Circular RNA circCTNNA1 is downregulated in osteoarthritis and sponges miR-29a to suppress LPS-induced apoptosis of synoviocytes

Abstract Objective CircRNA circCTNNA1 has been characterized as a critical player in cancer biology, while its role in other human diseases is unknown. This study was carried out to study the role of circCTNNA1 in osteoarthritis (OA). Materials and methods RNA was extracted from synovial fluid samples donated by OA patients (n = 62). RT-qPCRs were then performed to determine the expression of circCTNNA1 and miR-29a in these synovial fluid samples. The interaction between circCTNNA1 and miR-29a was predicted using an online program IntaRNA 2.0 and confirmed by RNA pull-down assay. Overexpression of circCTNNA1 and miR-29a was achieved in synoviocytes to analyze their effects on each other’s expression. The role of circCTNNA1 and miR-29a in regulating synoviocyte apoptosis was explored by cell apoptosis assay. Results CircCTNNA1 was downregulated in OA, while miR-29a was overexpressed in OA. CircCTNNA1 and miR-29a were not significantly correlated. RNA pull-down assay illustrated the direct interaction between circCTNNA1 and miR-29a. In synoviocytes, overexpression of circCTNNA1 and miR-29a failed to regulate the expression of each other. CircCTNNA1 overexpression suppressed the enhancing effects of miR-29a overexpression on cell apoptosis induced by LPS. Conclusions CircCTNNA1 is downregulated in OA, and its overexpression suppresses synoviocyte apoptosis via sponging miR-29a.


Background
As the most common type of arthritis, osteoarthritis (OA) is caused by chronic disruption of the protective joint cartilage that cushions bone ends [1,2]. OA mainly affects the older adult population, including more than 13% of women and 10% of men older than 60 years [3,4]. OA causes chronic pain, joint stiffness, and swelling [1,2]. At present, all available treatment approaches only control the symptoms, such as the relief of chronic pain [5,6]. Therefore, more effective treatment strategies are needed. However, the molecular mechanism of OA is still largely unknown.
Studies of OA in recent decades have shown that OA requires the participation of many molecular signaling pathways [7][8][9][10]. Some molecular players, such as Notch and NF-jB pathways, have been characterized as promising molecular targets to develop targeted therapies for OA treatment by regulating gene expressions [9]. However, targeted therapy is still under research. Particularly, the safe and effective molecular targets for OA treatment remain lacking. Circular RNAs encode no protein products, but they participate in OA by affecting protein synthesis [11,12]. Therefore, circRNAs may serve as potential therapeutic targets for OA. However, the role of most circRNAs in OA is unknown.
CircRNA circCTNNA1 has been characterized as a critical player in cancer biology [13], while its role in other human diseases is unknown. In this study, we performed bioinformatics analysis and analyzed the interaction between circCTNNA1 and miR-29a in OA. We found that circCTNNA1 could interact with miR-29a, a crucial player in synoviocyte apoptosis [14].

Research subjects and synovial fluid collection
The study was approved by the Ethics Committee of First Hospital of Qiqihar City. A total of 62 OA patients (knee OA) who were admitted to the first Hospital of Qiqihar City were enrolled in the study. In addition, 62 healthy volunteers who received routine physiological examinations at the same hospital were also included as the control group. Both OA and control group included 22 males and 40 females with an average age of 62.8 ± 5.9 years in the range of 57 and 71 years. OA patients were excluded if they were recurrent cases or had initiated therapy and other clinical disorders. All OA patients and healthy controls signed informed consent. Prior to therapy, synovial fluids (1.0 ml) were extracted from the affected sites of OA patients and the corresponding knees of the healthy controls.

Synoviocytes and transient transfections
Synoviocytes (type B, primary cells) derived from an adult OA patient were purchased from Sigma-Aldrich (Cat. 408OA-05A) and cultured in human synoviocyte media (Cell applications) at 37 C in an incubator with 5% CO 2 and 95% humidity. In some experiments, synoviocytes were treated with LPS at dosages of 0, 2, 4, 6, 8, and 10 lg/ml for 48 h prior to the subsequent analysis.
Overexpression of circCTNNA1 and miR-29a was achieved in synoviocytes by transfecting pcDNA3.1-circCTNNA1 vector (Invitrogen) or miR-29a (Invitrogen) using Lipofectamine 2000 (Invitrogen). In brief, transfection mixtures were prepared by mixing the expression vectors and miRNAs with transfection reagent and incubated with cells for 6 h. After that, cells were cultured in fresh media for 48 h and used in subsequent assays. Cells transfected with NC miRNA or empty pcDNA3.1 vector were used as negative control (NC), and untransfected cells were used as control (C) cells.

