Circ_0136474 and MMP‐13 suppressed cell proliferation by competitive binding to miR‐127‐5p in osteoarthritis

Abstract Osteoarthritis (OA) is a prevalent degenerative joint disease whose pathogenesis remains unclear. The research aims to investigate the roles of Circ_0136474/miR‐127‐5p/MMP‐13 axis in OA. Differentially expressed circRNAs and miRNAs in OA cartilage tissue were screened out and visualized by R project based on RNA‐seq data and microarray data respectively. qRT‐PCR was carried out for detection of relative expression levels of Circ_0136474, miR‐127‐5p, MMP‐13 and other inflammatory factors and Western blot analysis was conducted to detect the protein expression level of MMP‐13. CCK‐8 assay and flow cytometry were conducted to determine cell proliferation and cell apoptotic ability respectively. RNA‐fluorescence in situ hybridization (RNA‐FISH) experiments were conducted to confirm the immune‐localization of the Circ_0136474 and MMP‐13 in human tissues. Targeted relationships were predicted by bioinformatic analysis and verified by dual‐luciferase reporter assay. Our findings revealed that the expression levels of both Circ_0136474 and MMP‐13 in OA cartilage tissue were significantly higher than that in normal cartilage tissue. Circ_0136474 could suppress cell proliferation by facilitating MMP‐13 expression and suppressing miR‐127‐5p expression in OA. Overexpression of miR‐127‐5p negatively regulated MMP‐13 expression to enhance cell proliferation. Our study demonstrated that Circ_0136474 and MMP‐13 suppressed cell proliferation, while enhanced cell apoptosis by competitive binding to miR‐127‐5p in OA, which may well provide us with a new therapeutic strategy for osteoarthritis.


| INTRODUC TI ON
Osteoarthritis (OA) is one of the most common joint diseases mainly characterized by the degradation of the articular cartilage and osteophyte formation, 1,2 which is also regarded as the most common chronic and potentially irreversible disease that affects the joints globally. 3 OA is highly prevalent among the elder and also one of the three most frequent causes of disability in younger people. 4 Although OA therapy has been significantly upgraded, more optimal and integrated treatment has yet to be developed. 5 Hence, it is vital to study underlying mechanisms of OA pathogenesis, which will provide us with new therapeutic strategies for treatment of OA. Circular RNAs (circRNAs) are endogenous RNAs featuring for covalent loop structures with neither 5′ to 3′ polarity nor a polyadenylated tail. 6 It was reported that circRNAs regulate the gene expression in both transcriptional and post-transcriptional process. 7 Recently, circRNAs, novel noncoding RNAs with varied biological functions and pathological implications, have been shown to be closely related with various diseases, including osteoarthritis, atherosclerotic vascular disease risk, neurological disorders, Parkinson's disease, Alzheimer's disease, schizophrenia and so on. 8 However, the roles of circRNAs, as well as circRNA and miRNA axis in the development and progression of OA are little understood. In this study, our attention is mainly fixed on the function of Circ_0136474 in OA progression.
miRNA is one of the most eminent classes of ncRNAs and involved in several aspects of gene regulation in eukaryotes. miRNAs always serve as fine-tuning regulators such as onco-miRNAs or tumour-suppressor miRNAs for diverse biological processes, which is mainly dependent on its downstream genes. 9 Previous studies showed that miR-9 was up-regulated in late-stage OA cartilage and directly targeted MMP-13. 10 Besides, Okuhara et al reported that miR-146a could also directly target MMP-13 and was significantly higher during the early stages of OA than during the later stages. 11 Based on latest research, miR-127-5p regulated MMP-13 expression and IL-1β-induced catabolic response in human chondrocytes. 12 Matrix metalloproteinases (MMPs) family function in irreversible matrix degradation and subsequent joint destruction in OA. 13 Previous reports showed that Interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) are two most potent catabolic factors that increase the production of inflammatory mediators and the expression of MMPs in chondrocytes. 14 Among these MMPs, MMP-13 has caught a lot of attention in that it was significantly overexpressed in both joints and articular cartilage in OA patients and could hardly be detected in normal tissues. Besides, MMP-13 was known to function as an extracellular matrix (ECM)-degrading enzyme in OA joints. 15,16 In clinical samples, MMP-13 was abnormally expressed during different stages of OA process and was found to up-regulated during the early stage and down-regulated during the late stage in human OA cartilage, which indicated its central node in the cartilage degradation network and contribution of MMP-13 to the initiation/ onset of OA. 17,18 In addition, the activity of MMP-13 can be regulated at multiple levels, including epigenetic modification, transcriptional regulation and post-transcriptional regulation by ncRNAs. 19,20 Furthermore, it was revealed that MMP-3 and MMP-13 were both up-regulated in articular chondrocytes of OA patients. 21 Along with the illumination of circRNAs acting as endogenous miRNA sponges by competitive binding to miRNA response elements to suppress their expression, a new mode of action for cir-cRNAs was unveiled. 22 Interaction between miRNA and circRNAs has already been validated to be very significant in cases of osteoarthritis, gastric cancer, colorectal cancer and neurodegenerative pathologies. For instance, circRNA-CER (Circ_100876) serves as a miR-136 sponge, sequestering miRNAs and controlling the expression level of MMP13 and participating in the process of chondrocyte ECM degradation in OA. 23 Although quite a lot of miRNAs have been reported to play a pivotal role in OA, 24 the specific micro-environmental factors that are involved in this pathological process have not been fully elucidated. Therefore, researches on the effects of both miRNAs and their potential circRNA regulators on OA progression may help clarify the pathogenesis of OA.
Our study showed Circ_0136474 promoted MMP-13 expression via inhibiting miR-127-5p, which might well provide us with more potential and therapeutic strategies for treatment of OA.

| RNA-FISH assay
The FISH assay was performed according to the manufacturer's protocol (Sino Biological Inc, Beijing, China). Briefly, after the fixed slides being dehydrated, the probes specific to Circ_0136474 and MMP-13 were added to the slides and then pre-denatured at 78℃ for 5 minutes. Hybridization was then carried out at 42℃ overnight. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma). Images were examined with a Zeiss LSM 700 Meta confocal microscope (Jena, Germany).

| Cell culture and cell transfection
Human OA articular chondrocytes were isolated from articular cartilage of OA knee joints. In brief, articular cartilage was dissected and subjected to sequential digestion with both col-

| qRT-PCR
TRIzol TM Reagent (Thermo Fisher Scientific) was utilized for isolation of total RNA. RNase-R digestion treatment was conducted at 37°C by using RNase-R (Epicenter) 2 U/mg for 25 minutes.
Thereafter, RNA was reversely transcribed into cDNA with Reverse Transcription Kit (Takara, Dalian, China). Subsequently, qRT-PCR was carried out by SuperReal SYBR Green kit (Thermo Fisher Scientific) following manufacturer's instructions. The expression levels of mRNAs were normalized against GAPDH and relatively quantified using 2 −ΔΔCt method. The expression levels of miRNA and circRNA were normalized against U6 and relatively quantified using 2 −ΔΔCt method. The primers for qRT-PCR in this study are enlisted in Table 1. The relative luciferase activities should be measured 48 hours after transfection. for 1 hours. Primary antibodies were as follows: rabbit anti-MMP-13 (ab38012, 1:5000, Abcam) and rabbit anti-GAPDH (ab9485, 1:2500, Abcam). Primary antibodies were all purchased from Abcam. Images were obtained by a Tanon-4200 Chemiluminescent Imaging System (Tanon, China).

| Statistical analysis
ANOVA, along with the t test were adopted to test the statistical differences among multiple groups. Data were obtained from at least three independent tests. All statistical analysis was carried out using GraphPad Prism 6.0 software (La Jolla, CA, USA). The differences were deemed statistically significant with * P < 0.05.

| Circ_0136474 was up-regulated in OA cartilage tissues and could be targeted by miR-127-5p
Differentially expressed circRNAs between OA cartilage tissues and normal cartilage tissues were filtered out, top 20 of which were visualized in heat map in Figure 1A. Among them, Circ_0136474 showed a robust increase in OA. And we further detected the expression level of Circ_0136474 in OA tissues and normal tissues. Figure 1B revealed that relative expression of Circ_0136474 was largely higher in OA than in normal tissues. In general, RNase R can digest liner RNA but not circRNA. As shown in Figure 1C, the relative expression of GAPDH was greatly decreased after digestion by RNase R and the relative expression of Circ_0136474 was not affected, suggesting the cyclic structure of Circ_0136474.
Furthermore, differentially expressed miRNAs between OA cartilage tissues and normal tissues were filtered out from GSE105027 and visualized in heat map in Figure 2A.  Figure 2C showed us F I G U R E 1 Circ_0136474 expression was significantly higher in OA than in normal tissues. A, Heatmap contains expression profiles of 20 different circRNAs between normal tissues and OA cartilage tissues and Circ_0136474 showed a robust increase in OA. B, It was verified by qRT-PCR that Circ_0136474 was significantly higher in OA cartilage tissues than in normal cartilage tissues. C, Linear RNA can be digested by RNase R and GAPDH expression was significantly decreased after digested with RNase R while Circ_0136474 has not been affected significantly. **P < 0.01 and ***P < 0.001 meant that there was a significant difference compared with NC group that overexpression of Circ_0136474 tremendously increased Circ_0136474 expression, which was instead largely reduced with either si-Circ_0136474-1 or si-Circ_0136474-2 (P < 0.01).

