Hsa_circ_0007482 Promotes Proliferation and Differentiation of Chondrocytes in Knee Osteoarthritis

Abstract

Objective

Knee osteoarthritis (KOA) is a complex degenerative joint disease and a major cause of joint dysfunction. This study aimed to explore the function of hsa_circ_0007482 on inflammation, proliferation, differentiation, and apoptosis in KOA.

Design

Real-time quantitative polymerase chain reaction (PCR) was performed to detect the expression of circ_0007482, inflammatory factors, and differentiation-related molecules in KOA chondrocytes and interleukin (IL)-1β–stimulated chondrocytes. The correlation between the circ_0007482 expression and inflammatory factors was analyzed by the Pearson method. KOA cell model was established using IL-1β for 24 hours. The proliferation activity of chondrocytes was evaluated by CCK-8 assay, and cell apoptosis rate was assessed by flow cytometry. The downstream miRNA of circ_0007482 was validated using dual-luciferase reporter assay.

Results

The circ_0007482 expression was elevated in both KOA cartilage tissues and IL-1β–treated chondrocytes and positively correlated with inflammatory factors expression. In comparison to the control group, IL-1β treatment diminished chondrocyte proliferation abilities and increased cell apoptosis and inflammatory factors IL-6, IL-8, and tumor necrosis factor (TNF)-α mRNA expression. Inhibition of circ_0007482 partially improved IL-1β–induced inflammatory reaction. Circ_0007482 could negatively regulate the expression of miR-558.

Conclusions

Interfering of circ_0007482 might partially promote cell proliferation and differentiation, while inhibit cell apoptosis to improve joint injury by regulating miR-558 in IL-1β–treated chondrocyte cell model.

Introduction

Osteoarthritis (OA), also known as osteoarthropathy, is a common clinical degenerative disease of bone and joint swelling, pain, deformity, and complex pathogenesis.^1,2 OA can develop in any joint, but most commonly affects the knees, hips, hands, and feet. As a common chronic disease in the elderly population, knee osteoarthritis (KOA) is mainly characterized by degeneration of articular cartilage and subchondral bone hyperplasia. ³ The prevalence and incidence of KOA are increasing year by year due to many influence factors.^4,5 The typical pathological changes of KOA include destruction of cartilage, formation of osteophyte, and hyperplasia of synovial. ⁶ Cartilage is a major component of the joint and has a buffering effect to relieve stresses acting on the joint. Chondrocytes in cartilage can produce collagen and glycosaminoglycans to form a concentrated and highly coordinated extracellular matrix (ECM) that plays a key role in the progression of OA. ⁷ In addition, inflammation is a key feature associated with progression of OA. ⁸ At present, the treatment of KOA mainly focuses on improving the clinical symptoms of patients (relieving pain and controlling inflammation), and there is still a lack of effective treatment therapies. Therefore, understanding the pathogenesis of KOA is crucial for the treatment of KOA.

Circular RNA (circRNA) is a type of endogenous non–coding RNA that has a closed-loop structure, which can regulate the progression of a variety of diseases and participate in cellular activities, such as growth, differentiation, and apoptosis.^9,10 Numerous circRNAs are aberrantly expressed in OA and play their role through miRNA sponge, regulation of pathways, and direct targeting of protein molecules.^11-13 Hsa_circ_0007482, also known as circCOL5A1, is located at chr9:137582757-137593179 and generated by back-splicing of exons 13 to 19 of the Collagen type V alpha 1 chain (COL5A1) gene. COL5A1, one of the ECM-related genes, is more prevalent in OA subchondral bone tissues compared with normal tissues and may be a biomarker for OA. ¹⁴ Circ_0007482 was upregulated in aseptic loosening synovial tissues after total hip arthroplasty. ¹⁵ Whereas the expression and influence of circ_0007482 in KOA has not been reported.

circRNAs were indicated as functional sponges for miRNAs in diseases.^16,17 Circ_0007482 could interact with miR-27a-3p, miR-6512-3p, miR-4483, and miR-3918 in keloid ¹⁸ and other miRNAs in Ni et al. ¹⁵ miR-558 has regulatory role in the processes of many diseases, such as chronic obstructive pulmonary disease and OA.^19,20 miR-558 expression was found to be lower in OA tissues than in normal articular cartilage. ²¹ However, whether circ_0007482 has correlation with miR-558 remains unclear.

