartilage . of these circrnas , 44 and 60 were upregulated and downregulated , respectively , in the damaged tissue . 
 circrna - msr expression increased under mechanical stress in chondrocytes . 
 circrnas - msr were silenced using small interfering rna , and knockdown of circrnas - msr could suppress tumor necrosis factor alpha ( tnf- ) expression and increase extracellular matrix ( ecm ) formation . 
 our results demonstrated that circrnas - msr regulated tnf- expression and participated in the chondrocyte ecm degradation process . 
 we propose that the inhibition of circrnas - msr could inhibit the degradation of chondrocyte ecm and knockdown of circrnas - msr could be a potential therapeutic target for oa . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest