Hsa_circ_0032131 knockdown inhibits osteoarthritis progression via the miR-502-5p/PRDX3 axis

Osteoarthritis (OA) is a chronic disease characterized by progressive loss of cartilage and failure of the diarthrodial joint. Circular RNAs (circRNAs) are known to participate in the pathogenesis of multiple diseases, including OA. We investigated the functions of hsa_circ_0032131, a circRNA upregulated in OA, using CHON-001 cells and an in vivo OA rat model. CHON-001 cells were treated with interleukin (IL)-1β to mimic OA in vitro. IL-1β-induced inhibition of CHON-001 growth was reversed by silencing hsa_circ_0032131. In addition, hsa_circ_0032131 knockdown reversed IL-1β-induced activation of Trx1, Cyclin D and PRDX3, whereas overexpression of PRDX3, a direct target of miR-502-5p, reversed this effect. Hsa_circ_0032131 served as a competing endogenous RNA for miR-502-5p. Moreover, knockdown of hsa_circ_0032131 attenuated OA symptoms in vivo by inactivating the STAT3 signaling pathway. Thus, silencing of hsa_circ_0032131 inhibited the progression of OA by inactivating the miR-502-5p/PRDX3/Trx1/STAT3 axis, which highlights its potential as a therapeutic target for OA.


INTRODUCTION
Osteoarthritis (OA) results from progressive loss of joint cartilage that causes chronic pain and disability. It is known to affect approximately 50,000,000 adults in China [1][2][3]. Although the mainstay of OA treatment includes drugs to relieve the pain and surgery in advanced cases, the associated increase in annual hospitalization and ambulatory care visits results in a high socioeconomic burden [4]. Circular RNAs (circRNAs) are endogenous non-coding RNAs with a stable closed-loop structure that regulate several functions (protein synthesis, post-transcriptional modifications and others) [5,6]. In addition, circRNAs have been implicated in chondrocyte growth and inflammation in OA [7,8]. For example, Shen et al. indicated that overexpression of circSERPINE2 could alleviate the apoptosis in the OA cartilage tissues via sponging miR-1271 [9]. Zhou et al. found that circRNA.33186 could aggravate OA symptoms by sponging miR-127-5p [10]. In addition, hsa_circ_0032131 has been reported to be upregulated in OA [11]. Nevertheless, the role of hsa_circ_0032131 in OA remains largely unknown.
MiRNAs are small RNAs of approximately 20 to 25 nucleotides in length that regulate various biological processes by inhibiting the target mRNAs [12]. CircRNAs participate in development of OA in chondrocytes by directly binding to miRNAs to inhibit the translation of target mRNAs-a process known as sponging [9,13]. For example, circRNA CDR1 promoted OA progression by serving as a sponge for miR-641 [14]. Furthermore, miR-502-5p protected chondrocyte injury against IL-1β by inhibiting TRAF2, a critical signaling molecule of the NF-κB signaling [15]. This study investigated the function of hsa_circ_0032131 in OA pathogenesis and the correlation between hsa_circ_0032131 and miR-502-5p.

In vitro model of OA was successfully established
IL-1β plays a crucial role during the pathogenesis of OA [16]. Jiang et al. found that IL-1β significantly induced the apoptosis, inflammatory response and cartilage matrix destruction of chondrocytes [17]. Thus, to construct in vitro model of OA, CHON-001 cells were treated with IL-1β [18]. Western blotting was applied to detect the expression of protein biomarkers in OA [19,20]. As indicated in Figure 1A, 1C, IL-1β induced the level of MMP-1 and MMP-13, whereas it inactivated Aggrecan in cells ( Figure 1A, 1D). These data confirmed that the in vitro model of OA was successfully constructed.

Hsa_circ_0032131silencing rescued the antiproliferative effect of IL-1β
CHON-001 cells were transfected with shRNAs against hsa_circ_0032131. CHON-001 cells were more susceptible to hsa_circ_0032131 shRNA1 than hsa_circ_0032131 shRNA2 ( Figure 2A). Therefore, hsa_circ_0032131 shRNA1 was selected for subsequent experiments. The expression of hsa_circ_0032131 in CHON-001 cells was upregulated by IL-1β, whereas this effect was partially reversed after hsa_circ_0032131 knockdown ( Figure  2B). In addition, IL-1β decreased CHON-001 cell viability, whereas this effect was partially rescued by hsa_circ_0032131 shRNA1 ( Figure 2C). Ki67 cell proliferation assay further confirmed that hsa_circ_0032131 silencing restored IL-1β-induced suppression of cell proliferation ( Figure 2D, 2E). To sum up, the knockdown of hsa_circ_0032131 reversed the anti-proliferative effect of IL-1β on CHON-001 cells.