Therefore, in this study, 15 patients with meniscus injuries and 25 patients with KOA were selected to detect the expression of circ_0007482 in KOA tissue specimens. The functional implications and potential mechanism of circ_0007482 in modulating the pathophysiology of KOA were elucidated.

Materials and Methods

Tissue Specimens

All procedures were performed in line with the principles of the Declaration of Helsinki. This study was giving permission by the ethics committee of Zhejiang Provincial People’s Hospital, approval number: 2021(025), and informed consent was obtained from the participants. A total of 15 patients with meniscus injuries (normal control) and 25 patients with KOA admitted to our hospital from March 2021 to June 2023 were selected in this study. All patients met the inclusion criteria of this study: (1) patients with KOA were diagnosed according to the clinical diagnostic criteria of American College of Rheumatology; (2) patients with grade 3 meniscus injuries had no other underlying diseases except meniscus injury, (3) patients with other joint disease, such as rheumatoid arthritis, ankylosing spondylitis, joint involvement, infection, and tumor, were excluded. Articular cartilage tissue samples from the knee joint were collected after total knee arthroplasty surgery or arthroscopic resection surgery and were stored in liquid nitrogen.

Cell Line Culture and Cell Model Conduction

Human chondrocyte CHON-001 cell line was gained from ATCC and cultured in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA) with 10% fetal bovine serum (FBS) (Gibco). The culture condition was 37°C and 5% CO₂. The chondrocyte CHON-001 cells were treated with a medium containing 10 ng/ml interleukin (IL)-1β for different time intervals, which was recorded as the IL-1β group (KOA in vitro model). At the same time, the normal cultured chondrocytes were recorded as the control group.

Cell Transfection

CHON-001 chondrocytes in the logarithmic growth phase were collected and seeded in 6-well plates (1 × 10⁴ cells/well). Once cell growth confluence reached 60% to 70%, cell transfection was performed using Lipofectamine 3000 Transfection Reagent (Invitrogen, Waltham, MA). Circ_0007482 siRNA (si-circRNA-1, si-circRNA-2, si-circRNA-3), siRNA negative control (si-NC), miR-558 mimic, and mimic NC were synthesized by Sangon Biotech (Shanghai, China). After 24 hour culture of CHON-001 with 10 ng/ml IL-1β, either si-circRNA or si-NC was added. Cell transfection efficacy was measured after 48 hours.

Total RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction

RNA extraction experiments were performed in the absence of RNase. Tissue samples were washed 3 times with sterile phosphate-buffered saline (PBS) containing 1% double antibody. The normal cartilage tissues and KOA cartilage tissues (50 mg) were placed directly into a mortar, ground to a power under liquid nitrogen, ground by adding Trizol reagent, and the grinding solution was put into a 2 ml centrifuge tube for total RNA extraction. Reverse transcription was performed using Evo M-MLV RT Kit with gDNA Clean for quantitative polymerase chain reaction (qPCR) kit. SYBR Green Premix Pro Taq HS qPCR Kit was used in Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). The relative expression of target genes was calculated by 2^−ΔΔCt method.

RNase R Experiment

RNA was extracted from cells by the Trizol method. Total RNA (2 ng) was taken, one was used as control, the other was added 6 U RNase R (3 U/pg), placed in a 37°C, 5% CO₂ volume fraction incubator digestion for 15 minutes through reverse transcription to form cDNA. RT-qPCR was performed to detect the relative expression of circ_0007482 and COL5A1.

CCK-8 Assay

The transfected cells (2000 cells/well) were seeded in 96-well plates for cell proliferation detection using cell counting kit-8 (CCK-8; Dojindo, Japan). Over a period of 3 days, 10 μl of CCK-8 reagent was added to each well every 24 hours. After 1 hour of further cultivation, the absorbance at 450 nm was measured to assess the proliferation abilities.

