- Research article
- Open Access
LncRNA ZFAS1 protects chondrocytes from IL-1β-induced apoptosis and extracellular matrix degradation via regulating miR-7-5p/FLRT2 axis
Journal of Orthopaedic Surgery and Research volume 18, Article number: 320 (2023)
Increasing evidence suggested that long non-coding RNAs (lncRNAs) played vital roles in osteoarthritis (OA) progression. In this study, we aimed to reveal the protective roles of lncRNA ZFAS1 in osteoarthritis (OA) and further investigated its underlying mechanism.
The chondrocytes were stimulated by IL-1β to establish an in vitro OA model. Then, the expression of ZFAS1, miR-7-5p, and FLRT2 in chondrocytes was determined by qRT-PCR. Gain- and loss-of-function assays of ZFAS1, miR-7-5p and FLRT2 were conducted. CCK-8 assay and flow cytometry analysis were performed to detect cell viability and apoptosis rate. The expression levels of cartilage-related proteins, including MMP13, ADAMTS5, Collagen II, and Aggrecan, were measured by western blot analysis. The interaction between ZFAS1 and miR-7-5p, as well as miR-7-5p and FLRT2, was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay.
The expression of ZFAS1 and FLRT2 was down-regulated, while the expression of miR-7-5p was up-regulated in chondrocytes exposed to IL-1β. ZFAS1 overexpression promoted cell viability and suppressed apoptosis in IL-1β-treated chondrocytes. Besides, ZFAS1 overexpression suppressed the expression of MMP13 and ADAMTS5, but promoted the expression of Collagen II and Aggrecan to suppress ECM degradation. The mechanistic study showed that ZFAS1 sponged miR-7-5p to regulate FLRT2 expression. Furthermore, the overexpression of miR-7-5p could neutralize the effect of ZFAS1 in IL-1β-treated chondrocytes, and suppression of FLRT2 counteracted the miR-7-5p down-regulation role in IL-1β-treated chondrocytes.
ZFAS1 could promote cell viability of IL-1β-treated chondrocytes via regulating miR-7-5p/FLRT2 axis.
Trial registration Not applicable.
Osteoarthritis (OA), a chronic and degenerative joint disease, is the most common form of arthritis and a leading cause of deformity and disability in elderly individuals . OA is characterized by articular cartilage degradation, limited synovitis inflammation, osteophyte formation, and subchondral bone [2, 3]. Several risk factors for OA have been identified, including age, sex, obesity, genetics, prior joint injury, and abnormal joint shape [4, 5]. Although the etiology of OA is multifactorial, there is no therapeutic method available yet to modify OA progression. Some evidence has suggested that the synthesis and degradation of cartilage might be associated with the abnormal expression of specific genes in chondrocytes, which are the only cell type that dominates the degenerative process of mature cartilage [6, 7]. Therefore, investigations of new functional genes and molecular mechanisms in OA are of great importance.
So far, several epigenetic regulations have been investigated in OA pathogenesis, including DNA methylation, histone modifications, and non-coding RNAs . Particularly, recent evidences showed that non-coding RNAs played essential roles in cartilage development and OA [9,10,11,12]. Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with a length of more than 200 nucleotides; they have been reported to be the main regulators in the metastasis of tumors, the apoptosis of chondrocytes, and the metabolism of cartilage matrix [13, 14]. LncRNA ZFAS1 is reported to be an oncogene in hepatocellular carcinoma, gastric cancer, osteosarcoma, and glioma [15,16,17,18]. In studies of OA, the expression of ZFAS1 is down-regulated in OA chondrocytes compared with normal chondrocytes [19, 20]. And overexpression of ZFAS1 promotes the OA chondrocytes viability, proliferation, and migration . In addition, ZAFS1 has been shown to serve as a competitive endogenous RNA (ceRNA) in several human cancers, which sponges miRNAs to regulate tumor progression indirectly . In OA, Li et al. reported that ZFAS1 suppresses chondrocytes apoptosis through regulating miR-302d-3p/SMAD2 . Nevertheless, the role and mechanism of ZFAS1 in OA remain largely unknown. miRNAs are a type of small non-coding RNAs in a length of 18–22 nucleotides. The abnormal expression of miRNA has been found to be associated with the occurrence and development of OA . For example, miR-455 overexpression protects cartilage degeneration in a mouse OA model . Zhang et al. found that the miR-132 expression is decreased in OA patients, and miR-132 overexpression elevates cell proliferation and decreased apoptosis of chondrocytes . Here, we identified miR-7-5p as the downstream target of ZFAS1 in OA. miR-7-5p is the most investigated miRNA sequence in the miR-7 family. Previous studies have revealed the anti/pro-tumor effect of miR-7-5p in different human cancers [25, 26]. Another study by Huang et al. found the down-regulated expression of miR-7-5p in OA samples via bioinformatic analysis . However, the involvement of miR-7-5p in OA metastasis and the underlying regulatory between ZFAS1 and miR-7-5p remains to be elucidated.
