LincRNA-Cox2 targeting miR-150 regulating the proliferation and apoptosis of chondrocytes in osteoarthritis

Background: Osteoarthritis (OA) is a joint disease characterized by progressive cartilage degradation and inflammation, but the detailed pathogenesis of OA is still unclear. Here, we aimed to investigate the role of LincRNA-Cox2 in OA progression and the potential mechanism. Methods: OA mouse model and IL-1β-induced injury of mouse chondrocytes were conducted. Si-Cox2 was transfected into chondrocytes for elucidating the effect of LincRNA-Cox2 on OA. qR-TPCR was used to detect the expression of LincRNA-Cox2 and miR-150. Cell proliferation and apoptosis were analyzed by MTT assay and Annexin V/PI stain respectively. Western blot was used to evaluate the protein levels in chondrocytes. Results: High levels of LincRNA-Cox2 were observed in both cartilage tissues of OA and IL-1β-treated chondrocytes. Knockdown of LincRNA-Cox2 promoted the proliferation and inhibited apoptosis of chondrocytes. Mechanically, LincRNA-Cox2 directly target to miR-150, acting as a ceRNA, and the effect of si-Cox2 on proliferation and apoptosis in chondrocytes was reversed by miR-150 inhibitor. Moreover, LincRNA-Cox2 had ability to activate wnt/β-catenin pathway to regulate chondrocytes proliferation and apoptosis. Conclusion: Silencing LincRNA-Cox2 plays a protective role in OA by enhancing the proliferation and suppressing apoptosis of chondrocytes, which was related with increase of miR-150 and activation of Wnt/β-catenin pathway.

3 evident in OA. It is reported that lncRNAs, transcripts in length longer than 200 nucleotides, are involved in OA progression by regulating cartilage degradation [18]. Also, studies have demonstrated the therapeutic potential of noncoding RNAs including long noncoding RNAs (lncRNAs)in the treatment of OA [26]. Long intergenic noncoding RNAs (lincRNAs), a subclass of lncRNAs, are emerged as a type of key regulators of mammalian gene expression. Several thousand lincRNAs have been identified in the mouse genome [4,15]. Moreover, lincRNAs are reported to be associated with human inflammatory diseases and tumorigenesis [22,5]. LincRNA-Cox2 is one of the better characterized lincRNAs and were reported to regulate transcription of distinct classes of immune genes in the inflammatory response, thus are regarded as an immune-inducible lincRNA [2]. In the previous study, Elling R et al found that LincRNA-Cox2 could regulate critical innate immune genes, dependently or independently of Ptgs2 [6]. Moreover, Tong et al demonstrate a novel mechanism of epigenetic modulation by LincRNA-Cox2 on Il12b transcription, suggesting an important role for lincRNAs in regulation of intestinal epithelial inflammatory responses [23]. However, the role of LincRNA-Cox2 in cartilage degradation and the development of OA remains to be unclear.
Chondrocytes are the important type of cells in cartilage and their growth, differentiation, and apoptosis are regulated to maintain a dynamic equilibrium. Therefore, their dysfunction is responsible for OA development [17]. The apoptosis and growth of chondrocytes were also found to be modulated by lncRNA, such as lncRNA XIST, PVT1 and PART-1 [19,14,13]. In this study, we investigated the effect of LincRNA-Cox2 on the proliferation and apoptosis of chondrocytes supporting an important role for LincRNA-Cox2 in the development of OA.

Isolation and culture of primary mouse chondrocytes
Primary murine chondrocytes were isolated from newborn mice as described previously [8]. Briefly, 5day-old C57BL/6 mice were sacrificed by intraperitoneal injection of pentobarbital and then isolated articular cartilage. The isolated articular cartilage was digested with 3 mg/mL collagenase D for 90 min at 37 ℃ under 5% CO 2 and 0.5 mg/mL collagenase D overnight at 37 ℃. After centrifugation at 400 g for 10, the supernatant was discarded and cells precipitation was resuspended in DMEM 4 supplemented with 10% fetal bovine serum, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. Then seed chondrocytes on a culture dish at density of 8×10 3 cells per cm. Change the culture medium after 2 d of culture, and the isolated chondrocytes reach confluence by 6-7 d. Only passage 1 to 3 were used for further experiments.

Induction of OA mouse model
10-week-old C57BL/6J male mice were purchased from Animal Center of the Chinese Academy of Sciences (Shanghai, China) and housed in plastic cages with free access to drinking water and a pellet based diet. Experimental OA model was induced by surgical destabilization of the medial meniscus (DMM) as described previously [12]. Briefly, under general anesthesia, the medial collateral ligament and the medial meniscus of the right knee were resected under a microscope. After surgery, the mice were randomly divided into the following groups: sham group, OA group. 8 weeks after surgery, mice were sacrificed for detection of LincRNA-Cox2 expression.
Finally, the OD490 nm value was measured to evaluate the proliferation capability of chondrocytes.

