Circ_0116061 regulated the proliferation, apoptosis, and inflammation of osteoarthritis chondrocytes through regulating the miR-200b-3p/SMURF2 axis

Background Circular RNA (circRNA) has been shown to be associated with osteoarthritis (OA) progression. Circ_0116061 has been found to be highly expressed in OA cartilage tissues, but its role and mechanism in OA progression remain unclear. Methods Expression levels of circ_0116061, microRNA (miR)-200b-5p, and Smad ubiquitin regulatory factor 2 (SMURF2) were detected using quantitative real-time PCR. The proliferation and apoptosis of cells were measured using cell counting kit 8 (CCK8) assay, colony formation assay, and flow cytometry. Furthermore, the protein levels of proliferation-related marker, apoptosis-related markers, inflammatory factors, and SMURF2 were tested using western blot (WB) analysis. In addition, the interaction between miR-200b-3p and circ_0116061 or SMURF2 was examined using dual-luciferase reporter assay and biotin-labeled RNA pull-down assay. Results Circ_0116061 and SMURF2 were highly expressed, and miR-200b-3p was lowly expressed in OA cartilage tissues. Knockdown of circ_0116061 could promote the proliferation and inhibit the apoptosis and inflammation of OA chondrocytes. MiR-200b-3p could be sponged by circ_0116061, and its inhibitor could reverse the regulation of circ_0116061 silencing on the biological functions of OA chondrocytes. SMURF2 was a target of miR-200b-3p, and its expression was positively regulated by circ_0116061. Silencing of SMURF2 also could enhance the proliferation and suppress the apoptosis and inflammation of OA chondrocytes. Furthermore, the regulation of circ_0116061 silencing on the biological functions of OA chondrocytes also could be reversed by SMURF2 overexpression. Conclusion Our data showed that circ_0116061 might regulate the miR-200b-3p/SMURF2 axis to promote the progression of OA.


Introduction
Osteoarthritis (OA) is a chronic degenerative joint disease caused by articular cartilage degradation, subchondral bone sclerosis, osteophyte formation, synovial inflammation, meniscal degeneration, and inflammation and fibrosis of the infrapatellar fat pad [1][2][3][4][5]. OA has become a major public health problem, and the loss of joint function caused by it seriously affects the quality of life of the elderly [6,7]. The onset of OA is closely related to the apoptosis of chondrocytes and the persistent pathological inflammation in the joints [8,9]. Therefore, finding effective molecular targets for regulating chondrocyte apoptosis and inflammation is expected to offer new ideas for alleviating OA progression.
In recent years, the important role of non-coding RNA, including circular RNA (circRNA) and microRNA (miRNA), in a variety of diseases has been widely confirmed. Importantly, the function of circRNA as a miRNA sponge also provides a way to elucidate circRNA mechanism [10,11]. Many circRNAs have been identified as potential biomarkers for the diagnosis and treatment of OA, such as circRNA.33186 [12], circSERPINE2 [13], and circRNA-UBE2G1 [14]. Zhao et al. used highthroughput sequencing to find that circ_0136474 was highly expressed in OA cartilage tissues, and confirmed that it could promote OA progression by inhibiting the proliferation and promoting apoptosis and inflammation of OA chondrocytes [15]. In their study, they also screened that circ_0116061 was also an upregulated circRNA in OA cartilage tissues [15]. However, whether circ_0116061 is involved in the regulation of OA progression has not been studied.
MiR-200b-3p has been confirmed to participate in the regulation of cancer malignant progression as a tumor suppressor, including melanoma [16] and hepatocellular carcinoma [17]. It had been reported that miR-200b-3p was significantly low expressed in OA cartilage tissues and chondrocytes, which overexpression could increase OA chondrocyte viability and suppress apoptosis [18]. Therefore, miR-200b-3p might be the key miRNA that regulated the development of OA. Smad ubiquitin regulatory factor 2 (SMURF2) is a gene that is highly expressed in OA cartilage tissue and chondrocytes and has been shown to be closely related to OA progression [19,20].
The purpose of this study is to explore the role of circ_0116061 in the progression of OA and to reveal its underlying molecular mechanism through the hypothesis of circRNA/miRNA/mRNA axis. Our data showed circ_ 0116061 might play an active role in OA progression. Additionally, we found that there had been correlations among circ_0116061, miR-200b-3p, and SMURF2 expression in OA cartilage tissues. Further analysis revealed that circ_0116061 could regulate SMURF2 by sponging miR-200b-3p. Therefore, our research putted forward the hypothesis that circ_0116061 regulated OA progression via miR-200b-3p/SMURF2.