PRDX3 overexpression rescued the function of hsa_circ_0032131 shRNA1 in IL-1β-treated CHON-001 cells
The mechanism by which hsa_circ_0032131 participated in the progression of OA was explored. As indicated by the flow cytometry data shown in  Figure 6A, 6B, the anti-apoptotic effect of hsa_circ_ 0032131 downregulation on IL-1β-stimulated CHON-001 cells was rescued by PRDX3 overexpression. Moreover, IL-1β-induced G1 arrest of CHON-001 cells was rescued by hsa_circ_0032131 silencing, whereas this effect was partially reversed by PRDX3 overexpression ( Figure 6C, 6D). In summary, the overexpression of PRDX3 rescued the impacts of hsa_circ_0032131 shRNA1 on the growth of IL-1βstimulated cells.

Hsa_circ_0032131 knockdown attenuated OA symptoms in vivo
To further study the effects of hsa_circ_0032131 on OA, an in vivo model of OA was established in rats. As revealed in Figure 7A, cartilage destruction and articular chondrocyte cellularity loss in OA rats were reversed by hsa_circ_0032131 knockdown. The upregulated subchondral bone thickness was observed in OA rats, while hsa_circ_0032131 shRNA1 rescued this phenomenon ( Figure 7B). Additionally, hsa_circ_ 0032131 silencing reduced OARSI scores in OA rats ( Figure 7C, 7D). Moreover, the levels of TNF-α and IL-1β in the plasma of OA rats decreased following shRNA1-induced hsa_circ_0032131 silencing ( Figure 7E, 7F). Meanwhile, the level of hsa_circ_0032131 was notably increased in OA rats, whereas this effect was reversed following hsa_circ_0032131 knockdown ( Figure  7G). Altogether, the knockdown of hsa_circ_0032131 attenuated OA symptoms in vivo.

Hsa_circ_0032131 silencing alleviated OA progression in vivo by inhibiting the Trx1/STAT3 signaling
We next studied the expression of Trx1 and STAT3 in the tissues of rats by western blotting. The results AGING indicated that protein levels of PRDX3 and p-STAT3 were upregulated in OA rats, whereas this effect was rescued following hsa_circ_0032131 knockdown ( Figure 8A-8C). In contrast, Trx1 level in OA rats increased following hsa_circ_0032131 ( Figure 8A, 8D). In summary, the silencing of hsa_circ_0032131 alleviated the progression of OA in vivo by inhibiting Trx1/STAT3 signaling.

DISCUSSION
CircRNAs have been implicated in the progression of OA [7,21]. For instance, circRNA HIPK3 serves as a sponge of miR-124 and contributes to OA pathogenesis [22]. In addition, dysregulation of miRNAs is related with the occurrence of OA [23,24]. We found that hsa_circ_0032131 functioned as an endogenous AGING competing RNA for miR-502-5p in OA. Moreover, miR-502-5p directly targeted PRDX3, a downstream molecule of miR-502-5p, in CHON-001 cells, a finding contrary to that reported by Zhang et al. who found that miR-502-5p inhibited IL-1β-induced chondrocyte injury by targeting TRAF2 [15]. Nevertheless, because PRDX3 and TRAF2 are intricately associated with OA progression [25,26], AGING the difference in the results could be attributed to similar functions of these two proteins.
Oxidative stress plays a crucial role in OA development [27,28]. PRDX3, an antioxidative protein, can regulate cellular redox state [29]. Evidence has shown that antioxidant enzymes, such as dismutase 3 (SOD3), glutathione (GSH), were markedly decreased after OA development [30][31][32]. However, in this study, PRDX3 expression was significantly increased in IL-1β-treated CHON-001 cells as well as in OA rats, which was inconsistent with previous studies. The reason might be that PRDX3 expression in CHON-001 cells was compensatively elevated by IL-1β. These data indicated  AGING that the antioxidant capacity of CHON-001 cells was upregulated during IL-1β, and PRDX3 might play a protective role in IL-1β-treated CHON-001 cells.
Trx1 functions in the tumorigenesis of various cancers and inflammation [33,34]. We found that the knockdown of hsa_circ_0032131 markedly inactivated PRDX3 and activated Trx1 in IL-1β-induced CHON-001 cells. Jin et al. reported Trx1 as the downstream protein of PRDX3 and found that the expression of PRDX3 was upregulated and Trx-1 level was downregulated in CKI-treated AML cells [35]. In addition, Trx-1 has been shown to exhibit antiinflammatory and antiapoptotic effects [36]. These data indicated that knockdown of hsa_circ_0032131 attenuated the development of OA by inhibiting the miR-502-5p/PRDX3/Trx1 axis.
The STAT3 pathway participates in the regulation of inflammatory response and progression of OA [37,38]. Yao et al. indicated that IL-1β significantly upregulated the expression of p-STAT3 as well as induced the apoptosis of chondrocytes [39]. We found that silencing of hsa_circ_0032131 inactivated STAT3 signaling in IL-1β-treated CHON-001 cells. Furthermore, hsa_circ_ 0032131 shRNA inhibited the expression of Trx1. Lopez-Grueso et al. reported that Trx1 negatively regulated STAT3 signaling [40]. These data indicated that the knockdown of hsa_circ_0032131 alleviated OA symptoms via PRDX3/Trx1/STAT3 signaling.
The present study had certain limitations. First, it focused only on STAT3 signaling. Second, we failed to detect anti-apoptotic proteins. Because other pathways such as the PI3K/Akt signaling are also involved in the development of OA [41], future studies should focus on the effect of hsa_circ_0032131 on these pathways.
In summary, silencing of hsa_circ_0032131 inhibited the progression of OA by suppressing the miR-502-5p/PRDX3/Trx1/STAT3 axis. Thus, hsa_circ_0032131 could act as a new target for OA treatment.