FLRT2 is a fibronectin leucine-rich transmembrane protein family member, which encodes a small proteoglycan located in the extracellular matrix . Moreover, FLRT2 has been reported to function in receptor signaling and cell adhesion, and its overexpression in chondrogenic cells has been found to alter ERK phosphorylation levels . In this study, we investigated FLRT2, which has not been studied in OA, as a downstream regulator in the ZFAS1-related ceRNA network of OA progression.
Interleukin-1β (IL-1β) is a primary inflammatory factor that plays a central role in several pathological features of OA . In addition, the detrimental effects of IL-1β have been reported on the integrity of extracellular matrix (ECM) and chondrocyte function [30, 31]. In this study, OA chondrocytes induced by IL-1β were used as an in vitro model. We explored the role of ZFAS1 in promoting chondrocytes proliferation, inhibiting chondrocytes apoptosis and ECM degradation, and the underlying mechanism of regulating the miR-7-5p/FLRT2 axis. Our findings helped to clarify the mechanism of OA progression and provided a novel sight into OA treatment.
Cell lines and culture conditions
Human chondrocyte cell line CHON-001 was purchased from Genetimes ExCell Technology, Inc. (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Cells were incubated at 37℃ in a humidified atmosphere with 5% CO2.
To establish the OA model in vitro, chondrocytes were stimulated with IL-1β (R&D Systems, Minneapolis, MN, USA). In brief, when the cells reached a confluence of 70%, the cell culture medium was changed with the medium containing different quantities of IL-1β (0, 1, 5, 10, or 15 ng/mL) for 24 h, and the medium containing 10 ng/mL of IL-1β for different time (0, 6, 12, 24, and 48 h). In the functional studies, the chondrocytes were pre-exposed to ZFAS1 overexpression vector, short hairpin RNA (shRNA) targeting ZFAS1 (sh-ZFAS1), miR-7-5p mimics, miR-7-5p inhibitor, small interfering RNA (siRNA) targeting FLRT2 (si-FLRT2), and their negative controls followed IL-1β (10 ng/mL) for 24 h. All the oligonucleotides or vectors were bought from GenePharma (Shanghai, China), and the transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 48 h transfection, the cells were collected and then used for further experiments. The oligonucleotides are listed in Additional file 1: Table S1.
Quantitative real-time PCR (qRT-PCR)
Total RNAs were isolated from chondrocytes by using TRIzol reagent (Invitrogen, USA) following the manufacturer’s protocol. Then, the RNA was reverse-transcribed into cDNA by using the HiScript II One-Step RT-PCR Kit (Vazyme, Nanjing, China) or miScript Reverse Transcription Kit (Qiagen, Frankfurt, Germany). qRT-PCR was performed on a 7500 Real-Time PCR system (Applied Biosystem) with SYBR Premix Dimer Eraser Kit (Takara, Japan). U6 and GAPDH were used as endogenous controls. The relative quantification was determined by the 2−ΔΔCt method. All used primer sequences are listed in Additional file 1: Table S1.
Cell viability assay
Cell Counting Kit-8 (CCK-8; Dojindo) was applied in this assay to analyze the viability of chondrocytes according to the manufacturer’s protocol. In brief, cells were inoculated into 96-well plates (2000 cells/well) and incubated for 0, 24, 48, 72, and 96 h, respectively. Then, 10 μL of CCK-8 reagent was added into the cell culture and incubated for another 2 h at 37 °C. The OD450nm value was measured by a Microplate Reader (Bio-Rad Laboratories).
The cell apoptosis rate was measured by using the Annexin V-FITC/PI Apoptosis Detection Kit (Sigma). In brief, cells were collected and washed twice with PBS, resuspended in binding buffer, and then stained with Annexin V-FITC and PI staining solutions for 30 min at room temperature. A FACSCalibur flow cytometer (BD Bioscience) was applied to analyze the apoptosis rate. Apoptosis rate results were judged as follows: Annexin V-FITC on the horizontal axis and PI on the vertical axis. The left upper quadrant was the necrotic cells; the right upper quadrant was the late apoptotic cells; the left lower quadrant was the normal cells; the right lower quadrant was the early apoptotic cells. Apoptosis rate was defined as both early apoptotic cells in the right lower quadrant and late apoptotic cells in the right upper quadrant.