| miR-127-5p could directly target MMP-13 and suppress its expression in OA
After analysing the sequence of MMP-13, targeted relationship was uncovered between miR-127-5p seed region and MMP-13 3′UTR region based on circular RNA Interactome. Relative luciferase activity in that group co-transfected with miR-127-5p mimics and MMP-13 WT was lower than that of group with cotransfection of both miR-127-5p NC and MMP-13 WT, suggesting the direct binding between miR-127-5p and MMP-13 in OA ( Figure 3A, P < 0.01). Thereafter, qRT-PCR was adopted for validation of transfection efficiency. Overexpression of miR-127-5p tremendously increased miR-127-5p expression, which was largely reduced with inhibitor of miR-127-5p as shown in Figure 3B. Based on Figure 3C, inhibition of miR-127-5p greatly increased both mRNA expression and protein expression of MMP-13, which was largely decreased with overexpression of miR-127-5p (P < 0.01).
To summarize, miR-127-5p could directly target MMP-13 and suppress its expression in OA. Thereafter, the relative expression of miR-127-5p and MMP-13 in both OA cartilage tissues and normal cartilage tissues was measured as shown in Figure 3D,E respectively. miR-127-5p expression was significantly down-reg- Circ_0136474 were co-localized in the cytoplasm ( Figure 3F). As shown in Figure 3G, overexpression of Circ_0136474 or inhibition of miR-127-5p greatly increased MMP-13 protein expression, which was largely reduced with si-Circ_0136474-2 or miR-127-5p mimics (P < 0.01). In addition, si-Circ_2 alone suppressed protein expression of MMP-13, which could be rescued to normal expression level after co-transfection with miR-127-5p inhibitor. Besides, p-circ alone increased protein expression of MMP-13, which could be retrieved to normal expression level after co-transfection with

| Overexpression of Circ_0136474 promoted cell apoptosis yet suppressed cell proliferation by promoting the expression of MMP-13 yet suppressing miR-127-5p expression
To further investigate the roles of Circ_0136474/miR-127-5p in OA progression, flow cytometry and CCK-8 assay were adopted for assessment of cell apoptotic rate and cell proliferation rate respectively. As shown in Figure 4A,B, either si-Circ_0136474-2 or miR-127-5p mimics could restrain cell apoptosis, which could be instead enhanced with the overexpression of Circ_0136474 or miR-127-5p inhibitor. Figure 4C reveals that the overexpression of F I G U R E 4 Circ_0136474 affected cell apoptosis and proliferation by down-regulation of miR-127-5p. A, B, Si-circ and miR-127-5p mimics reduced the cell apoptotic rate in OA cartilage tissues, while p-circ and inhibitor enhanced the cell apoptotic rate markedly. C, The results of cell proliferation showed that si-circ and miR-127-5p mimics could promote the cell proliferation and p-circ or miR-127-5p inhibitor could suppress the cell proliferation while it was reverted by co-transfection of si-circ and inhibitor. D, E, The detection of expression level of apoptosis-related proteins was included to ensure the effects on cell numbers in cell apoptosis were direct. *P < 0.05 and ** P < 0.01 showed there was a significant difference compared with NC group

| Silencing of MMP-13 suppressed cell apoptosis yet enhanced cell proliferation via down-regulation of IL-1β, TNF-α and IL-17 and up-regulation of Collagen type II
As shown in Figure 6A,B, si-MMP-13-1 and si-MMP-13-2 could up-regulate cell proliferation rate yet decrease cell apoptotic rate, which could be reversed with co-transfection with miR-127-5p inhibitor (P < 0.01). In addition, knockdown of MMP13 largely decreased MMP13 expression, which was retrieved to NC after co-transfection with miR-127-5p inhibitor (P < 0.01) as shown in Figure 6C. Figure 6D showed that silencing MMP-13 increased the expression of Collagen type II, which was rescued to NC after co-transfection with inhibitor of miR-127-5p (P < 0.01). Based on

| D ISCUSS I ON
It was unveiled by this study that both Circ_0136474 and MMP-13 were significantly up-regulated, whereas miR-127-5p expression was tremendously reduced in OA cartilage tissues compared with normal cartilage tissues. The overexpression of Circ_0136474 progression of several types of diseases, such as atherosclerosis and nervous system disorders. 25 Hence, circRNAs might well be selected as therapeutic targets, as well as new biomarkers.
The circRNAs could regulate the expression of the mRNA by reducing the inhibitory effect of miRNAs. 26 27 Based on latest research, miR-27b was identified as a potential regulator of MMP-13 expression in human chondrocytes by demonstrating its modulation by IL-1β in human chondrocytes. 28 Furthermore, it was reported that IL-1β and TNF-α were the two most potent catabolic factors that increased the production of inflammatory mediators and the expression of MMPs in chondrocytes. 29  In addition, relative expression change in ASH2L mRNA and protein between OA cartilage tissues and normal cartilage tissues was not illuminated, which was not enough for the elucidation of the relationships between ASH2L and Circ_0136474 in OA. However, this part of experiment will be involved in further studies for further validation of roles of Circ_0136474/miR-127-5p/MMP-13 in OA progression. Last, diagnosis and treatment of OA is far more sophisticated than our expectations. Therefore, it really does matter to identify new circRNAs, miRNAs and mRNAs, as well as interpret the mechanisms underlying. In short, Circ_0136474 could suppress cell proliferation yet enhance cell apoptosis by inhibiting miR-127-5p expression and enhancing MMP-13 expression in OA.

E THI C S APPROVAL AND CON S ENT TO PARTI CIPATE
This study was authorized by Peking University First Hospital and written informed consents were obtained from all the participants.

CO N FLI C T O F I NTE R E S T
No conflict of interest exits in the submission of this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.