Circ_0005526 contributes to interleukin-1β-induced chondrocyte injury in osteoarthritis via upregulating transcription factor 4 by interacting with miR-142-5p

ABSTRACT Circular RNAs (circRNAs) can regulate the progression of osteoarthritis (OA) via serving as competing endogenous RNAs (ceRNAs). This work was performed for functional research of circ_0005526 in Interleukin-1β (IL-1β)-induced OA injury. Circ_0005526, microRNA-142-5p (miR-142-5p) or transcription factor 4 (TCF4) expression was measured via reverse transcription-quantitative polymerase chain reaction assay. Cell analysis was performed by Cell Counting Kit-8 assay for cell viability, EdU assay for cell proliferation and flow cytometry for cell apoptosis. The protein level detection was conducted using western blot. Target analysis was carried out via dual-luciferase reporter assay and RNA pull-down assay. Circ_0005526 was upregulated in OA cartilage tissues and IL-1β-exposed chondrocyte cells. IL-1β inhibited cell viability and proliferation but enhanced cell apoptosis and inflammation, then these damages were attenuated after downregulation of circ_0005526. Circ_0005526 interacted with miR-142-5p, and circ_0005526 knockdown suppressed IL-1β-induced OA progression through upregulating miR-142-5p. TCF4 was regulated by circ_0005526 via targeting miR-142-5p. The function of circ_0005526 was also achieved by upregulation of TCF4. These results unraveled that circ_0005526 promoted IL-1β-induced chondrocyte injury in OA via suppressing miR-142-5p binding to TCF4.


Introduction
Osteoarthritis (OA) is a common joint disease with destruction of articular cartilages [1]. OA has seriously affected the life quality of human beings, and there is no effective strategy for treatment of OA [2]. Chondrocyte dysfunction is the leading cause of cartilage degeneration and OA progression [3]. The previous studies have indicated that the pathogenesis of OA is related to chondrocyte apoptosis and inflammatory response [4,5]. Interleukin-1β (IL-1β) signaling is a risk factor of OA, and IL-1β induction is commonly used to establish OA cell model [6]. Investigating the molecular mechanism in IL-1β-induced OA progression is important. Circular RNAs (circRNAs) are known as competing endogenous RNAs (ceRNAs) to inhibit the binding between microRNAs (miRNAs) and downstream genes, consequently regulating the pathogenic development of human diseases [7]. Chen et al. uncovered that circRNA-9119 suppressed IL-1β-induced chondrocyte apoptosis via interacting with miR-26a to result in upregulation of phosphatase and tensin homolog (PTEN) [8]. Wu et al. elucidated that circ_0134111 aggravated inflammatory reaction and extracellular matrix degradation of chondrocytes in OA through downregulating suppressor of cytokine signaling 1 (SOCS1) by targeting miR-515-5p [9]. Circ_0005526 has been shown to function as miRNA sponges in OA [10], but the regulatory function of circ_0005526 remains unknown.
Herein, circ_0005526 was hypothesized to be a miR-142-5p sponge and TCF4 was assumed to be a downstream target for miR-142-5p. The goals of this study were to explore the biological function and regulatory mechanism of circ_0005526 in IL-1β-induced chondrocyte injury.

Human tissues
Tissue acquisition was performed after informed consent files were provided by all participators. OA cartilage tissues (n = 39) and normal cartilage tissues (n = 21) were respectively collected from OA patients who underwent total knee arthroplasty and amputees who had no OA at the Sixth Affiliated Hospital of Xinjiang Medical University. The clinical data of OA patients were shown in Table 1. These samples were stored in liquid nitrogen until further use. Additionally, this research was approved by Ethics Committee of the Sixth Affiliated Hospital of Xinjiang Medical University.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay
Tissues and cells were lysed with Trizol reagent (Invitrogen) for total RNA extraction. Then 2 μg RNA/sample was used for synthesis of complementary DNA (cDNA) through ReverTra Ace® qPCR RT Kit (Toyobo, Kita-Ku, Osaka, Japan), followed by quantification reaction via SYBR® Green Realtime PCR Master Mix (Toyobo) using specific primers. The sequences were shown in Table 2. For level normalization, beta-actin (β-actin) for circ_0005526/TCF4 and U6 for miR-142-5p were applied as internal references. Then, relative expression was calculated by data analysis via 2 −∆∆Ct method [17].

Cell viability assay
1 × 10 5 cells were transplanted into 48-well plates, followed by IL-1β treatment and cell transfection. 48 h later, cells were added with 10 μL/well CCK-8 solution (Beyotime, Shanghai, China). The plates were performed with 2 h of incubation at 37°C, then absorbance at 450 nm was tested by a microplate reader. Cell viability = viable cells/total cells × 100%.