Cartilage tissues
Our research was approved by Rizhao Central Hospital. Knee cartilage tissues were collected from OA patients (n = 37) and healthy controls (n = 19, only fractures) in Rizhao Central Hospital. For this study, all patients signed an informed consent. The clinical parameters of healthy controls and OA patients are shown in Table 1.

Colony formation assay
OA chondrocytes (200 cells) were seeded in the cell culture dish and cultured for 14 days. The cloned cells were fixed with ethanol (KeyGen, Jiangsu, China) and stained with crystal violet (KeyGen). Cell cloning number was counted under a microscope (Olympus, Tokyo, Japan).

Flow cytometry
Cell apoptosis was determined using Annexin V-FITC Apoptosis Detection Kit (Dojindo). In brief, OA chondrocytes were collected and suspended with binding buffer at a concentration of 1 × 10 6 cells/mL. Then, cell suspensions were stained with Annexin V-FITC and propidium iodide. The apoptosis was analyzed by a flow cytometer (BD Biosciences, San Jose, CA, USA).

Dual-luciferase reporter assay
Based on the binding sites of miR-200b-3p in circ_ 0116061 and SMURF2 3′UTR, the wild-type (wt) and mutant-type (mut) sequences of circ_0116061 and SMURF2 3′UTR were inserted into the psi-CHECK-2 vector (Promega, Madison, WI, USA), respectively. HEK293 cells (Procell, Wuhan, China) were inoculated into a 24-well plate (5 × 10 4 cells/well). After the cells were grown to 60% confluences, the reporter vectors were transfected into the cells with miR-200b-3p mimic or miR-NC for 48 h. Using the Dual-Luciferase Reporter Assay System Kit (Promega), the relative luciferase activity was detected.

Biotin-labeled RNA pull-down assay
The bio-miR-200b-3p probe and control probe (bio-miR-NC) were synthesized by Sangon. After transfecting with bio-miR-200b-3p or bio-miR-NC, OA chondrocytes were harvested and lysed. The cell lysates were incubated with Dynabeads M-280 Streptavidin (Invitrogen), and then the purified RNA was conducted to measure circ_0116061 expression.

Statistical analysis
GraphPad Prism 6.0 software (GraphPad Inc., La Jolla, CA, USA) was used to conduct the statistical analysis. All data were presented as mean ± standard deviation from 3 independent experiments. Results from different groups were analyzed by one-way analysis of variance or Student's t-test. Pearson correlation analysis was used for analyzing the correlations among circ_0116061, miR-200b-3p, and SMURF2. P < 0.05 was considered statistically significant. CRP, mean (mg/L) 5.2 ± 2.6 13.2 ± 4.9 12.8 ± 5.6 13.6 ± 4.5 13.9 ± 3.9 OA osteoarthritis, KL Kellgren-Lawrence, BMI body mass index, ESR erythrocyte sedimentation rate, CRP C-reactive protein

Results
The expression of circ_0116061, miR-200b-3p, and SMURF2 in OA cartilage tissues Using qRT-PCR, we measured the expression of circ_ 0116061, miR-200b-3p, and SMURF2 in the cartilage tissues of OA patients and healthy controls. Compared to the cartilage tissues of healthy controls, we found that circ_0116061 was notably enhanced (Fig. 1a), miR-200b-3p was significantly decreased (Fig. 1b), and SMURF2 was markedly promoted (Fig.  1c) in the cartilage tissues of OA patients. Therefore, we speculated that circ_0116061, miR-200b-3p, and SMURF2 might play key roles in the development of OA.