Western blot analysis
Total proteins from chondrocytes were extracted by using radioimmunoprecipitation assay lysis (RIPA) and then quantified by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The protein extracts were separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (Bio-Rad). After that, 5% skim milk was used to block the membrane for 30 min at room temperature. Next, the blots were incubated with primary antibodies obtained from Abcam: anti-FLRT2 (1/500, ab154023), anti-matrix metalloproteinase 13 (MMP13) (1/5000, ab39012), anti-ADAMTS5 (1/250, ab41037), anti-Collagen II (1/1000, ab188570), anti-Aggrecan (1 μg/mL, ab3778), and anti-β-actin (1/2000, ab8227), overnight at 4℃. Membranes were then incubated with the secondary antibody (ab7090) for 1 h at room temperature. The protein bands were measured using the enhanced chemiluminescence system (Millipore).
Dual-luciferase reporter assay
The wild type and mutant sequences of ZFAS1 and FLRT2 containing the binding site of miR-7-5p were designed by Sangon Biotech (Shanghai, China) and cloned into pmirGLO luciferase reporter vector (Promega, USA). The reporter vector was co-transfected with miR-7-5p mimics or the negative control into chondrocytes by Lipofectamine 2000 (Invitrogen), respectively. After 48 h of transfection, the relative luciferase activity of the chondrocytes from each group was determined by the Dual-Luciferase Reporter Assay System (Promega).
To investigate the subcellular localization of ZFAS1 in chondrocytes, a fluorescent in situ hybridization kit (RiboBio) was used following the instructions. Briefly, the probe targeting ZFAS1 was obtained from GenePharma (Shanghai, China). Then the anti-ZFAS1 probe was labeled with Alexa Fluor 594 and incubated with CHON-001 cells at 55 °C for 18 h. Cell nuclei were visualized by staining with 4ʹ,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 30 min. The subcellular localization of ZFAS1 could be observed under a fluorescence microscope (Nikon, Japan).
Isolation of cytoplasmic and nuclear RNA
Cytoplasmic and nuclear RNA was isolated and purified by the Cytoplasmic & Nuclear RNA Purification Kit (Norgen) according to the manufacturer’s protocol. U6 and GAPDH were taken as the internal references.
RNA immunoprecipitation assay (RIP)
The EZ Magna RNA Immunoprecipitation Kit (Millipore) was used to confirm the interaction between ZFAS1 and miR-7-5p under the manufacturer’s instructions. In brief, the normal human chondrocyte cells were lysed using RIP lysis buffer. Then, 5 μg of anti-Ago2 antibody (Abcam) or anti-IgG antibody (Abcam) was added into the cell lysate and incubated with magnetic beads overnight at 4℃ for immunoprecipitation. IgG served as a negative control. Finally, the co-precipitated RNA fraction was purified by RIP assay and detected by qRT-PCR.
All statistical analyses were conducted using GraphPad 9.1.1. All data were presented as the mean ± standard deviation (SD). The significant difference between groups was determined by Student’s t-test or one-way ANOVA. P < 0.05 was considered statistically significant.
ZFAS1 expression was down-regulated in IL-1β-stimulated chondrocytes
Increasing evidence suggested that lncRNAs played vital roles in OA progression. A previous study based on microarray analysis reported four differently expressed lncRNAs (SNHG5, ZFAS1, GAS5, and DANCR) involved in OA cartilage . Here, we detected the expression of the four lncRNAs in IL-1β-treated (10 ng/mL, 24 h) chondrocytes. As shown in Additional file 2: Fig. S1, the expression of GAS5 and DANCR was significantly up-regulated after IL-1β stimulation (P < 0.01), while the expression of SNHG5 and ZFAS1 was significantly down-regulated after IL-1β stimulation (P < 0.01). In the two significant down-regulated lncRNAs, ZFAS1 showed a higher fold change; thus, we selected ZFAS1 in our subsequent studies. We detected the ZFAS1 expression in chondrocytes stimulated with IL-1β at different times or with different IL-1β concentrations. qRT-PCR results showed that ZFAS1 expression was down-regulated with the time increases (Fig. 1A, P < 0.01). Moreover, with the concentration increasing, the ZFAS1 expression was gradually down-regulated (Fig. 1B, P < 0.05). Overall, these results suggested that ZFAS1 was down-regulated in the in vitro OA model.
