CircMAPK9 promotes the progression of fibroblast-like synoviocytes in rheumatoid arthritis via the miR-140-3p/PPM1A axis

Background Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, and fibroblast-like synoviocytes (FLSs) are key effector cells in RA development. Mounting evidence indicates that circular RNAs (circRNAs) participate in the occurrence and development of RA. However, the precise mechanism of circRNA mitogen-activated protein kinase (circMAPK9) in the cell processes of FLSs has not been reported. Methods The expression levels of circMAPK9, microRNA-140-3p (miR-140-3p), and protein phosphatase magnesium-dependent 1A (PPM1A) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assay. Cell proliferation was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis and cycle distribution were assessed by flow cytometry. Cell migration and invasion were tested by transwell assay. All the proteins were inspected by western blot assay. Inflammatory response was evaluated by enzyme-linked immunosorbent assay (ELISA). The interaction between miR-140-3p and circMAPK9 or PPM1A was verified by dual-luciferase reporter assay. Results CircMAPK9 and PPM1A were upregulated and miR-140-3p was downregulated in RA patients and FLSs from RA patients (RA-FLSs). CircMAPK9 silence suppressed cell proliferation, migration, invasion, inflammatory response, and promoted apoptosis in RA-FLSs. MiR-140-3p was a target of circMAPK9, and miR-140-3p downregulation attenuated the effects of circMAPK9 knockdown on cell progression and inflammatory response in RA-FLSs. PPM1A was targeted by miR-140-3p, and circMAPK9 could regulate PPM1A expression by sponging miR-140-3p. Furthermore, miR-140-3p could impede cell biological behaviors in RA-FLSs via targeting PPM1A. Conclusion CircMAPK9 knockdown might inhibit cell proliferation, migration, invasion, inflammatory response, and facilitate apoptosis in RA-FLSs via regulating miR-140-3p/PPM1A axis, offering a new mechanism for the comprehension of RA development and a new insight into the potential application of circMAPK9 in RA treatment.


Introduction
Rheumatoid arthritis (RA) is a common chronic autoimmune disorder that mainly influences the synovial joints [1,2]. Fibroblast-like synoviocytes (FLSs), one class of dominating cells in synovial tissues, are reported to serve a vital role in the pathogenesis of RA [3,4]. Although several treatment options are available for the management of RA patients, there is no cure for RA [5]. Thus, it is essential to explore the mechanism of FLSs progression in order to find new targets for RA treatment.
Circular RNAs (circRNAs) are a special class of noncoding RNAs possessing continuous covalently closed loops that are produced via back-splicing of precursor mRNAs [6,7]. Increasing evidence supported that cir-cRNAs are concerned with the onset and development of multiple human diseases [8]. Meanwhile, numerous circRNAs played vital part in the progression of autoimmune diseases, including RA [9]. CircRNA mitogenactivated protein kinase (circMAPK9), also known as hsa_circ_0001566, is derived from back-splicing of MAPK9 transcript and has been reported to be highly expressed in peripheral blood mononuclear cells (PBMCs) from RA patients [10], whereas the exact role and regulatory mechanism of circMAPK9 in FLSs progression is indistinct.
MicroRNAs (miRNAs) are defined as small noncoding molecules that can regulate gene expression through combining with the 3′untranslated regions (3′UTRs) of target mRNAs [11]. Generally, circRNAs are known to work as miRNA molecular sponges to inhibit miRNA activity by competitively binding to miRNAs [12]. Plentiful miRNAs have been found to be dysregulated and may serve as biomarkers or therapeutic targets in RA [13][14][15]. Furthermore, miR-140-3p abundance was declined in synovial tissue and FLSs from RA patients (RA-FLSs) and from mice in arthritis models, and miR-140-3p overexpression in FLSs inhibited cell proliferation and migration [16]. Moreover, Lee et al. reported that protein phosphatase magnesium-dependent 1A (PPM1A) was involved in the development of RA [17]. Nevertheless, the relationships among circMAPK9, miR-140-3p, and PPM1A in the pathogenesis of RA are undiscovered.
In this research, circMAPK9 abundance was measured in RA patients and RA-FLSs. Then, we explored the effects of circMAPK9 on cell growth, transferability, and inflammation in RA-FLSs. Besides, we uncovered the regulatory network of circMAPK9/miR-140-3p/PPM1A in RA-FLSs.

