LncRNA CASC19 accelerates chondrocytes apoptosis and proinflammatory cytokine production to exacerbate osteoarthritis development through regulating the miR-152-3p/DDX6 axis

Background Osteoarthritis (OA) is one kind of degenerative joint disease that happens in articular cartilage and other joint tissues. Long non-coding RNAs (lncRNAs) have been reported to serve as pivotal regulators in many diseases, including OA. However, the role and relevant regulatory mechanisms of CASC19 in OA remain unknown. Methods The expression levels of CASC19, miR-152-3p, and DDX6 were identified by reverse-transcription polymerase chain reaction (RT-qPCR). Cell viability and apoptosis were determined by Cell Counting Kit-8 (CCK-8) and flow cytometry assays, respectively. The relationship between miR-152-3p and CASC19 or DDX6 was predicted by bioinformatics tools and verified by the dual-luciferase reporter assay. Results CASC19 was verified to exhibit higher expression in OA tissues and cells. Moreover, inhibition of CASC19 weakened proinflammatory cytokine (IL-6, IL-8, and TNF-α) production and cell apoptosis but facilitated cell viability. Experiments of the ceRNA mechanism elucidated that miR-152-3p was a sponge for CASC19, and miR-152-3p targeted DDX6, suggesting that CASC19 sponged miR-152-3p to release DDX6. Finally, results from rescue assays proved that the impacts of CASC19 silencing on chondrocytes apoptosis and proinflammatory cytokine production could be reversed by DDX6 overexpression. Conclusions It was concluded that lncRNA CASC19 accelerated chondrocytes apoptosis and proinflammatory cytokine production to exacerbate osteoarthritis development through regulating the miR-152-3p/DDX6 axis. These findings may offer an effective biological target for OA treatment.


Introduction
Osteoarthritis (OA), one complicated chronic arthropathy characterized by local inflammation and articular cartilage damage and degradation, is one primary cause of disability in the elderly [1,2]. At present, OA has become a great threat to public health worldwide, and almost 10% of the population and 50% of people aged above 65 suffer from OA [3,4]. Hence, it is urgent to extend the knowledge of OA pathology and develop more effective therapeutic strategies. Aberrant apoptosis, extracellular matrix, and inflammatory response of chondrocytes are related to cartilage degradation in OA [5,6]. Therefore, in-depth exploration of chondrocytedysfunction-related mechanisms may help to enhance understanding of OA pathogenesis.
In this study, we intended to investigate the functions and potential regulatory mechanism of CASC19 in OA. Findings in this study revealed that CASC19 accelerated chondrocyte apoptosis and proinflammatory cytokine production to exacerbate OA development through regulating miR-152-3p/DDX6 axis, suggesting that CASC19 might be a new target for OA treatment.

Patients and specimens
A total of 40 samples were respectively collected from 20 patients with OA and 20 trauma patients (undergoing lower-extremity amputation) without OA. Patients meeting the diagnostic criteria for OA were included in the study, while patients complicated with other diseases, such as history of joint surgery or rheumatoid arthritis were excluded. The general clinical characteristics of 20 OA patients and 20 trauma patients without OA (control group) were illustrated in Table 1. According to the data acquired, OA and control groups were homogeneous in age, gender, and BMI. For OA patients, total knee arthroplasty was performed and the cartilage of knee joints was taken. For trauma patients, the normal articular cartilage of knee joints was also obtained after lower-extremity amputation according to relevant studies [25,26]. Informed consent was acquired from all participators. This study was supported by the Human Ethics Committee of Changzhou Hospital of Traditional Chinese Medicine.

Reverse-transcription polymerase chain reaction (RT-qPCR)
RNAs were extracted from C28/I2 cells or OA tissues using Trizol reagent (Invitrogen, CA, USA). Synthesis of cDNA was performed with the PrimeScript® RT reagent Kit (Takara, Dalian, China). The PCR was conducted with SYBR Green PCR kit (TaKaRa, Dalian, China) using GAPDH/U6 as endogenous control. The data were processed through the 2 −ΔΔCt method.

Flow cytometry assay
Cell apoptosis was assessed through the Annexin V-FITC Apoptosis Detection Kit (Abcam, Cambridge, UK). Generally, after being rinsed twice with cold PBS solution, C28/I2 cells were resuspended. Afterwards, Annexin V-FITC and propidium iodide (PI) were mixed and incubated. Finally, the apoptosis rate was examined under the flow cytometer (BD Biosciences, San Jose, CA, USA).

RNA immunoprecipitation (RIP) assay
RIP assay was implemented via Magna RIP RNA-Binding Protein Immunoprecipitation Kit. Cell lysate, RIP buffer, and magnetic beads coupled to Ago2 antibody or normal IgG antibody (as negative control) were mixed. The immunoprecipitated RNA was analyzed through RT-qPCR to quantify gene expression.