Preparation RNA samples
Total RNAs were isolated using RNAzol (Sigma-Aldrich) from both synoviocytes and synovial fluid samples from both OA and control groups and treated with DNase I (Invitrogen) to remove genomic DNA. After that, RNA integrity was analyzed on 5% urea-PAGE gels, and RNA purity was determined based on the ratio of OD260/280. RNA samples with a ratio close to 2.0 were used for subsequent analyses.

RT-qPCRs
A total 1 mg RNA from each sample was converted into cDNA. To determine circCTNNA1 expression level, cDNA was used as templated for qPCRs using SYBR Green Master Mix (Bio-Rad) with 18S rRNA as the endogenous control. To determine mature miR-29a expression level, cDNA was subjected to poly (A) addition, followed by miRNA reverse transcriptions and qPCRs with poly (T) as the reverse primer. All steps were completed using All-in-One TM miRNA qRT-PCR Detection Kit (GeneCopoeia). U6 was used as the internal control of miR-29a. The primer sequences were 5 0 -TGCGT AGACAGCTCCGCAAAG-3 0 (forward) and 5 0 -ATCAATTTGTTGG CATGTTC-3 0 (reverse) for circCTNNA1; 5 0 -TAACCCGTTGAACCCC ATT-3 0 (forward) and 5 0 -CCATCCAATCGGTAGTAGC3'(reverse) for 18S rRNA. MiR-29a forward primer was 5 0 -ACTGATTTCTTT TGGTG-3 0 . MiR-29a reverse primer and U6 primers were from the kit. PCR reaction conditions were 95 C for 3 min followed by 40 cycles of 95 C for 10 s and 58 C for 45 s. The 2 ÀDDCt method was used to normalize the Ct values.

RNA-RNA interaction prediction and RNA pulldown assay
The interaction between circCTNNA1 and miR-29a was predicted using the online program IntaRNA2.0 with circCTNNA1 as the long sequence, miR-29a as the short sequence, and other parameters set as default.
Biotinylated NC (Bio-NC) and circCTNNA1 RNA (Bio-circCTNNA1) were synthesized by Sangon Biotech (Shanghai, China). In addition, the predicted binding sites of miR-29a on circCTNNA1 were mutated through site-directed mutagenesis, followed by biotin labeling to prepare mutated Bio-circCTNNA1, which was named Bio-circCTNNA1(mut). These Biotinylated RNAs were transfected into synoviocytes using the method described above. After transfection, cells were lysed and incubated with streptavidin magnetic beads (Invitrogen) to pull down Bio-NC, Bio-circCTNNA1 and Bio-circCTNNA1(mut). MiR-29a level in precipitated samples was determined by RT-qPCRs.

Western blot
Protein samples were isolated using RIPA solution and quantified using BCA Kits (Invitrogen). After denaturation, protein samples were separated on 5% SDS-PAGE gels and transferred onto PVDF membranes. The membranes were incubated in turn with an anti-caspase antibody (ab2302, Abcam) and anti-rabbit IgG-HRP secondary antibody (1:1000, MBS435036, MyBioSource). Signals were produced with ECL Western Blotting Substrate (Thermo Fisher Scientific).

Apoptosis assay
Synoviocytes collected at 48 h after transfections were transferred into a 6-well cell culture plate with 2 ml media containing 40,000 cells per well. Cells were treated with LPS (10 lg/ml) for 48 h with three replicates for each treatment. After that, cells were subjected to Annexin-V FITC and propidium iodide (PI) staining in the dark for 20 min, and cell apoptosis was analyzed using flow cytometry.

Statistical analysis
Comparisons between control and OA groups were performed by unpaired t test. ANOVA (one-way or two-way) Tukey's test was used to compare multiple groups. Correlations were analyzed by Pearson's correlation coefficient analysis. A p < .05 value was considered statistically significant.

Altered expression of circCTNNA1 and miR-29a was observed in the OA group
Expression of circCTNNA1 and miR-29a in synovial fluid samples was determined using RT-qPCR. CircCTNNA1 was significantly downregulated in OA (Figure 1(A), p < .001) while miR-29a was significantly overexpressed in OA (Figure 1(B), p < .001) compared with the control group, indicating that miR-29a overexpression and circCTNNA1 downregulation might participate in OA. It is worth noting that circCTNNA1 and miR-29a expression levels in OA patients were closely correlated with OA stages (both p < .05) but not with patients' age, gender, body mass index (BMI), and smoking and drinking habits.