Apoptosis assay
Apoptosis of chondrocytes was determined using Annexin V FITC Apoptosis Detection Kit (BD Bioscience, NJ, USA). After IL-1β treatment and/or relevant transfection, cells were collected, washed with phosphate-buffered saline (PBS) and resuspended in 1 Binding Buffer at a concentration of 1×10 6 cells/mL. Then 5 µl of annexin V FITC and PI were added. After incubation for 15 min at room temperature in the dark, quantification of apoptotic cells was analyzed by flow cytometry (BD Bioscience, NJ, USA). Data was analyzed using FlowJo software (Tree Star, Ashland, OR).

Western blotting
After the indicated treatment, chondrocytes were collected and washed with PBS, then lysed on ice with RIPA lysis buffer supplemented with 10 mM of PMSF (Beyotime, Nanjing, China) for 15 min. Total protein was quantified using the BCA Protein Assay Kit (Solarbio, Beijing, China). Then, proteins in equal amounts were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, CA, USA). After blocking with 5% skimmed milk for 2 h at room temperature, membranes were incubated with specific primary antibodies against Ki67, PCNA, Bax, Capase-3, Capase-9, GSK-3β, p-GSK-3β (ser9), β-catenin, cynlin D1, c-Myc, MMP-7 and GAPDH (Santa Cruz, CA, USA) overnight at 4 °C. Then membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature, and protein bands were visualized using electrochemiluminescence (ECL) plus (GE Healthcare; Buckinghamshire, England, UK) according to the manufacturer's instructions. Densitometry analysis of bands was performed using Image J software (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as an endogenous protein for normalization

Cell transfection
The miR-150 mimic, miR-150 inhibitor and their negative control (Scramble and anti-NC) were synthesized by GenePharma Co (Shanghai, China). The full-length wide-type LincRNA-Cox2 sequences 6 was constructed into the pEX-2 plasmid (GenePharma, Shanghai, China). An empty pEX-2 plasmid was transfected as a negative control. siRNA specific for LincRNA-Cox2 was constructed into U6/Neo plasmid (GenePharma, Shanghai, China). An empty U6/Neo plasmid with non-targeting sequences was transfected as a negative control (NC). Cell transfections were conducted using Lipofectamine 3000 reagent (Invitrogen, CA, USA) depending on the manufacturer's descriptions. After 48 h, the transfection efficiency was detected by qRT-PCR.

Dual luciferase activity assay
The 3'UTR target site was generated by PCR and the luciferase reporter constructs with the LincRNA-Cox2 sequences carrying a putative miR-150-binding site into pMiR-report vector were amplified by PCR. Cells were co-transfected with the reporter construct, control vector and miR-150 or scramble using Lipofectamine 3000 (Life Technologies, USA). Reporter assays were done using the dualluciferase assay system (Promega) following to the manufacturer's information.

Statistical analyses
All results were observed from at least three independent experiments. Statistical analysis was carried out using SPSS 19.0 and data were presented as the mean ± SD as indicated. Statistical differences between two groups were determined by two-tailed Student's t-test. Differences among more than two groups in the above assays were estimated by one-way ANOVA. The linear relationship among levels of LincRNA-Cox2and miR-150 in OA mice was analyzed by Spearman's correlation coefficient. P< 0.05 was considered statistically significant.

LincRNA-Cox2 expression is up-regulated in cartilage tissues of OA and IL-1βinducedchondrocytes
To detect the expression of LincRNA-Cox2 in cartilage tissues of OA, qRT-PCR was performed in cartilage specimens from 10 OA mice and 10 normal mice. The results demonstrated that the expression of LincRNA-Cox2 was markedly higher in OA cartilage tissues than that in normal tissues (P< 0.05, Fig. 1A). Moreover, it was also observed that the expression of LincRNA-Cox2 was significantly up-regulated in chondrocytes stimulated by IL-1β at 10 and 20 ng/mL(P< 0.05, Fig. 1B).

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To explore whether LincRNA-Cox2 exerted its function by miR-150 in chondrocytes, the rescue experiments were performed. As shown in Fig. 4A and B, knockdown of LincRNA-Cox2 enhanced the proliferation of IL-1β-treated chondrocytes and the expression of Ki67 and PCNA, while suppression of miR-150 blocked the effect effectively (P<0.05). Moreover, the anti-apoptosis effect of LincRNA-Cox2 knockdown was also evidently reversed by miR-150 inhibitor (P< 0.05, Fig. 4C and D).

