LncRNA CYTOR Regulates Chondrocyte Apoptosis in Osteoarthritis by Acting as a CeRNA for miR-10a-5p

Background: It has been reported that lncRNA CYTOR suppresses LPS-induced inammation, which plays crucial roles in osteoarthritis (OA). This study was therefore performed to analyze the role of CYTOR in OA. Methods: Expression of CYTOR and miR-10a-5p in OA patients and chondrocytes as well as healthy patients and chondrocytes was determined by RT-qPCR. The interaction between CYTOR and miR-10a-5p was predicted by IntaRNA online program and conrmed by dual luciferase activity assay. The role of CYTOR and miR-10a-5p in regulating the apoptosis of chondrocytes were analyzed by cell apoptosis assay. Results: CYTOR was under-expressed in OA patients and chondrocytes, and miR-10a-5p was overexpressed in OA patients and chondrocytes. After treatment, CYTOR was upregulated and miR-10a-5p was downregulated. CYTOR was predicted to interact with miR-10a-5p, and the interaction between them was conrmed by dual luciferase reporter assay. Overexpression of CYTOR and miR-10a-5p failed to affect the expression of each other. Cell apoptosis analysis showed that CYTOR overexpression reduced the enhancing effects of miR-10a-5p overexpression on the apoptosis of OA chondrocytes. Conclusion: CYTOR may suppress the development of OA by serving as a ceRNA for miR-10a-5p.

overexpressed in OA patients and chondrocytes. After treatment, CYTOR was upregulated and miR-10a-5p was downregulated. CYTOR was predicted to interact with miR-10a-5p, and the interaction between them was con rmed by dual luciferase reporter assay. Overexpression of CYTOR and miR-10a-5p failed to affect the expression of each other. Cell apoptosis analysis showed that CYTOR overexpression reduced the enhancing effects of miR-10a-5p overexpression on the apoptosis of OA chondrocytes.
Conclusion: CYTOR may suppress the development of OA by serving as a ceRNA for miR-10a-5p.

Background
Osteoarthritis (OA), also known as degenerative joint disease, is the most common form of arthritis that mainly affects the protective cartilage around joints [1]. The highest prevalence of OA is observed in the age group > 60 years, it is estimated that more than 13% of females and 10% of males in this age group are suffering for OA [2,3]. At present, there is no cure for OA [4]. Current therapeutic approaches main focus on the relief of symptoms, such as the use of Acetaminophen to reduce mild to moderate pain [5]. However, medications may not be helpful for severe cases. In addition, long-term use of Acetaminophen may cause liver damage [6]. Therefore, novel therapies are needed.
OA is closely correlated with aging [7]. Besides that, the development and progression of OA also require the involvement of molecular pathways, such as Notch and NF-κB signaling [8]. With the increased elucidation of the molecular mechanism of OA, novel therapies, such as targeted therapy to treat OA by regulating gene expression, have been developed [9,10]. However, safe and effective treatments for OA remain lacking. LncRNAs are not directly responsible for protein-coding but they regulate gene expression network to participate in human diseases including OA, suggesting that they are potential targets for OA treatment [11]. However, the function of most lncRNAs in OA remains to be elucidated. In a recent study, lncRNA CYTOR was reported to play critical roles in LPS-induced cell injury [12], which can promote OA development [13]. We performed RNA-RNA interaction and found that CYTOR could interact with miR-10a-5p, which may promote the apoptosis of miR-10a-5p to participate in OA [14]. We then explore the interactions between CYTOR and miR-10a-5p in OA.