ZFAS1 suppressed IL-1β-induced ECM degradation and apoptosis of chondrocytes and promoted cell proliferation
We then investigated the effects of ZFAS1 on cell viability and apoptosis of IL-1β-stimulated chondrocytes. The cells were transfected with ZFAS1 overexpression vector and conducted to the CCK-8 assay and flow cytometry apoptosis analysis. As shown in Fig. 2A, transfection with ZFAS1 overexpression vector significantly increased the ZFAS1 expression in chondrocytes (P < 0.01). Then, CCK-8 assay results showed that the cell viability of IL-1β-stimulated chondrocytes was remarkably lower than the control group (Fig. 2B, P < 0.01). Moreover, ZFAS1 overexpression in IL-1β-stimulated chondrocytes significantly enhanced the cell viability rate compared to the IL-1β + vector group (Fig. 2B, P < 0.05). In addition, flow cytometry results indicated that apoptosis rate was significantly increased after IL-1β treatment, while the ZFAS1 overexpression significantly reduced the cell apoptosis rate (Fig. 2C, P < 0.01). As MMP13 and ADAMTS5 were positively correlated with ECM degradation, and Collagen II and Aggrecan were negatively correlated with ECM degradation in chondrocytes. We then detect the expression of MMP13, ADAMTS5, Collagen II, and Aggrecan with western blot assay. As expected, the expression of MMP13 and ADAMTS5 were significantly up-regulated after IL-1β stimulation, while the expression of Collagen II and Aggrecan was significantly decreased after IL-1β stimulation (Fig. 2D, P < 0.01). And the expression changes in response to IL-1β stimulation were abolished with ZFAS1 overexpression (Fig. 2D, P < 0.05). In addition, knockdown of ZFAS1 was conducted for further verification. As shown in Fig. 3A–D, knockdown of ZFAS1 significantly reduced the cell viability rate while increasing cell apoptosis rate after IL-1β treatment (P < 0.05). In short, these results demonstrated that overexpression of ZFAS1 could promote cell viability and repress cell apoptosis and ECM degradation in IL-1β treated chondrocytes.
ZFAS1 directly targeted miR-7-5p in chondrocytes
lncRNAs have been reported to sponge miRNAs and serve as ceRNAs . Hence, we detected the expression distribution of ZFAS1 in chondrocytes. The results showed that ZFAS1 was mainly located in the cytoplasm of chondrocytes (Fig. 4A). FISH assay further confirmed the cytoplasm location of ZFAS1 in chondrocytes (Fig. 4B). With the starBase webtool, we found that ZFAS1 had binding sites to miR-7-5p (Fig. 4C). The dual-luciferase reporter assay was performed to confirm the targeting relationship. Transfection of miR-7-5p mimics significantly promoted the miR-7-5p expression in chondrocytes (Fig. 4D, P < 0.01). Then, the luciferase activity of the ZFAS1-Wt luciferase reporter vector was decreased with miR-7-5p overexpression, while the luciferase activity in the ZFAS1-Mut luciferase vector had no obvious changes with miR-7-5p overexpression (Fig. 4E, P < 0.01). For further confirmation, we performed the RIP assay, and the results showed that both ZFAS1 and miR-7-5p were enriched in Ago2 immunoprecipitation compared to IgG immunoprecipitation (Fig. 4F, P < 0.01), indicating that ZFAS1 and miR-7-5p had a targeting relationship in chondrocytes. Moreover, the qRT-PCR results manifested that miR-7-5p expression was remarkably increased in IL-1β-treated chondrocytes (Fig. 4G, P < 0.01). Additionally, the overexpression of ZFAS1 could down-regulate the expression of miR-7-5p in chondrocytes (Fig. 4H, P < 0.01). The above results demonstrated that ZFAS1 could sponge miR-7-5p and suppress its expression in chondrocytes.
miR-7-5p could neutralize the effects of ZFAS1 overexpression on viability, apoptosis, and ECM degradation in IL-1β-stimulated chondrocytes
In order to confirm the regulatory function of the ZFAS1/miR-7-5p axis in IL-1β-stimulated chondrocytes, rescue experiments were performed. As shown in Fig. 5A, miR-7-5p expression was suppressed in chondrocytes with ZFAS1 overexpression (P < 0.01), and this down-regulation was then compensated by increasing miR-7-5p expression via miR-7-5p mimics co-transfection (P < 0.01). Subsequently, the results of CCK-8 assay and flow cytometry analysis indicated that the promoting effect of ZFAS1 overexpression on cell viability and its inhibitory effect on cell apoptosis could be partly neutralized by co-transfection with miR-7-5p mimics (Fig. 5B–D, P < 0.05). We next analyzed the effects of miR-7-5p on the ZFAS1-mediated modulation of the ECM in chondrocytes. The results showed that ZFAS1 overexpression led to MMP13 and ADAMTS5 down-regulation and Collagen II and Aggrecan up-regulation, which was overall blocked by increasing miR-7-5p expression in chondrocytes (Fig. 5E, P < 0.05). Rescue experiments were also performed with sh-ZFAS1 and miR-7-5p inhibitor, and the results indicated that the effects of ZFAS1 knockdown on IL-1β-treated chondrocytes could be partly neutralized by co-transfection with miR-7-5p inhibitor (Fig. 5A–E, P < 0.05). To conclude, these results suggested a ZFAS1/miR-7-5p interaction in regulating OA chondrocytes viability, apoptosis, and ECM degradation.