Discussion
OA is a chronic, progressive, and degenerative disease, affecting multiple joint tissues, and results in great suffering including pain, stiffness, movement difficulty, and even progressive disabilities. The main characteristics of OA are degeneration of articular cartilage and chronic inflammatory response.
However, despite the diverse etiology and pathogenesis of OA, the detailed pathogenesis has not yet been elucidated. Recent focus of the epigenetic regulating mechanisms of OA have revealed that numerous lncRNAs were served important functions in the development of inflammation related diseases including OA, such as lncRNA XIST and PVT1 [19,14]. LincRNA-Cox2, a class of lncRNA localized to both thecytosolic and nuclear compartments, has ability to affect the expression of hundreds of inflammatory genes and regulate inflammatory response [2]. Besides, LincRNA-Cox2 has capacity to mediate neuroinflammation by regulating NLRP3 inflammasome and autophagy [24].
However, whether LincRNA-Cox2 involved in the pathogenesis of OA remains unclear. In this study, we found that the expression of LincRNA-Cox2 was markedly up-regulated both in vivo and in vitro OA model, indicating that LincRNA-Cox2 may play a role in OA development.
Chondrocytes are the only cells found in the cartilage and their dynamic equilibrium between growth, differentiation, and apoptosis was crucial to maintain the appropriate cycles of the biosynthesis and 9 degradation of the cartilaginous matrix [17]. Here, we investigated the role of LincRNA-Cox2 in viability of chondrocytes using IL-1β-induced chondrocytes, and the results demonstrated that LincRNA-Cox2 inhibited the viability of chondrocytes. In addition, the protein levels of Ki67 and PCNA, two main proliferation related proteins, were also suppressed by LincRNA-Cox2. Apoptosis is an important processes associated cell viability, and activation of Bax, c-Caspase 3 and c-Caspase 9 are responsible for monitor of cell apoptosis [25,1]. In this study, we found that both the apoptosis cells and the expression of Bax, c-Caspase 3 and c-Caspase 9 are reduced after knockdown of LincRNA-Cox2, suggesting an important pro-apoptotic role of LincRNA-Cox2 in OA chondrocytes.
Recently, competing endogenous RNA (ceRNA) hypothesis attains more and more attentions as an alternative function for lncRNAs [21]. As an novel regulatory mechanism, the crosstalk between LncRNAs and miRNAs has been identified in various diseases including OA [10]. Zhang et al found that LncRNA MALAT1 promotes osteoarthritis by competing with miR-150-5p [27]. We hypothesized that LincRNA-Cox2 may act as an ceRNA to sponge miRNAs through which promoted the OA development.
Our current study predicted a direct binding site between LincRNA-Cox2 and miR-150, and confirmed this prediction by luciferase activity assay. Furthermore, we found that the expression of miR-150 was decreased after silence of LincRNA-Cox2, while LincRNA-Cox2 can also negatively regulate miR-150 expression. Also, an inverse correlation between LincRNA-Cox2 and miR-150 was observed in OA cartilage tissues. These findings illustrated that LincRNA-Cox2 exerted its functions on the proliferation and apoptosis of chondrocyte by sponging miR-150. However, such effect of LincRNA-Cox2 was reversed by miR-150 inhibitor. To the best of our knowledge, we firstly revealed LincRNA-Cox2/miR-150 axis mediated OA progression.
Previous study indicated that Wnt/β-catenin pathway play a pivotal role in regulation of inflammation processes in various mammalian non-neuronal cells [20], and was also involved in OA development [9]. Moreover, silencing lncRNA HOTAIR regulated synoviocyte proliferation and apoptosis in osteoarthritis through inhibiting Wnt/β-catenin signaling pathway [16]. In this study, we found that knockdown of LincRNA-Cox2 inhibited β-catenin nuclear accumulation as well as other related proteins expression, while these proteins levels were return to increase by miR-150 inhibitor. Our data demonstrated LincRNA-Cox2 triggered the activity of wnt/β-catenin pathway by sponging miR-150 in chondrocytes, suggesting a new sight in OA progression.

Conclusion
In conclusion, LincRNA-Cox2 was up-regulated in OA cartilage tissues and IL-1β-induced chondrocytes.
The overexpressed LincRNA-Cox2 reduced the viability, enhanced apoptosis and aggravated chondrocytes injury, suggesting it could act as a useful marker and potential therapeutic target in OA.