Results
Expression of CYTOR and miR-10a-5p was altered in both OA patients and chondrocytes.
Expression of CYTOR and miR-10a-5p in OA patients (synovial uid) and chondrocytes as well as healthy patients (synovial uid) and chondrocytes was determined by RT-qPCR. Compared to controls, CYTOR was signi cantly under-expressed in OA patients and chondrocytes (Fig. 1A, p < 0.01). In contrast, miR-10a-5p was signi cantly overexpressed in OA patients and chondrocytes compared to corresponding controls (Fig. 1B, p < 0.01). Therefore, altered expression of CYTOR and miR-10a-5p may participate in OA.
Treatment increased the expression of CYTOR and decreased the expression of miR-10a-5p in OA patients Expression of CYTOR and miR-10a-5p in synovial uid collected from OA patients before and 1 and 2 months after the initiation of treatment. Heatmaps were plotted using Heml 1.0 software to present the changes in the expression levels of CYTOR and miR-10a-5p during treatment. Compared to pre-treatment levels, CYTOR was upregulated and miR-10a-5p was downregulated during treatment.
CYTOR and miR-10a-5p directly interacted with each other in two types of chondrocytes The potential interaction between CYTOR and miR-10a-5p was predicted by IntaRNA2.0. It was observed that CYTOR and miR-10a-5p may form basepairing with each other (Fig. 3A). Dual luciferase reporter assay was performed to con rm the interaction between CYTOR and miR-10a-5p. Compared to the CYTOR luciferase vector + NC miRNA group (NC group), luciferase activity was signi cantly lower in CYTOR luciferase vector + miR-10a-5p mimic group (miR-10a-5p group), suggesting the direct interaction between them (Fig. 3B, p < 0.05).
Overexpression of CYTOR and miR-10a-5p failed to alter the expression of each other To further explore the interaction between CYTOR and miR-10a-5p, OA and control chondrocytes were transfected with either CYTOR expression vector or miR-10a-5p mimic, and the expression of them was detected by RT-qPCR every 24 h until 96 h. It was observed that CYTOR and miR-10a-5p were signi cantly overexpressed between 48 h and 96 h (Fig. 4A, p < 0.05). However, at different time points, overexpression of CYTOR failed to signi cantly affect the expression of miR-10a-5p (Fig. 4B, p < 0.05), and overexpression of miR-10a-5p also showed no signi cant effect on the expression of CYTOR (Fig. 4C). Therefore, CYTOR is unlikely a target of miR-10a-5p.
CYTOR overexpression suppressed the apoptosis of OA chondrocytes through miR-10a-5p The role of CYTOR and miR-10a-5p in regulating the apoptosis of OA and control chondrocytes was analyzed by cell apoptosis assay. CYTOR overexpression decreased the apoptosis of OA chondrocytes, and miR-10a-5p overexpression increased OA chondrocytes. Moreover, CYTOR overexpression reduced the enhancing effects of miR-10a-5p overexpression on the apoptosis of OA chondrocytes (Fig. 5A, p < 0.05). In contrast, overexpression of CYTOR and miR-10a-5p failed to signi cantly affect the expression of control chondrocytes (Fig. 5B). Therefore, CYTOR may suppress the apoptosis of OA chondrocytes by serving as a ceRNA of miR-10a-5p.

Discussion
The involvement of CYTOR in OA and its interaction with miR-10a-5p were analyzed in this study. We found that CYTOR was downregulated in OA and overexpression of CYTOR may suppress the apoptosis of OA chondrocytes by serving as a ceRNA of miR-10a-5p.
CYTOR has been characterized as an oncogenic lncRNA in many types of cancers [15,16]. In cancers, CYTOR is overexpressed to promote cancer development by increasing cell proliferation and suppressing cell apoptosis [15,16]. In this study we found that CYTOR was downregulated in both OA patients and OA chondrocytes. Moreover, overexpression of CYTOR decreased the apoptosis of OA chondrocytes. Therefore, CYTOR may play protective roles in OA, and overexpression of CYTOR may serve as a target for the treatment of OA. However, in vivo animal model experiments and clinical trials are needed to test our hypothesis. It is also worth noting that CYTOR suppress cell apoptosis in both cancers [15,16] and OA. Therefore, CYTOR may play similar roles in cell apoptosis in different human diseases.
It has been reported that miR-10a-5p is overexpressed in OA and targets HOXA1 to promote the apoptosis of chondrocytes [14]. Consistently, we also observed the overexpression of miR-10a-5p in OA and its enhancing effects on the apoptosis of OA chondrocyte. However, the upstream regulator or miR-10a-5p in OA is unclear. Our study showed that PTV1 and miR-10a-5p could interact with each other, while they failed to affect the expression of each other. Moreover, CYTOR overexpression suppressed the role of miR-10a-5p in enhancing OA chondrocyte apoptosis. Therefore, CYTOR may suppress the apoptosis of OA chondrocytes by serving as a ceRNA of miR-10a-5p.
Interestingly, CYTOR and miR-10a-5p could directly interact with each other in both OA and control chondrocytes, while they only regulate the apoptosis of OA chondrocytes, but not control chondrocytes. Therefore, other pathological factors may interact with CYTOR/miR-10a-5p to participate in the apoptosis of chondrocytes in OA.

Conclusion
In conclusion, CYTOR is under-expressed in OA and may promote the apoptosis of OA chondrocytes by serving as the ceRNA of miR-10a-5p.

OA patients and healthy controls
This study enrolled 52 OA patients (20 males and 32 females, severe stages) and 52 healthy controls (20 males and 32 females) at A liated Hospital of Guizhou Medical University between March 2019 and June 2020. This study was approved by Ethics Committee of this hospital. Age ranges of patients and control were both 52 to 66 years, with a median of 59 years. All OA patients were diagnosed for the rst time and no severe clinical disorders were diagnosed. Initiated therapy was excluded from the patients.
All healthy controls showed normal physiological functions during systemic physiological health test. All patients signed informed consent. According to affected sites, the 52 patients included 20 cases of knee, 14 cases of hip, and 18 cases of feet.