miR-7-5p directly targeted FLRT2
miRNA could bind to the 3'UTR of target mRNAs and subsequently induce their degradation . In the current study, we used miRanda and TargetScan to predict the targets of miR-7-5p and interacted the results with the down-regulated mRNAs in GSE110606 (Fig. 6A, Additional file 3: Table S2). Among the three candidate mRNAs (SSX2IP, FLRT2, and NREP), FLRT2 was previously reported to promote cellular proliferation and inhibit cell adhesion during chondrogenesis . Thus, we selected FLRT2 as the potential targeted gene of miR-7-5p. The binding sites between miR-7-5p and FLRT2 are shown in Fig. 6B. Dual-luciferase reporter assay confirmed the targeting relationship that miR-7-5p mimics significantly decreased the luciferase activity of FLRT2-Wt reporter, but showed no significant effects on the luciferase activity of FLRT2-Mut reporter (Fig. 6B). In addition, FLRT2 mRNA and protein expression levels decreased remarkably in IL-1β-treated chondrocytes (Fig. 6C, D, P < 0.01). Furthermore, qRT-PCR and western blot results confirmed that miR-7-5p overexpression could remarkably suppress FLRT2 expression in chondrocytes (Fig. 6E, F, P < 0.01). Besides, ZFAS1 overexpression led to high expression of FLRT2 IL-1β-treated chondrocytes, and this was rescued by co-transfection with miR-7-5p mimics (Fig. 7A, B, P < 0.01). In addition, knockdown of ZFAS1 resulted in down-regulation of FLRT2 in IL-1β-treated chondrocytes, and this was rescued by co-transfection with miR-7-5p inhibitors (Fig. 7A, B, P < 0.05). Thus, it was indicated that FLRT2 was downstream of ZFAS1/miR-7-5p in chondrocytes.
FLRT2 suppression could neutralize the effects of miR-7-5p inhibition on IL-1β-treated chondrocytes
Since the targeting relationship between FLRT2 and miR-7-5p was confirmed, we next wanted to explore whether the effects of miR-7-5p inhibition on the cell viability, apoptosis, and ECM degradation could be restored by FLRT2 suppression in IL-1β-treated chondrocytes. miR-7-5p inhibition markedly promoted FLRT2 expression in chondrocytes, and this effect was restored by co-transfection with FLRT2 siRNA (Fig. 8A, P < 0.05). In CCK-8 assay, we found that the inhibition of miR-7-5p could markedly increase the viability of IL-1β-treated chondrocytes (Fig. 8B, P < 0.01). However, the effect of miR-7-5p inhibition was diminished after the co-transfection with FLRT2 siRNA (Fig. 8B, P < 0.01). On the other hand, the apoptosis rates of IL-1β-treated chondrocytes were significantly repressed in miR-7-5p inhibited group, while the repression was neutralized when co-transfected with FLRT2 siRNA (Fig. 8C-D, P < 0.01). Subsequently, western blot results showed that the expression of MMP13 and ADAMTS5 was decreased obviously with miR-7-5p inhibition, while the co-transfection of miR-7-5p inhibitor and FLRT2 siRNA could restore the MMP13 and ADAMTS5 expression (Fig. 8E, P < 0.01). Oppositely, the expression of Collagen II and Aggrecan was up-regulated with miR-7-5p inhibition, and this up-regulation was abrogated by co-transfection with miR-7-5p inhibitor and FLRT2 siRNA (Fig. 8E, P < 0.01). In brief, the above results implied a miR-7-5p/FLRT2 axis in regulating OA chondrocytes viability, apoptosis, and ECM degradation.
OA as a degenerative joint disease shows a high incidence in middle-aged and elderly individuals. It is characterized by the destruction of articular cartilage and can lead to joint stiffness and loss of motor ability . Previous studies have reported the association between the pathogenesis and development of OA and lncRNAs. For instance, lncRNA-CIR, related to cartilage injury, is able to induce in vitro degradation of cartilage extracellular matrix ; lncRNA HOTAIR overexpression could contribute to IL-1β-induced chondrocyte apoptosis and matrix metalloproteinases overexpression . However, the expression profile of lncRNAs and their potential targets as well as biological functions in terms of OA development remain elusive. In this study, the effect of lncRNA ZFAS1 in OA development was explored.