Apoptosis Assay

Cells in the logarithmic growth phase were selected, washed 3 times with precooled PBS, resuspended in 500 μl PBS, added with 5 μl Annexin V-fluorescein isothiocyanate and 5 μl propidium iodide, and incubated at room temperature for 10 minutes. Then, the apoptosis rate was detected by the FACS Calibur flow cytometry.

Dual-Luciferase Reporter Assay

Wild type (WT) or mutant (MUT) circ_0007482 was cloned into the pmirGLO plasmid receptor, and miR-558 mimics or mimic NC was transferred into CHON-001 at the same time. After 48 hours of co-culture, the dual-luciferase reporter assay system was used to measure the dual-luciferase activity.

Statistical Analysis

GraphPad Prism 9.0 software was used for statistical analysis; the experimental data were expressed as mean ± SD. Two independent sample Student t tests were used for comparison between 2 groups. One-way analysis of variance (ANOVA) was used for comparison among multiple groups. If the data set was normally distributed (measured by Kolmogorov-Smirnov test), the Pearson correlation analysis was used for correlation analysis. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.

Results

Circ_0007482 Expression in KOA Cartilage Tissue

To comprehend the expression characteristics of circ_0007482 in articular cartilage tissue of KOA patients, the expression of circ_0007482 in KOA and normal articular cartilage tissues was detected. As shown in Figure 1A , circ_0007482 expression in cartilage tissues of KOA patients was increased in contrast to that in normal cartilage tissues. Furthermore, the COL5A1 mRNA was also measured and showed an elevated expression in KOA cartilage tissues in contrast with normal cartilage tissues ( Fig. 1B ).

Figure 1.

Circ_0007482 and Collagen Type V alpha 1 chain (COL5A1) mRNA expression in articular cartilage tissues was measured by quantitative real-time PCR (RT-qPCR). (A) circ_0007482 expression was higher in knee osteoarthritis (KOA) tissues compared with normal tissues. (B) COL5A1 mRNA levels were higher in KOA tissues than in normal tissues. ***P < 0.001.

Correlation Between Upregulated Circ_0007482 and Inflammatory Factors

To explore the expression level of inflammatory genes in articular cartilage tissues of patients with KOA, this study measured the IL-6, IL-8, and tumor necrosis factor (TNF)-α mRNA levels in cartilage tissues of KOA patients and in normal articular cartilage tissues of patients in the control group by RT-qPCR. The results in Figure 2A-C displayed that the mRNA levels of IL-6, IL-8, and TNF-α were higher in KOA tissues than in normal cartilage tissues.

Figure 2.

There is a correlation between upregulated circ_0007482 and inflammatory factors. (A-C) Inflammatory factors IL-6, IL-8, and TNF-α mRNA expression were increased in KOA tissues compared with normal tissues. ***P < 0.001. (D-F) A positive correlation was observed between circ_0007482 expression and IL-6 (r = 0.6920, P < 0.001), IL-8 (r = 0.7456, P < 0.001), and TNF-α mRNA levels (r = 0.6512, P < 0.001), respectively. ***P < 0.001.

The Pearson correlation analysis was used to assess the relationship between circ_0007482 and KOA-related inflammatory factors. As depicted in Figure 2D-F , circ_0007482 was positively correlated with the mRNA levels of IL-6, IL-8, and TNF-α.

Silencing of Circ_0007482 Enhanced Chondrocyte Viability and Repressed Apoptosis After Interleukin-1β Injury