Materials and methods
Patient tissue collection RA synovial tissues were collected from RA patients (n = 22) who underwent knee replacement surgery. Normal synovial tissues were obtained from patients with traumatic knee and no history of autoimmune diseases (n = 22). All subjects were recruited from Ganzhou People's Hospital, The Affiliated Ganzhou Hospital of Nanchang University, and they all signed the written informed consent. After surgical resection, these tissues were preserved at -80°C until usage. This research was permitted by the Ethical Committee of Ganzhou People's Hospital, The Affiliated Ganzhou Hospital of Nanchang University. The clinical characteristics of RA and trauma patients are listed in Table 1.

Cell culture
Fibroblast-like synoviocytes from RA patients (RA-FLSs) or healthy subjects (H-FLSs) were separated as formerly mentioned [18]. Briefly, synovial tissue samples were cut into small debris and digested using 2 mg/mL of collagenase (type II, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 2 h to isolate synoviocytes. FLSs were cultured in HFLS growth medium (Cell Applications, San Diego, CA, USA) plus 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA) in a 37°C incubator containing 5% CO 2 . FLSs were separated from all healthy donors and RA patients for detecting the circMAPK9 expression. Two sets of RA-FLSs were selected for functional assays. Set 1 of RA-FLSs was acquired from three random-selected RA patients and set 2 of RA-FLS was acquired from another three random-selected RA patients. Then, the same number of RA-FLSs was mixed from these three RA patients. The RA-FLSs were cultured, and set 1 of RA-FLSs was utilized for mechanistic investigation. In these experiments, cells at passage 3 were used.

RNase R treatment
For detecting the stability of circMAPK9, total RNA (2 μg) was reacted with RNase R (3 U/μg, Geneseed, Guangzhou, China) at 37°C for 0.5 h to digest linear RNA. After that, the treated RNA was used for qRT-PCR to survey the RNA abundance of circMAPK9 and linear MAPK9.

Flow cytometry
Flow cytometry analysis was executed for the detection of cell apoptosis and cycle distribution. For cell apoptosis detection, Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) was utilized. Briefly, following transfection for 48 h, 1 × 10 5 RA-FLSs were collected and resuspended in Annexin binding buffer. Then, cells were stained with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) in a dark place at 4°C for 15 min. Subsequently, the apoptotic cells were analyzed by a flow cytometer (BD Biosciences). For cell cycle distribution detection, RA-FLSs (1 × 10 5 ) after 48-h transfection were collected and re-suspended in PBS, then added with PI (BD Biosciences) staining solution in dark place at 37°C for 10 min after 70% ethanol fixation. The distribution of different cell cycle phases (G0/G1, S and G2/M) was assessed utilizing a flow cytometer (BD Biosciences).

Transwell assay
Transwell chambers (Costar, Corning, NY, USA) were used to detect cell migratory and invasive abilities. 5 × 10 4 RA-FLSs or 1 × 10 4 RA-FLSs after 48-h transfection was re-suspend in 200 μL of serum free HFLS medium and then added into the upper chamber coated with or without Matrigel (BD Biosciences) to identify cell invasion and migration, respectively. The lower chamber was added with 0.6 mL complete HFLS medium with 10% FBS. The non-migrating or non-invading cells were removed after incubation for 24 h, and migratory and invasive cells on the bottom membrane were fastened by paraformaldehyde (4%, Beyotime) and dyed by crystal violet solution (0.1%, Beyotime) for 30 min. The migratory or invasive cells were observed and counted using a microscope (Olympus, Tokyo, Japan) at × 100 magnification.