IL-1β was commonly used to simulate chondrocytes affected by OA. As reported, IL-1β could promote the secretion of pro-inflammatory cytokines and suppress collagen synthesis, thereby resulting in the biological dysfunction of chondrocytes and the degradation of articular cartilage . Here, we found that IL-1β treatment decreased the cell viability and increased the apoptosis of chondrocytes. We also found that IL-1β treatment promoted MMP-13 and ADAMTS5 expression and suppressed Collagen II and Aggrecan expression to stimulate ECM destruction in chondrocytes, which confirmed the effect of IL-1β on aggravating OA development.
ZFAS1 has been reported as an essential player in promoting chondrocyte proliferation and suppressing matrix synthesis in OA, and it works through Wnt3a . In consist with the previous study, we found that ZFAS1 expression was decreased in IL-1β-treated chondrocytes. Functionally, overexpression of ZFAS1 promoted the viability and suppressed the apoptosis of IL-1β-treated chondrocytes. Furthermore, overexpression of ZFAS1 could inhibit IL-1β-induced ECM degradation, which has not been reported before. Besides, we found that ZFAS1 is mainly located in the cytoplasm of chondrocytes, suggesting a potential of ZFAS1 as a ceRNA. Li et al. found ZFAS1 sponged miR-302d-3p to regulate SMAD2 expression in OA . In this study, we identified miR-7-5p as a downstream target of ZFAS1, which was also reported by Peng et al.  and Mo et al. .
According to the qRT-PCR results, miR-7-5p was highly expressed in IL-1β-treated chondrocytes. Chen et al. reported that miR-7-5p expression is up-regulated in OA knee cartilage tissues and IL-1β-stimulated OA chondrocytes . Zhou et al. also found an up-regulated expression of miR-7 in chondrocytes induced by IL-1β . Our result was consistent with the previous studies. Furthermore, we found that the overexpression of miR-7-5p could notably reduce the function of ZFAS1 overexpression with respect to cell viability, apoptosis and ECM degradation in IL-1β-stimulated chondrocytes. All these results suggested that ZFAS1 regulated cell viability, apoptosis, and ECM degradation by functioning as a sponge for miR-7-5p in chondrocytes stimulated by IL-1β.
We then identified FLRT2 as the downstream target of miR-7-5p. FLRT2 belonged to the fibronectin leucine-rich transmembrane protein family, which are essential factors in guiding several biological processes, such as neural, vascular, and early embryonic development [43,44,45]. It was reported that FLRT2 played an essential role in regulating cell adhesion and cell–matrix interactions during early chondrogenesis [46, 47]. In addition, Xu et al.  identified that FLRT2 was overexpressed in ATDC5 cells and could promote cell proliferation and reduce intercellular adhesion at early chondrogenesis. In this study, dual-luciferase reporter assay confirmed the targeting relationship between miR-7-5p and FLRT2. In addition, FLRT2 suppression could neutralize the viability, apoptosis, and matrix synthesis of OA chondrocytes, which were mediated by miR-7-5p inhibition. More importantly, the FLRT2 expression was significantly increased in OA chondrocytes following ZFAS1 overexpression, indicating that FLRT2 was regulated by ZFAS1. Therefore, ZFAS1 might regulate the FLRT2 expression indirectly via sponging miR-7-5p, and we found the regulatory network of ZFAS1/miR-7-5p/FLRT2 in OA.
In summary, our study demonstrated that ZFAS1 could enhance proliferation and inhibit apoptosis and matrix synthesis in IL-1β-treated chondrocytes via regulating miR-7-5p/FLRT2 axis. ZFAS1 functioned as a novel ceRNA might serve as a potential therapeutic target for OA treatment. Nevertheless, the regulatory effects of ZFAS1 on OA development still require further demonstration by in vivo studies with animal models.
Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Cell Counting Kit-8
Competitive endogenous RNA
Dulbecco’s modified Eagle’s medium
Fetal bovine serum
Long non-coding RNAs
Matrix metalloproteinase 13
RNA immunoprecipitation assay
Short hairpin RNA
Small interfering RNA
Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum. 2012;64:1697–707.
Goldring MB, Goldring SR. Osteoarthritis. J Cell Physiol. 2007;213:626–34.
Felson DT, Lawrence RC, Dieppe PA, et al. Osteoarthritis: new insights. Part 1: the disease and its risk factors. Ann Intern Med. 2000;133:635–46.
Blagojevic M, Jinks C, Jeffery A, Jordan K. Risk factors for onset of osteoarthritis of the knee in older adults: a systematic review and meta-analysis. Osteoarthritis Cartil. 2010;18:24–33.