RNase R treatment experiments were used to confirm the cyclization of circ_0007482. The relative expression of circ_0007482 did not significantly change in the control and RNase R-treated chondrocytes CHON-001, whereas the COL5A1 mRNA levels were downregulated in the RNase R-treated group ( Fig. 3A ). CHON-001 cells were cultured with 10 ng/ml IL-1β to stimulate an OA condition in vitro. Circ_0007482 levels gradually increased with the extension of incubation time and the difference attained the highest level after 24 hours of culture ( Fig. 3B ). Circ_0007482 siRNA-3 exhibited the highest transfection efficiency in CHON-001 cells, which was selected for following experiments ( Fig. 3C ). After IL-1β treatment, circ_0007482 levels were increased, and circ_0007482 siRNA repressed the increased circ_0007482 induced by IL-1β ( Fig. 3D ). Furthermore, CCK-8 assay displayed that the cell viabilities of CHON-001 cell were decreased after IL-1β treatment compared with control. Whereas compared with IL-1β+si-NC group, circ_0007482 knockdown can attenuate the inhibitory effect of IL-1β on chondrocyte viability ( Fig. 3E ). Flow cytometry assay data showed that compared with the control group, the apoptosis rate of CHON-001 in IL-1β treatment group was increased, whereas silencing circ_0007482 could attenuate the promoting effect of IL-1β on chondrocyte apoptosis ( Fig. 3F ).

Figure 3.

Effect of circ_0007482 on IL-1β–treated CHON-001 cell proliferation and apoptosis. (A) The cyclization verification of circ_0007482. (B) Gradually upregulated circ_0007482 in CHON-001 treated with 10 ng/ml IL-1β with the extension of incubation time. (C) Transfection efficiency of circ_0007482 siRNAs. (D) The circ_0007482 expression in IL-1β–treated CHON-001 after circ_0007482 siRNA transfection. (E) Interfering of circ_0007482 promoted IL-1β-inhibited cell proliferation. (F) Cell apoptosis was promoted by IL-1β and inhibited by si-circ_0007482. *P < 0.05, **P < 0.01, ***P < 0.001.

Effect of Circ_0007482 Downregulation on the Inflammatory Factors and Differentiation of Chondrocytes

The inflammatory factors including IL-6, IL-8, and TNF-α mRNA expression were measured in KOA cell model. IL-1β–treated chondrocytes exhibited higher IL-6, IL-8, and TNF-α mRNA levels compared with the control group. In contrast with the IL-1β + si-NC group, silencing circ_0007482 decreased the inflammatory factors expression ( Fig. 4A-C ). After IL-1β treatment, the expression levels of differentiation-related molecules bone morphogenetic protein 2 (BMP2) and Runx2 mRNA were decreased, whereas inhibition of circ_0007482 increased BMP2 and Runx2 mRNA expression ( Fig. 4D and E ).

Figure 4.

Low expression of circ_0007482 repressed inflammatory factors and promoted differentiation of CHON-001 chondrocytes. (A-C) Inhibition of circ_0007482 restained the mRNA expression of inflammatory molecules IL-6, IL-8, and TNF-α. (D and E) Cell differentiation–related molecules bone morphogenetic protein 2 (BMP2) and Runx2 mRNA expression levels were detected by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001.

Circ_0007482 Targeted and Bound to miR-558 in Chondrocytes CHON-001 Cells

The potential circ_0007482-targeted miRNAs were predicted through the CircInteractome website (http://circinteractome.nia.nih.gov). Based on the binding score over 90, 7 miRNAs were selected and measured in CHON-001 cells transfected with circ_0007482 siRNA. Among these miRNAs, miR-558 displayed the most significant expression changes ( Fig. 5A ), which was validated in further experiments. Figure 5B shows the binding sites between circ_0007482 and miR-558. The dual-luciferase reporter assay revealed that the activities of dual-luciferase were reduced in CHON-001 transfected with WT-circ_0007482 and miR-558 mimic ( Fig. 5C ). In addition, the decreased expression miR-558 was observed in cells treated with IL-1β and increased expression of miR-558 was tested in cells after transfected with circ_0007482 siRNA ( Fig. 5D ).

Figure 5.

miR-558 was a potential target of circ_0007482. (A) The CircInteractome predicted potential miRNAs expression was detected by RT-qPCR. (B) The binding sites between circ_0007482 and miR-558 were identified. (C) Dual-luciferase reporter assay verified the target relationship between circ_0007482 and miR-558. (D) The miR-558 expression was downregulated in IL-1β–treated CHON-001 and upregulated by si-circ_0007482. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

This study indicated that circ_0007482 expression was elevated in both KOA tissues and the OA cell model. Silencing circ_0007482 could facilitate chondrocyte cell proliferation and differentiation and repress cell apoptosis and chondrocyte cell inflammation. In addition, miR-558 may be the target miRNA of circ_0007482.