Statistical analysis
Statistical analysis was executed using GraphPad Prism 6 (GraphPad Inc., La Jolla, CA, USA). All data from at least 3 independent biological replications were displayed as mean ± standard deviation (SD). Difference was analyzed using Student's t-test (between 2 groups) or one-way analysis of variance followed by Tukey test (among multiple groups) in specific circumstances. Statistical significance was considered when P-value < 0.05.

Results
CircMAPK9 was upregulated in RA patients and RA-FLSs CircMAPK9 (hsa_circ_0001566) was located on chr5: 179688683-179707608 of chromosome and derived from exon 16-21 of MAPK9 genome (Fig. 1A). To explore the potential roles of circMAPK9 in RA, its expression pattern was detected by qRT-PCR in synovial tissues from RA patients (n = 22) and normal patients (n = 22). The results showed that circMAPK9 abundance was greatly increased in RA patients compared to normal patients (Fig. 1B). Then, the expression of circMAPK9 in RA-FLSs or normal subjects (H-FLSs) was measured. The results demonstrated that circMAPK9 level was higher in RA-FLSs more than triple than control group (Fig.  1C). Furthermore, the stability of circRNA was evaluated by RNase R digestion assay. As displayed in Fig. 1D, linear mRNA (MAPK9) was obviously decreased after digestion by RNase R while circMAPK9 expression was not affected, indicating the cyclic structure of cir-cMAPK9. These data indicated that increased expression of circMAPK9 might be associated with RA progression.
Knockdown of circMAPK9 inhibited cell proliferation, migration, invasion, inflammation, and promoted apoptosis in RA-FLSs To study the effect of circMAPK9 on RA progression, loss-of-function experiments were performed in RA-FLSs transfected with siRNAs to knock down cir-cMAPK9. As displayed in Fig. 2A, relative to si-NC group, the expression of circMAPK9 was signally declined in RA-FLSs transfected with si-circMAPK9#1, si-circMAPK9#2, or si-circMAPK9#3, especially in si-circMAPK9#2 group. Therefore, si-circMAPK9#2 was chosen for further study. Next, the impacts of cir-cMAPK9 on cell proliferation, apoptosis, cycle distribution, and invasiveness were investigated. MTT assay indicated that knockdown of circMAPK9 restrained cell proliferation in RA-FLSs (Fig. 2B). Flow cytometry assay showed that circMAPK9 silence evidently promoted cell apoptosis and induced cell cycle arrest at G0/G1 phase in RA-FLSs (Fig. 2C, D). Moreover, circMAPK9 deficiency markedly restrained the migratory and invasive abilities of RA-FLSs using transwell analysis (Fig. 2E, F). Besides, western blot assay exhibited that circMAPK9 interference significantly increased the level of proapoptotic protein Bax, and decreased the expression of anti-apoptotic protein Bcl-2, migration and invasionrelated proteins (MMP2 and MMP9), further supporting the effects of circMAPK9 silence on cell apoptosis and invasiveness (Fig. 2G). Additionally, the inflammatory response was analyzed in RA-FLSs via ELISA, which presented that circMAPK9 knockdown visibly reduced the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in RA-FLSs (Fig. 2H-J). These results indicated that circMAPK9 downregulation could suppress cell proliferation, migration, invasion, inflammatory response, and accelerate apoptosis of RA-FLSs.

CircMAPK9 acted as a sponge of miR-140-3p
To analyze the potential mechanism of circMAPK9, the potential target miRNAs of circMAPK9 were predicted using CircInteractome (https://circinteractome.nia.nih. gov/mirna_target_sites.html). The results showed that miR-140-3p had putative binding sequence for cir-cMAPK9 (Fig. 3A). To validate the relationship between circMAPK9 and miR-140-3p, dual-luciferase reporter assay was performed through constructing circMAPK9 WT and circMAPK9 MUT. The results displayed that miR-140-3p overexpression remarkably decreased the luciferase activity of circMAPK9 WT but not that of circMAPK9 MUT when the binding sites were mutated (Fig. 3B). Furthermore, the level of miR-140-3p in RA patients and RA-FLSs was observed. Results showed that the abundance of miR-140-3p was evidently decreased in synovial tissues from RA patients and RA-FLSs (Fig. 3C,  D). In addition, the expression of miR-140-3p was promoted after knockdown of circMAPK9 (Fig. 3E). The above evidence verified that miR-140-3p was a direct target of circMAPK9.