Felson DT. Osteoarthritis of the knee. N Engl J Med. 2006;354:841–8.
Kraus V, Jordan J, Doherty M, et al. The genetics of generalized osteoarthritis (GOGO) study: study design and evaluation of osteoarthritis phenotypes. Osteoarthritis Cartil. 2007;15:120–7.
Aigner T, Söder S, Gebhard PM, McAlinden A, Haag J. Mechanisms of disease: role of chondrocytes in the pathogenesis of osteoarthritis—structure, chaos and senescence. Nat Clin Pract Rheumatol. 2007;3:391–9.
Reynard LN, Loughlin J. Genetics and epigenetics of osteoarthritis. Maturitas. 2012;71:200–4.
Gargano G, Oliva F, Oliviero A, Maffulli N. Small interfering RNAs in the management of human rheumatoid arthritis. Br Med Bull. 2022;142:34–43.
Gargano G, Oliviero A, Oliva F, Maffulli N. Small interfering RNAs in tendon homeostasis. Br Med Bull. 2021;138:58–67.
Oliviero A, Della Porta G, Peretti GM, Maffulli N. MicroRNA in osteoarthritis: physiopathology, diagnosis and therapeutic challenge. Br Med Bull. 2019;130:137–47.
Giordano L, Porta GD, Peretti GM, Maffulli N. Therapeutic potential of microRNA in tendon injuries. Br Med Bull. 2020;133:79–94.
Chen C, Xu G, Yuan K, et al. Transcriptional analysis of long non-coding RNAs in facet joint osteoarthritis. RSC Adv. 2018;8:33695–701.
Hajjari M, Salavaty A. HOTAIR: an oncogenic long non-coding RNA in different cancers. Cancer Biol Med. 2015;12:1.
Li T, Xie J, Shen C, et al. Amplification of long noncoding RNA ZFAS1 promotes metastasis in hepatocellular carcinoma. Cancer Res. 2015;75:3181–91.
Zhou H, Wang F, Chen H, et al. Increased expression of long-noncoding RNA ZFAS1 is associated with epithelial-mesenchymal transition of gastric cancer. Aging. 2016;8:2023–38.
Liu G, Wang L, Han H, et al. LncRNA ZFAS1 promotes growth and metastasis by regulating BMI1 and ZEB2 in osteosarcoma. Am J Cancer Res. 2017;7:1450–62.
Gao K, Ji Z, She K, Yang Q, Shao L. Long non-coding RNA ZFAS1 is an unfavourable prognostic factor and promotes glioma cell progression by activation of the Notch signaling pathway. Biomed Pharmacother. 2017;87:555–60.
Ye D, Jian W, Feng J, Liao X. Role of long noncoding RNA ZFAS1 in proliferation, apoptosis and migration of chondrocytes in osteoarthritis. Biomed Pharmacother. 2018;104:825–31.
Li J, Liu M, Li X, Shi H, Sun S. Long noncoding RNA ZFAS1 suppresses chondrocytes apoptosis via miR-302d-3p/SMAD2 in osteoarthritis. Biosci Biotechnol Biochem. 2021;85:842–50.
Tay Y, Rinn J, Pandolfi PP. The multilayered complexity of ceRNA crosstalk and competition. Nature. 2014;505:344–52.
Nugent M. MicroRNAs: exploring new horizons in osteoarthritis. Osteoarthritis Cartil. 2016;24:573–80.
Ito Y, Matsuzaki T, Ayabe F, et al. Both microRNA-455-5p and -3p repress hypoxia-inducible factor-2alpha expression and coordinately regulate cartilage homeostasis. Nat Commun. 2021;12:4148.
Zhang W, Hu C, Zhang C, Luo C, Zhong B, Yu X. MiRNA-132 regulates the development of osteoarthritis in correlation with the modulation of PTEN/PI3K/AKT signaling. BMC Geriatr. 2021;21:175.
Kalinowski FC, Brown RA, Ganda C, et al. microRNA-7: a tumor suppressor miRNA with therapeutic potential. Int J Biochem Cell Biol. 2014;54:312–7.
Yu Z, Ni L, Chen D, et al. Identification of miR-7 as an oncogene in renal cell carcinoma. J Mol Histol. 2013;44:669–77.
Huang PY, Wu JG, Gu J, et al. Bioinformatics analysis of miRNA and mRNA expression profiles to reveal the key miRNAs and genes in osteoarthritis. J Orthop Surg Res. 2021;16:63.
Lacy SE, Bönnemann CG, Buzney EA, Kunkel LM. Identification of FLRT1, FLRT2, and FLRT3: a novel family of transmembrane leucine-rich repeat proteins. Genomics. 1999;62:417–26.