Statistical analysis
Statistical analysis was performed via SPSS 20.0 (SPSS, Inc., Chicago, IL, USA). The data were shown as mean ± SD. Statistical differences were analyzed through Student's t-test or one-way analysis of variance. P < 0.05 was supposed as statistically significant. [14][15][16], but its role in OA keeps unknown. To probe whether CASC19 is altered in OA, RT-qPCR assay was conducted to test CASC19 expression in OA tissues, and the results indicated that CASC19 exhibited higher expression in OA samples (Fig. 1a). Similarly, CASC19 exhibited higher expression in OA cell model (Fig. 1b). To sum up, CASC19 exhibited higher expression in OA tissues and cell model.

Inhibition of CASC19 inhibited proinflammatory cytokine production and cell apoptosis
Next, the function of CASC19 in OA progression was explored. CASC19 expression was markedly reduced after suppressing CASC19 in C28/I2 cells mediated with IL-1β (Fig. 2a). The IL-6, IL-8, and TNF-α levels were Fig. 3 MiR-152-3p was a sponge for CASC19. a The miRNAs sponged with CASC19 were predicted through starBase website with the condition of CLIP Data: high stringency (≥ 3). The binding ability between CASC19 and miRNAs was tested by RNA pull-down assay. b The miR-152-3p expression was verified through RT-qPCR assay. c The overexpression efficiency of miR-152-3p was identified through RT-qPCR assay. d The binding ability between CASC19 and miR-152-3p was confirmed through luciferase reporter assay. e The binding ability between CASC19 and miR-152-3p was assessed through RIP assay. *P < 0.05, **P < 0.01, and ***P < 0.001 reduced through silencing CASC19 (Fig. 2b-d). In addition, the cell viability was increased by CASC19 knockdown (Fig. 2e). Through flow cytometry analysis, it was confirmed that CASC19 suppression retarded cell apoptosis (Fig. 2f). These findings suggested that inhibition of CASC19 inhibited proinflammatory cytokine production and cell apoptosis in OA.

CASC19 regulated proinflammatory cytokine production and cell apoptosis through DDX6
To explore whether CASC19 regulated proinflammatory cytokine production and cell apoptosis through DDX6, rescue assays were performed. The upregulated expression of DDX6 was verified after overexpressing DDX6 (Fig. 5a). The reduced IL-6, IL-8, and TNF-α levels Fig. 4 MiR-152-3p targeted DDX6. a The Venn diagram displayed these mRNAs which could combine with miR-152-3p. b The selected mRNAs expression was detected after overexpressing miR-152-3p. c The DDX6 expression was examined through RT-qPCR assay. d The binding capacity between miR-152-3p and DDX6 was tested through luciferase reporter assay. e The interaction between miR-152-3p and DDX6 was evaluated through RIP assay. **P < 0.01 and ***P < 0.001 caused by repressing CASC19 could be reversed by upregulating DDX6 (Fig. 5b-d). As displayed in Fig. 5e, overexpression of DDX6 could offset the increased cell viability induced by CASC19 inhibition. In addition, the inhibitive effects of CASC19 suppression on cell apoptosis could be rescued by DDX6 overexpression (Fig. 5f). These results suggested that CASC19 regulated proinflammatory cytokine production and cell apoptosis through DDX6.

Discussion
With the fast development of science and technology, an increasing number of lncRNAs have been identified as crucial regulators in various diseases [27,28]. CASC19 has been uncovered to be implicated in various cancers [14][15][16], but its function in OA remains indistinct. In this work, CASC19 exhibited higher expression in OA tissues and cell model. Moreover, inhibition of CASC19 weakened proinflammatory cytokine production and cell apoptosis.
It has been reported that DEAD-box protein 6 (DDX6) plays an important role in various diseases. For example, RNA helicase DDX6 upregulates c-Myc expression by serving as an oncogene in gastric cancer [36]. Additionally, DDX6 exhibited higher expression and modulated by miR-124 in colon cancer [37]. MiR-130 family modulates P-body protein DDX6 to modulate the hypoxia response signal [38]. In our study, results from rescue assays certified that DDX6 overexpression

Conclusions
This study was the first to discover the function and related ceRNA regulatory mechanism of CASC19 in OA. Our findings revealed that CASC19 accelerated chondrocytes apoptosis and proinflammatory cytokine production to exacerbate OA development through regulating miR-152-3p/DDX6 axis, indicating CASC19 may be a promising target for OA treatment and providing a novel direction of improving OA therapeutic methods for orthopedists. Although our study highlighted a CASC19-regulated regulatory mechanism in osteoarthritis progression, it is still limited because of the small sample size. In the future, this study will be further improved by expanding the sample size and adding more in vitro and in vivo experiments.