Cell proliferation assay
Cell proliferation was evaluated by EdU-positive cells [18]. EdU Proliferation Kit (Beyotime) was used in this study. According to the user's manuals, cells were incubated with 100 µL EdU solution and nuclei were stained with diamidinyl phenylindole (DAPI; Beyotime). The micrographs were taken by a fluorescence microscope (Olympus, Tokyo, Japan). Then, EdU-positive cells were counted as merged cells by EdU and DAPI staining.

Statistical analysis
The experiments were independently repeated by three times with three parallels (n = 3). Data were shown as mean ± standard deviation and processed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA), then Student's t-test or analysis of variance (ANOVA) followed by Tukey's test was exploited to analyze the difference. P < 0.05 demonstrated that difference was significant.

Circ_0005526 was upregulated in OA
CircRNA dysregulation is associated with OA progression and circRNA function is related to miRNA/mRNA network. The aim of this research was to investigate functional mechanism of circ_0005526 in OA. Circ_0005526 was considered to have regulatory effect on TCF4 by targeting miR-142-5p. Firstly, expression analysis of circ_0005526 was conducted by RT-qPCR. Relative to normal cartilage tissues and control cells, circ_0005526 level was significantly upregulated in OA cartilage tissues (Figure 1(a)) and 10 ng/mL IL-1β-treated CHON-001 cells (Figure 1(b)). Circ_0005526 is produced by back-splicing of runt-related transcription factor 2 (RUNX2) in chr6: 45459677-45460699with spliced sequence of 1022 bp (Figure 1(c)). The high expression of circ_0005526 implied the potential regulation in OA progression.

IL-1β-induced inhibition of cell viability and proliferation but promotion of cell apoptosis and inflammation were relieved by circ_0005526 knockdown
IL-1β-treated CHON-001 cells were transfected with si-circ_0005526 or si-NC to investigate circ_0005526 function in OA. The transfection efficiency was assessed using RT-qPCR. As shown in Figure 2(a), IL-1β-induced upregulation of circ_0005526 was significantly attenuated by si-circ_0005526. Cell viability by CCK-8 assay (Figure 2(b)) and cell proliferation by EdU assay (Figure 2(c)) were reduced by treatment of IL-1β, then si-circ_0005526 partly eliminated these effects. The result of flow cytometry revealed that cell apoptosis rate was decreased in IL-1β +si-circ_0005526 group compared to IL-1β+si-NC group (Figure 2(d)). The protein detection by western blot (Figure 2(e)) demonstrated that circ_0005526 inhibition reversed IL-1β-mediated downregulation of PCNA, Cyclin D1 and Bcl2, as well as upregulation of Bax in CHON-001 cells (Figure 2(f-i)). IL-6 and TNF-α protein levels were elevated after exposure to IL-1β, which was obviously restored by downregulation of circ_0005526 (Figure 2(j-l)). Overall, circ_0005526 knockdown inhibited IL-1βinduced chondrocyte injury.

Discussion
The previous study indicated that circ_0005526 was upregulated in serum of OA patients, and had The miR-142-5p level was detected via RT-qPCR in OA tissues (c) and CHON-001 cells exposed to 10 ng/mL IL-1β (C). (d-e) Dual-luciferase reporter assay (d) and RNA pull-down assay (e) were used for identification of circ_0005526 and miR-142-5p binding. ***P < 0.001.
clinical significance in OA [10]. Our evidences unraveled that circ_0005526 knockdown inhibited chondrocyte injury in IL-1β-induced OA model through mediating miR-142-5p/TCF4 axis. Summary of the article search process was shown in Supplementary  Fig. 1.
CircRNAs are identified to be associated with the pathogenesis of OA [22]. Hsa_circ_0045714 level downregulation reduced IL-1β-evoked OA chondrocyte injury [23]. Silencing circ_0001846 alleviated cell apoptosis and inflammation in IL-1βinduced OA progression [24]. CircRNA_0001236 contributed to chondrogenesis and inhibited cartilage degradation in OA [25]. Thus, circRNAs paly different roles in the development of OA. In the current research, circ_0005526 overexpression was detected in cartilage tissues from OA patients. Furthermore, circ_0005526 elevated cell viability and proliferation ability but suppressed cell apoptosis and inflammatory reaction in IL-1βestablished OA model. These findings implied that circ_0005526 aggravated the progression of OA in vitro.
This study still has some limitations. For example, the results were acquired from cell experiments. There is no data from in vivo model due to the limited condition and fund. The further   exploration in vivo may be performed in future study. In addition, circRNAs can act on many miRNAs and miRNAs can target different genes. It is necessary to explore whether circ_0005526 function in OA is related to other miRNA/mRNA axes.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.

Ethics approval and consent to participate
Written informed consents were obtained from all participants and this study was permitted by the Ethics Committee of the Sixth Affiliated Hospital of Xinjiang Medical University.