Discussion
RA is a common form of inflammatory multisystem disease with undiscovered etiology [2]. CircMAPK9 was highly expressed in PBMCs from RA patients and might act as a possible diagnostic biomarker for RA [10]. FLSs play a pivotal role in RA etiology by regulating inflammatory response and cartilage destruction [19,20]. Thereby, investigating the tumor-like biologic behaviors of RA-FLSs is indispensable to develop novel therapies for RA patients. Meanwhile, noncoding RNAs (ncRNAs) including circRNAs might serve as promising biomarkers for RA [21]. In this report, we aimed to study the biological role of circMAPK9 and explore the underlying mechanism in the advancement of RA-FLSs. Through the verification of functional experiment, we first clarified that circMAPK9 knockdown repressed were tested by qRT-PCR and western blot assays, respectively. J, K PPM1A mRNA and protein levels were detected in RA-FLSs with transfection of si-NC, si-circMAPK9#2, si-circMAPK9#2 + anti-miR-NC, or si-circMAPK9#2 + anti-miR-140-3p by qRT-PCR and western blot assays, respectively. *P < 0.05 proliferation, invasiveness, and inflammation of RA-FLSs via circMAPK9/miR-140-3p/PPM1A regulatory network.
The deregulation of circRNAs is identified to be closely related to the occurrence and development of autoimmune diseases including RA [22]. For instance, circ_0088036 promoted the proliferative and migratory capacities of FLSs via the circ0088036/miR-140-3p/ SIRT1 axis in RA [23]. In keeping with previously report [10], we also verified that circMAPK9 level was enhanced in synovial tissues from RA patients and RA-FLSs. Thus, we speculated that the disordered level of circMAPK9 might be connected with RA evolution. Through implementing loss-of-function experiment in RA-FLSs, it was evidenced that circMAPK9 silence could repress cell proliferation, migration, invasion, and accelerated apoptosis of RA-FLSs. Many cytokines are associated with RA progression, including TNF-α, IL-1, IL-6, and IL-17 [24,25]. Furthermore, this report also attested that circMAPK9 knockdown lessened the inflammatory response of RA-FLSs by decreasing the release of TNF- Fig. 6 The influence of miR-140-3p and PPM1A on cell malignant progression and inflammatory response in RA-FLSs. RA-FLSs were transfected with miR-NC, miR-140-3p, miR-140-3p + vector, or miR-140-3p + PPM1A. A Western blot assay was performed to detect the protein expression of PPM1A. Cell proliferation (B), apoptosis (C) and cycle distribution (D), migration (E), and invasion (F) were measured by MTT assay, flow cytometry analysis, or transwell assay, respectively. G Western blot was carried out to examine the protein expression of Bax, Bcl-2, MMP2, and MMP9. H-J The levels of TNF-α, IL-1β, and IL-6 were analyzed by ELISA. *P < 0.05 α, IL-1β, and IL-6. Hence, we deemed that circMAPK9 might contribute to RA malignant development by facilitating cell proliferation, migration, invasion, inflammatory response, and hindering cell apoptosis of RA-FLSs.
Accumulating reports have revealed that circRNAs could modulate the progression of multifarious diseases via acting as miRNA sponges [26]. As well, miRNAs have been certified to serve pivotal part in FLSs of RA [27]. For instance, miR-20a was involved in the modulation of pro-inflammatory cytokines release by controlling ASK1 expression in RA-FLSs [28]. To validate whether miRNAs were implicated in circMAPK9-mediated RA evolution, CircInteractome database was applied to forecast the possible miRNAs of circMAPK9. The prediction result indicated that miR-140-3p was targeted by cir-cMAPK9 in RA-FLSs, and the dual-luciferase reporter assay verified the interacting effect between them furtherly. Previous studies have demonstrated the suppressive role of miR-140-3p in the progress of bladder cancer [29], colorectal cancer [30], and so on. Yin et al. pointed out that the decline of miR-140-3p was correlated with increased osteoarthritis