Apoptosis analysis
CHON-001 cells were centrifuged and resuspended in binding buffer. After that, 5 μL Annexin V-fluorescein isothiocyanate (FITC) and PI were added to the cells at 4° C for 15 min. The cell apoptosis was analyzed by flow cytometry (Becton, Dickinson and Company, Franklin Lake, NJ, USA).

Prediction of downstream targets
The target gene of hsa_circ_0032131 was predicted using a publicly available program (StarBase, http:// starbase.sysu.edu.cn/). TargetScan and miRDB were used to predict the target of miR-502-5p.

Dual-luciferase reporter assay
The partial sequences of hsa_circ_0032131 and the PRDX3 3'-UTR containing the sites of miR-502-5p were synthesized by GenePharma. The aforementioned sequences were cloned into the pmirGLO vectors (Promega, Madison, WI, USA) for establishment of wild-type (WT) or mutant (WT) reporter hsa_circ_ 0032131 and PRDX3 vectors. The WT or MUT miR4435-2HG vector was transfected into cells along with miR-502-5p agomir using Lipofectamine 2000 reagent. The data were quantified and normalized to Renilla luciferase activity.

Fluorescence in situ hybridization
The co-localization of miR-502-5p and hsa_circ_ 0032131 in the cytoplasm was detected using FISH, as described previously [45].

In vivo model of OA
Fifteen Wistar rats (12-week-old) were obtained from the Chinese Academy of Sciences (Shanghai, China). Rats were injected with saline (control and OA group, 200 μL) or hsa_circ_0032131 shRNA1 (OA + hsa_circ_0032131 shRNA1; 2 × 10 7 plaque-forming units (PFUs), 200 μL, twice a week) via joint cavity. To induce OA in vivo, Rats in OA or OA + hsa_circ_0032131 shRNA1 group were treated with medial meniscus (DMM) surgery [46]. To mimic OA in vivo, rats were intraperitoneally injected with 40 mg/kg pentobarbital (2%), and then the joint capsule of the right knee was incised medially, as described previously [46]. Afterward, microsurgical scissors were used to transect the medial meniscotibial ligament [46]. Rats were sacrificed at the end of the study for collection of plasma and knee joint tissues. The protocols for animal care and use of laboratory animals were approved by Tianjin Hospital (No. 20200221).

Histopathological analysis
Safranin O and Fast Green was used to stain tissue specimens. The morphology of the subchondral bone and cartilage was observed under a microscope. The tibial plateau and medial femoral condyle were evaluated by OARSI scoring system [47]. In addition, hematoxylin and eosin (H&E) staining was performed to investigate the status of cartilage destruction.

Enzyme-linked immunosorbent assay
The levels of IL-1β and TNF-α in the plasma of rats were assessed using an ELISA kit (MultiSciences Lianke Biotech Co., Ltd, Hangzhou, China).

Statistical analysis
Data are presented as the mean ± SD. One-way analysis of variance and Tukey's post hoc tests were used for comparisons between ≥3 groups. Student's ttest was used for comparisons between tumor tissues and adjacent normal tissues of the same patients, while an unpaired Student's t-test was used for comparisons between unpaired groups. P<0.05 was considered to indicate a statistically significant difference.

Ethics approval statement
This study was approved by the Ethics Committee of Tianjin Hospital (No. 20200221).

AUTHOR CONTRIBUTIONS
Xinlong Ma conceived and supervised the study. Jin Xu designed and performed the experiments. Both authors collected the data, reviewed the results, and approved the final version of the manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.

FUNDING
Our experiments were funded by Science and Technology project of Tianjin Health Committee (No. #ZC20068).