Circ_0116061 silencing promoted the proliferation and inhibited the apoptosis and inflammation of OA chondrocytes
To explore the role of circ_0116061 in OA, we silenced circ_0116061 expression using si-circ_0116061. The decreased expression of circ_0116061 indicated the transfection efficiency of si-circ_0116061 in OA chondrocytes was good (Fig. 2a). CCK8 results suggested that circ_0116061 knockdown could increase the viability of OA chondrocytes (Fig. 2b). Moreover, silenced circ_ 0116061 also markedly promote the cell cloning number in OA chondrocytes (Fig. 2c). The results of flow cytometry showed that the apoptosis rate of OA chondrocytes was obviously repressed in the presence of circ_0116061 silencing (Fig. 2d). In addition, we also measured the protein levels of Cyclin D1, Bax, Cleaved-casp3, and Bcl2 and found that circ_0116061 knockdown remarkably increased the protein levels of Cyclin D1 and Bcl2, while decreasing the protein levels of Bax and Cleaved-casp3 in OA chondrocytes (Fig. 2e-i). Furthermore, the protein levels of inflammatory factors (IL-1β, IL-6, IL-1α, and TNFα) were obviously reduced in OA chondrocytes downregulating circ_0116061 (Fig. 2j-n). These data confirmed that circ_0116061 knockdown could alleviate OA progression. Cell viability, the cell cloning number, and apoptosis rate were determined using CCK8 assay (b), colony formation assay (c), and flow cytometry (d). e-n WB analysis was used to examine the protein levels of Cyclin D1, Bcl2, Bax, Cleaved-casp3, IL-1β, IL-6, IL-1α, and TNFα. **P < 0.01, ***P < 0.001

Circ_0116061 could serve as a sponge for miR-200b-3p
Using the bioinformatics software analysis (Circinteractome: https://circinteractome.nia.nih.gov/), we discovered that miR-200b-3p had complementary binding sites with circ_0116061 (Fig. 3a). The results of the dualluciferase reporter assay indicated that miR-200b-3p overexpression could repress the luciferase activity of circ_0116061-wt reporter vector, while there was no effect on the luciferase activity of circ_0116061-mut reporter vector (Fig. 3b). Biotin-labeled RNA pull-down assay suggested that the expression of circ_0116061 was notably enriched in the bio-miR-200b-3p probe compared to the bio-miR-NC probe (Fig. 3c). Meanwhile, we also discovered that circ_0116061 silencing could enhance the expression of miR-200b-3p in OA chondrocytes (Fig. 3d), and there was a negative correlation between circ_0116061 and miR-200b-3p in OA cartilage tissues (Fig. 3e).

MiR-200b-3p inhibitor reversed the regulation of circ_0116061 silencing on the proliferation, apoptosis, and inflammation of OA chondrocytes
Subsequently, we constructed the miR-200b-3p inhibitor and confirmed that anti-miR-200b-3p could inhibit miR-200b-3p expression in OA chondrocytes (Fig. 4a). Then, si-circ_0116061 and anti-miR-200b-3p were co-transfected into OA chondrocytes to confirm whether circ_0116061 regulated OA progression by sponging miR-200b-3p. Using CCK8 and colony formation assay, we found that miR-200b-3p inhibitor could reverse the promotion effect of circ_0116061 silencing on the viability and the cell cloning number in OA chondrocytes (Fig. 4b, c). Moreover, the suppressive effect of circ_0116061 knockdown on the apoptosis rate of OA chondrocytes also could be abolished by a miR-200b-3p inhibitor (Fig. 4d). Additionally, the increasing effect of circ_0116061 silencing on the protein levels of Cyclin D1 and Bcl2, as well as the decreasing effect on the protein levels of Bax, Cleaved-casp3, IL-1β, IL-6, IL-1α, and TNFα, also could be reversed by the miR-200b-3p inhibitor (Fig. 4e-n). All data indicated that circ_0116061 might sponge miR-200b-3p to regulate OA progression.