Wei K, Xu Y, Tse H, Manolson M, Gong S-G. Mouse FLRT2 interacts with the extracellular and intracellular regions of FGFR2. J Dent Res. 2011;90:1234–9.
Daheshia M, Yao JQ. The interleukin 1beta pathway in the pathogenesis of osteoarthritis. J Rheumatol. 2008;35:2306–12.
Jenei-Lanzl Z, Meurer A, Zaucke F. Interleukin-1β signaling in osteoarthritis—chondrocytes in focus. Cell Signal. 2019;53:212–23.
Xiao K, Yang Y, Bian Y, et al. Identification of differentially expressed long noncoding RNAs in human knee osteoarthritis. J Cell Biochem. 2019;120:4620–33.
Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33.
Xu Y, Wei K, Kulyk W, Gong SG. FLRT2 promotes cellular proliferation and inhibits cell adhesion during chondrogenesis. J Cell Biochem. 2011;112:3440–8.
Takagi S, Omori G, Koga H, et al. Quadriceps muscle weakness is related to increased risk of radiographic knee OA but not its progression in both women and men: the matsudai knee osteoarthritis Survey. Knee Surg Sports Traumatol Arthrosc. 2018;26:2607–14.
Liu Q, Zhang X, Dai L, et al. Long noncoding RNA related to cartilage injury promotes chondrocyte extracellular matrix degradation in osteoarthritis. Arthritis Rheumatol. 2014;66:969–78.
Zhang C, Wang P, Jiang P, et al. Upregulation of lncRNA HOTAIR contributes to IL-1β-induced MMP overexpression and chondrocytes apoptosis in temporomandibular joint osteoarthritis. Gene. 2016;586:248–53.
Wojdasiewicz P, Poniatowski ŁA, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis. Mediat Inflamm 2014;2014.
Peng J, Liu F, Zheng H, Wu Q, Liu S. IncRNA ZFAS1 contributes to the radioresistance of nasopharyngeal carcinoma cells by sponging hsa-miR-7-5p to upregulate ENO2. Cell Cycle. 2021;20:126–41.
Mo D, Liu W, Li Y, Cui W. Long Non-coding RNA zinc finger antisense 1 (ZFAS1) regulates proliferation, migration, invasion, and apoptosis by targeting MiR-7-5p in colorectal cancer. Med Sci Monit. 2019;25:5150–8.
Yang M, Wang A, Li C, et al. Methylation-induced silencing of ALDH2 facilitates lung adenocarcinoma bone metastasis by activating the MAPK pathway. Front Oncol. 2020;10:1141.
Li B, Fang J, He T, et al. Resistin up-regulates LPL expression through the PPARgamma-dependent PI3K/AKT signaling pathway impacting lipid accumulation in RAW264.7 macrophages. Cytokine. 2019;119:168–74.
Yamagishi S, Hampel F, Hata K, et al. FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons. EMBO J. 2011;30:2920–33.
Tai-Nagara I, Yoshikawa Y, Numata N, et al. Placental labyrinth formation in mice requires endothelial FLRT2/UNC5B signaling. Development. 2017;144:2392–401.
Maretto S, Müller P-S, Aricescu AR, Cho KW, Bikoff EK, Robertson EJ. Ventral closure, headfold fusion and definitive endoderm migration defects in mouse embryos lacking the fibronectin leucine-rich transmembrane protein FLRT3. Dev Biol. 2008;318:184–93.
Flintoff KA, Arudchelvan Y, Gong S-G. FLRT2 interacts with fibronectin in the ATDC5 chondroprogenitor cells. J Cell Physiol. 2014;229:1538–47.
Seiradake E, del Toro D, Nagel D, et al. FLRT structure: balancing repulsion and cell adhesion in cortical and vascular development. Neuron. 2014;84:370–85.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1: Table S1.
The sequences of oligonucleotides and primers
Additional file 2: Figure S1.
lncRNAs expression changes in chondrocytes treated with IL-1β. A–D The expression levels of four lncRNAs were determined by qRT-PCR analysis. The chondrocytes were treated with 10 ng/mL IL-1β for 24 h. **P < 0.01, compared with the control group
Additional file 3: Table S2.
The list of the down-regulated genes in GSE110606
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Han, J., Luo, Z., Wang, Y. et al. LncRNA ZFAS1 protects chondrocytes from IL-1β-induced apoptosis and extracellular matrix degradation via regulating miR-7-5p/FLRT2 axis. J Orthop Surg Res 18, 320 (2023). https://doi.org/10.1186/s13018-023-03802-9