severity [31]. Moreover, Zhong et al. illuminated the participation of miR-140-3p in the proliferative and migratory processes of RA-FLSs via SIRT1 in RA [23]. In this research, low expression of miR-140-3p was observed in synovial tissues from RA patients and RA-FLSs, which was in agreement with previous work [16]. Simultaneously, miR-140-3p could restrain cell propagation, transferability, and inflammatory response of RA-FLSs. Besides, miR-140-3p silence restored the influence of circMAPK9 deficiency on cell progression and inflammation in RA-FLSs. Therefore, these findings confirmed that circMAPK9 could modulate the aggressive phenotype of RA-FLSs by sponging miR-140-3p.
The circRNA/miRNA/mRNA network has been identified in diversiform diseases, such as hepatocellular carcinoma [32], gastric cancer [33], and systemic lupus erythematosus [34]. To explore the downstream mRNAs of circMAPK9/miR-140-3p network in RA, the possible targets of miR-140-3p were sought. Through identification, PPM1A was sponged by miR-140-3p. Philippe et al. attested that miR-19a/b could act as negative regulators in RA-FLSs by controlling TLR2 expression [35]. Nevertheless, whether miR-140-3p could regulate PPM1A level to affect RA progression is still ill-defined. In different cancers, PPM1A has been evinced to serve as a tumor suppresser or promoter [36,37]. However, the precise function of PPM1A in RA progression has not been expounded. Lee et al. has disclosed that PPM1A was highly expressed in RA, and PPM1A expression was positively correlated with pro-inflammatory cytokine TNF level in RA synovial fluid [17]. In this study, the data showed that PPM1A enrichment was elevated in synovial tissue from RA patients and RA-FLSs, manifesting that PPM1A might be involved in RA progression. Interestingly, the rescue experiments indicated that PPM1A overexpression could abolish the impacts of miR-140-3p introduction on cell progression and inflammation in RA-FLSs, hinting that miR-140-3p could regulate the malignant development of RA via targeting PPM1A, which was parallel with the previous report [35]. Moreover, circMAPK9 was attested to positively regulate PPM1A expression by the crosstalk of miR-140-3p. Collectively, these data indicated that circMAPK9 might promote RA progression by regulating miR-140-3p/PPM1A axis.
This research conducted the in vitro experiments using the primary RA-FLSs, which represented the physiological function of RA patients. Furthermore, the involvement of circMAPK9/miR-140-3p/PPM1A network in RA-FLSs dysfunction was firstly confirmed, implying the significance and clinical expectation of this axis in RA advancement and therapy. Nevertheless, some limitations were still subsistent in the current study. For example, a larger number of RA patients and animal studies are needed in a further study in consideration of the limited number of patients and the restriction of in vitro experiments in this study. Besides, nanotechnology plays significant role in the area of bone-related therapy through providing attractive carrier options for delivery of therapeutic agents [38,39]. The progressive damage of articular bone and cartilage was developed in RA patients, which might cause disability over time [40]. Recent evidence has suggested that bioengineered composite scaffolds and magnetic nanoparticles are effective promising therapeutic tools for RA remedy [41,42]. Therefore, the combination of nanotechnology and molecular targeted drugs might be a most effective method for RA treatment.

Conclusion
In conclusion, circMAPK9 interference constrained RA-FLSs proliferation, migration, invasion, inflammatory response, and expedited apoptosis possibly by enhancing miR-140-3p and lessening PPM1A expression. Our study first elucidated the circMAPK9/miR-140-3p/PPM1A regulatory network in RA-FLSs, offering a new perception about RA-FLSs progression, and offering a novel possible target for RA therapy.