Knockdown of SMURF2 increased the proliferation, while suppressing the apoptosis and inflammation of OA chondrocytes
In addition, we also determined the regulation of SMURF2 on the biological functions of OA chondrocytes. After transfecting with si-SMURF2 into OA chondrocytes, we found that the protein level of SMURF2 was remarkably reduced (Fig. 6a). Through measuring OA chondrocyte viability, cell cloning numbers, and apoptosis rate, we confirmed that silenced SMURF2 could promote the proliferation and restrain the apoptosis of OA chondrocytes (Fig. 6b-d). Besides, knockdown of SMURF2 also enhanced the protein levels of Cyclin D1 and Bcl2, while reducing the protein levels of Bax and Cleaved-casp3 in OA chondrocytes (Fig. 6ei). Furthermore, the protein levels of IL-1β, IL-6, IL-1α, and TNFα in OA chondrocytes also were decreased by silencing SMURF2 (Fig. 6j-n). These results revealed that the role of SMURF2 in OA progression was similar to circ_0116061.
Overexpressed SMURF2 reversed the regulation of circ_0116061 silencing on OA progression To further evaluate whether circ_0116061 regulated OA progression via regulating SMURF2 expression, we performed the rescue experiments. The transfection of OE-SMURF2 in OA chondrocytes could significantly promote the expression of SMURF2, which confirmed the good transfection efficiency of OE-SMURF2 (Fig. 7a). Then, si-circ_0116061 and OE-SMURF2 were co-transfected into OA chondrocytes. Function experiments revealed that the addition of OE-SMURF2 could effectively reverse the promotion effect of circ_0116061 knockdown on the viability and the cloning number (Fig. 7b, c), as well as the inhibition effect on the apoptosis rate of OA chondrocytes (Fig.  7d). In addition, the protein levels of Cyclin D1 and Bcl2 enhanced by circ_0116061 silencing, and the protein levels of Bax, Cleaved-casp3, IL-1β, IL-6, IL-1α, and TNFα repressed by circ_0116061 knockdown also could be reversed by the overexpression of SMURF2 (Fig. 7e-n). These results showed that circ_0116061 mainly regulated SMURF2 to mediate the progression of OA.

Discussion
The occurrence of OA is a complex process involving multiple factors, and its etiology and pathogenesis are still not very clear. A great amount of evidence shows that the degeneration of articular cartilage is considered to be the most important pathological link that causes OA [21,22]. Chondrocytes, as the only cellular component of cartilage, and their biological characteristics are closely related to the development of OA [23,24]. Here, we explored the role of a new circRNA, circ_0116061, in the progression of OA by assessing its function in the biological function of OA chondrocytes. Consistent with the previous study [15], our data found that circ_ 0116061 was overexpressed in OA cartilage tissues and chondrocytes. Interference of circ_0116061 could increase the proliferation and reduce the apoptosis and inflammation of OA chondrocytes, indicating that circ_ 0116061 silencing might be an effective strategy to alleviate OA progression. The competitive endogenous RNA (ceRNA) hypothesis provides a new mechanism for the interaction between RNAs. A large number of studies have shown that circRNA can be used as a ceRNA of miRNA to indirectly regulate mRNA expression [25,26]. For example, circPSMC3 had anti-proliferation and anti-metastasis functions in gastric cancer, which were mainly realized by sponging miR-296-5p [27]. Circ-GRB10 could act as a ceRNA for miR-328-5p to suppress nucleus pulposus cell apoptosis [28]. In addition to being a tumor suppressor, miR-200b-3p had also been found to be related to the progression of endometriosis [29], and its downregulation could also inhibit vascular smooth muscle cell calcification [30]. Here, we discovered that miR-200b-3p was underexpressed in OA cartilage tissues and chondrocytes, which was similar with the past research [18]. MiR-200b-3p inhibitor could reverse the proproliferation, anti-apoptosis, and anti-inflammatory functions of circ_0116061 knockdown on OA chondrocytes, confirming that circ_0116061 indeed mediated OA progression via targeting miR-200b-3p.
Additionally, our data showed that SMURF2 was a target of miR-200b-3p and circ_0116061 sponged miR-200b-3p to regulate SMURF2. SMURF2 is a kind of E3 ubiquitin ligase, which can participate in the regulation of TGF-β signaling pathway through the ubiquitinribosome pathway [31]. Current research confirmed that SMURF2 was related to the progression of multiple fibrotic diseases, including liver fibrosis [32] and kidney fibrosis [33]. Chen et al. showed that SMURF2 could suppress OA chondrocyte proliferation, while accelerating apoptosis and ECM degradation [19]. Studies had suggested that the high expression of SMURF2 could induce the degeneration of articular chondrocytes and produce the OA-like phenotype [34,35]. Here, we silenced SMURF2 expression and confirmed that downregulated SMURF2 could facilitate OA chondrocyte proliferation and restrain apoptosis and inflammation. Besides, the reversal effect of SMURF2 on circ_0116061 silencing illuminated that circ_0116061 might positively regulate SMURF2 to mediate OA progression.
Combined with all the results, we proposed that circ_ 0116061 promoted OA progression through the miR-200b-3p/SMURF2 axis. The pro-proliferation, antiapoptosis, and anti-inflammatory functions of circ_ 0116061 silencing on OA chondrocytes suggested that circ_0116061 knockdown might be a beneficial method to alleviate OA progression, which had an important clinical significance.