Treatment and synovial uid preparation
Synovial uid (about 1.0 ml) was extracted from the affected sites of patients and the corresponding sites of controls prior to therapy. All patents were treated with acetaminophen and physical therapy.
Synovial uid (about 1.0 ml) was also extracted from the affected sites of patients at 1 and 2 months after the initiation of therapy. All synovial uid samples were kept in liquid nitrogen storage prior to subsequent assays.

Two types of chondrocytes
Chondrocytes derived from an adult OA patient (402OA-05A) and a healthy control (402-05A) were both from Sigma-Aldrich. Chondrocyte growth medium (PromoCell) was used to cultivate cells at 37°C, 5% CO 2 and 95% humidity.
Transfections and dual luciferase reporter assay CYTOR expression vector was constructed using pcDNA3.1 vector (Invitrogen) as backbone. Mimic of miR-10a-5p and miRNA negative control (NC) were the products from Sigma-Aldrich. Two types of chondrocytes were transfected with either 1µg CYTOR expression vector or 50 nM miRNA using lipofectamine 2000 (Invitrogen). NC cells (empty vector-or NC miRNA-transfected cells) and Control (C, untransfected cells) cells were included.
CYTOR luciferase vector was established with pGL3 luciferase vector (Promega) as backbone.

RNA isolation and process
Trizol reagent (Invitrogen) was used to isolate total RNA from both synovial uid samples and chondrocytes, followed by genomic DNA removal performed by incubating with DNase I (Invitrogen) for 2h. Electrophoresis performed using 5% urea-PAGE gels was used to check RNA integrity. OD 260/280 ratios were determined to re ect RNA purity and a ratio close to 2.0 indicates pure RNA.

RT-qPCRs
PrimeScript RT Reagent Kit (Takara Bio) was used for the preparation of cDNA samples with total RNA samples as template. QuantiTect SYBR Green PCR Kit (QIAGEN) was used to perform all qPCRs with 18S rRNA as an internal control to analyze the expression of CYTOR. Expression of miR-10a-5p was analyzed by The All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia) with U6 as an internal control. Each PCR was performed in three technical replicates and 2 −ΔΔCt method was used to normalize Ct values of target genes to corresponding internal controls.
Cell apoptosis assay Two types of chondrocytes were subjected to cell apoptosis assay at 48h post-transfection. In a 6-well cell culture plate, chondrocytes (20,000 cells in 2ml medium per well) were cultivated for further 48h. After that, cells were washed with ice-cold PBS and resuspended in binding buffer. Following Annexin-V FITC and propidium iodide (PI) staining, FACSCalibur instrument was used to analyze cell apoptosis.

Statistical analysis
Unpaired t test was used to compare the expression levels of CYTOR and miR-10a-5p between patients and controls. Heatmaps were plotted using Heml 1.0 software to present the changes in the expression levels of CYTOR and miR-10a-5p during treatment. Data of cell transfection experiments was expressed as mean±SD values of three biological replicates and ANOVA Tukey's test was used for data comparison. P<0.05 was statistically signi cant.

Declarations
Ethics approval and consent to participate This study was approved by Ethics Committee of A liated Hospital of Guizhou Medical University.

Not applicable
Availability of data and materials The datasets used analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.
Funding Figure 1 Expression of CYTOR and miR-10a-5p was altered in both OA patients and chondrocytes. Expression of CYTOR (A) and miR-10a-5p (B) in OA patients (synovial uid) and chondrocytes as well as healthy patients (synovial uid) and chondrocytes was determined by RT-qPCR. Expression data was expressed as average values of three technical replicates. **, p<0.01.

Figure 2
Treatment increased the expression of CYTOR and decreased the expression of miR-10a-5p in OA patients Expression of CYTOR (A) and miR-10a-5p (B) in synovial uid collected from OA patients before and 1 and 2 months after the initiation of treatment. Heatmaps were plotted using Heml 1.0 software to present the changes in the expression levels of CYTOR and miR-10a-5p during treatment. CYTOR and miR-10a-5p directly interacted with each other in two types of chondrocytes The potential interaction between CYTOR and miR-10a-5p was predicted by IntaRNA2.0 (A). To con rm the interaction between them, OA and control chondrocytes were co-transfected with CYTOR luciferase vector+NC miRNA (NC group) or CYTOR luciferase vector+miR-10a-5p mimic (miR-10a-5p group), luciferase activity was con rmed at 48h post-transfection. *, p<0.05.

Figure 4
Overexpression of CYTOR and miR-10a-5p failed to alter the expression of each other To further explore the interaction between CYTOR and miR-10a-5p, OA and control chondrocytes were transfected with either CYTOR expression vector or miR-10a-5p mimic, and the expression of them was detected by RT-qPCR every 24h until 96h (A). The effects of the overexpression of CYTOR on miR-10a-5p (B, and the effects of the overexpression of miR-10a-5p on CYTOR (C) at different time points were also analyzed by RT-qPCR. *, compared to NC group, p<0.05.