CircCTNNA1 and miR-29a directly interact with each other in synoviocytes
The interaction between circCTNNA1 and miR-29a was predicted using an online program IntaRNA2.0. The results showed that circCTNNA1 could form strong base pairing with  The interaction between circCTNNA1 and miR-29a was predicted by IntaRNA2.0 (A) and analyzed using RNA pull-down assay (B). The interaction between circCTNNA1 (mut) and miR-29a was also analyzed by RNA pull-down assay (C). Ã p < .05. miR-29a (Figure 2(A)). RNA pull-down assay was applied to analyze their interaction in synoviocytes. The results showed that miR-29a level was significantly higher in the precipitates of the bio-circCTNNA1 group than in the bio-NC group (Figure 2(B), p < .05), indicating that circCTNNA1 and miR-29a could interact with each other in synoviocytes. In contrast, no significant differences in levels of miR-29a were observed between bio-NC and bio-circCTNNA1 (mut) groups, further confirming the interaction between circCTNNA1 and miR-29a (Figure 2(C)).

CircCTNNA1 and miR-29a failed to regulate the expression of each other in synoviocytes
To further explore whether the interaction between circCTNNA1 and miR-29a could affect their expression, circCTNNA1 and miR-29a overexpression were achieved in synoviocytes after 24 h to 96 h of post-transfection. Interestingly, circCTNNA1 and miR-29a overexpression failed to affect each other's expression (Figure 3(B)). Moreover, circCTNNA1 and miR-29a levels were not significantly correlated across OA (Figure 3(C)) and control ( Figure  3(D)) samples.

CircCTNNA1 may sponge miR-29a to reduce LPS-induced synoviocyte apoptosis
Synoviocytes were treated with LPS at dosages of 0, 2, 4, 6, 8, and 10 lg/ml for 48 h. LPS significantly downregulated circCTNNA1 (Figure 4(A), p < .05) and upregulated miR-29a (Figure 4(B), p < .05) in synoviocytes. The role of circCTNNA1 and miR-29a in regulating apoptosis of synoviocytes induced by LPS (10 lg/ml for 48 h) was analyzed using cell apoptosis assay. The results showed that circCTNNA1 decreased cell apoptosis while miR-29a increased cell apoptosis. In addition, circCTNNA1 suppressed the enhancing effect of miR-29a on cell apoptosis induced by LPS (Figure 4(C), p < .05). Moreover, active (cleaved) caspase-3 levels in different cell apoptosis groups analyzed by Western blot were in accordance with the level of cell apoptosis (Figure 4(D)). The role of circCTNNA1 (mut) in cell apoptosis was also analyzed. The mutated circCTNNA1 failed to affect cell apoptosis and also failed to suppress the role of miR-29a in cell apoptosis (Figure 4(E)). Therefore, the role of circCTNNA1 in cell apoptosis is mainly mediated by miR-29a.

Discussion
The crosstalk between circCTNNA1 and miR-29a was explored in OA. We found that circCTNNA1 was downregulated in OA. In addition, circCTNNA1 may sponge miR-29a to reduce the enhancing effect of miR-29a on LPS-induced synoviocyte apoptosis.
CircCTNNA1 has been characterized as a critical player in colorectal cancer [13]. It is reported that circCTNNA1 is overexpressed in colorectal cancer and promotes cancer progression by upregulating FOXM1 via sponging miR-149-5p [13]. However, the role of circCTNNA1 in other human diseases is unknown. In this study, we observed that circCTNNA1 was downregulated in OA, suggesting it is potentially involved in OA progression. Type B synoviocytes are responsible for the production of glycoproteins in synovial fluid, an essential joint lubricant. However, under disease conditions, the activation of type B synoviocytes by proinflammatory cytokines may enhance the production of erosive enzymes, such as collagenase, GM-CSF, aggrecanase, and metalloproteinases in these cells to increase joint destruction and cartilage degradation, which in turn aggregate the conditions of OA [15,16]. In this study we showed that circCTNNA1 decreased the apoptosis of type B synoviocytes induced by LPS. Therefore, circCTNNA1 may participate in OA by regulating cell apoptosis.
MiR-29a plays a critical role in rheumatoid arthritis (RA) by inducing apoptosis and inhibiting proliferation of synoviocytes [14]. Interestingly, our study also observed the overexpression of miR-29a in OA and its enhancing effect on the apoptosis of OA-derived synoviocytes [17]. Therefore, miR-29a may play opposite pathological roles in OA.
Interestingly, our study showed that circCTNNA1 and miR-29a could directly interact with each other in synoviocytes. However, overexpression of circCTNNA1 and miR-29a failed to significantly affect the expression of each other. Instead, circCTNNA1 overexpression reduced the enhancing effect of miR-29a on cell apoptosis. Therefore, we speculated that circCTNNA1 could sponge miR-29a to suppress its effects on OA.

Conclusion
CircCTNNA1 is under-expressed in OA and may sponge miR-29a to reduce the apoptosis of type B synoviocytes induced by LPS.