With the in-depth study of genetics and the development of advanced biotechnology, such as RNA sequencing and bioinformatics methods, numerous non-coding RNAs have been identified.^22,23 Research increasingly confirmed that circRNA has crucial biological functions, including miRNA sponging, functional protein trapping, and translation functions, and that the dysfunction of circRNA is involved in the occurrence and progression of various diseases.^24,25 Increasing studies identified circRNAs in OA progression and the regulation of disease state, such as circRNA HIPK3 and circ_0128846.^26,27 The aberrant expression of circ_0007482 was involved in the pathogenesis of pterygium and keloid scarring.^18,28 In this study, overexpressed circ_0007482 was detected in the cartilage tissues of KOA cases, which presented that circ_0007482 might act as a promoting factor in the progression of KOA.

IL-6, IL-8, and TNF-α are inflammatory factors associated with OA and are the main inflammatory mediators in the pathogenesis of KOA. ²⁹ The levels of inflammatory factors can reflect the degree of OA lesions to a certain extent. There is a synergistic effect between the inflammatory factors, which can lead to the destruction of articular cartilage and accelerate the process of OA by mediating the inflammatory response together. ³⁰ The mRNA expression of IL-6, IL-8, and TNF-α in KOA articular cartilage was higher than that in normal articular cartilage, which verified the correlation between inflammatory factors and the progression of KOA. Furthermore, circ_0007482 was positively correlated with the trend of inflammatory factors, which hinted that circ_0007482 might be significantly correlated with the KOA progression.

Abnormal proliferation and differentiation of chondrocytes are the main characteristics affecting joints in KOA diseases. ³¹ CircRNA is differentially expressed in OA. For instance, circ_0002715 was upregulated in OA tissues, and interfering with circ_0002715 could inhibit inflammation and apoptosis in OA chondrocytes induced by IL-1β by sponging miR-127-5p. ³² This study explored the cell function using CHON-001 cells and IL-1β act on CHON-001 cells to mimic the KOA cell model in vitro. BMP2 and Runx2, which are crucial for chondrocyte differentiation, have increased expression when circ_0007482 is downregulated. These data suggest that the downregulation of circ_0007482 could promote cell differentiation. Interference with circ_0007482 not only facilitated IL-1β-stimulated CHON-001 cell proliferation and differentiation, but also repressed inflammation and apoptosis. Similarly, some circRNAs, such as ciRS-7, circSERPINE2, and circ_0045714, are able to inhibit the pathogenesis of OA. ³³ These data confirmed the crucial role of circRNAs in the pathogenesis of OA.

The online CircInteractome website predicted the potential targets of circ_0007482. miR-558 was verified to be a target miRNA of circ_0007482. miR-558 could be targeted by circ_0043610 or circ_0083756 and take part in the progression of several diseases, such as preeclampsia or intervertebral disc degeneration.^34,35 Importantly, miR-558 expression was found to be lower in OA tissues than in normal articular cartilage. ²¹ Another study indicated that circTBX5 could bind to miR-558 to mitigate IL-1β–stimulated chondrocyte apoptosis, ECM degradation, and inflammation. ³⁶ Consistently, herein, miR-558 was downregulated in IL-1β–induced chondrocyte CHON-001 cells. In addition, circ_0007482 could negatively regulate miR-558 expression. These data revealed that circ_0007482 might participate in regulating chondrocyte proliferation, differentiation, and apoptosis of KOA by modulating miR-558 expression. However, the precise regulatory role and specific mechanism of circ_0007482 in the onset and progression of KOA require further verification in future in vivo studies. Besides, whether inhibition of circ_0007482 has effect on the other circRNAs remains unknown, which also need to be explored in future.

In summary, circ_0007482 can partially ameliorate the IL-1β–stimulated chondrocyte injuries by mediating miR-558. This study explored the pathogenesis of KOA from the perspective of circRNA dysregulation, providing the basis for circ_0007482 as a valuable marker for KOA.