Mesenchymal stem cell-originated exosomal lncRNA HAND2-AS1 impairs rheumatoid arthritis fibroblast-like synoviocyte activation through miR-143-3p/TNFAIP3/NF-κB pathway

Background Long non-coding RNA heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1) was found to be elevated in rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLSs). However, whether HAND2-AS1 functions as an exosomal lncRNA related to mesenchymal stem cells (MSCs) in RA progression is unknown. Methods The expression of HAND2-AS1, microRNA (miR)-143-3p, and tumor necrosis factor alpha-inducible protein 3 (TNFAIP3) was detected using quantitative real-time polymerase chain reaction and Western blot. Cell proliferation, apoptosis, migration, and invasion were detected using cell counting kit-8, flow cytometry, and wound healing and transwell assays. The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL)-6 were analyzed using enzyme-linked immunosorbent assay. The level of phosphorylated-p65 was examined by Western blot. The binding interaction between miR-143-3p and HAND2-AS1 or TNFAIP3 was confirmed by the dual-luciferase reporter and RIP assays. Exosomes were isolated by ultracentrifugation and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Results HAND2-AS1 was lowly expressed in RA synovial tissues, and HAND2-AS1 re-expression suppressed the proliferation, motility, and inflammation and triggered the apoptosis in RA-FLSs via the inactivation of NF-κB pathway. Mechanistically, HAND2-AS1 directly sponged miR-143-3p and positively regulated TNFAIP3 expression, the target of miR-143-3p. Moreover, the effects of HAND2-AS1 on RA-FLSs were partially attenuated by miR-143-3p upregulation or TNFAIP3 knockdown. HAND2-AS1 could be packaged into hMSC-derived exosomes and absorbed by RA-FLSs, and human MSC-derived exosomal HAND2-AS1 also repressed above malignant biological behavior of RA-FLSs. Conclusion MSC-derived exosomes participated in the intercellular transfer of HAND2-AS1 and suppressed the activation of RA-FLSs via miR-143-3p/TNFAIP3/NF-κB pathway, which provided a novel insight into the pathogenesis and treatment of RA.


Introduction
Rheumatoid arthritis (RA) is a common systemic and chronic autoimmune disease, highlighted by hyperplasia, hypertrophy, and functional disability of joint structure, leading to incredibly high mortality and morbidity [1,2]. Growing evidence has indicated that RA fibroblast-like synoviocytes (RA-FLSs) play key roles in the regulation of inflammatory response and joint destruction [3]. Targeting the activation of FLSs has been explored as a therapeutic strategy for RA treatment [4].
Long non-coding RNAs (lncRNAs) are RNA transcripts longer than ∼ 200 nucleotides in length, which can modulate gene expression by a wide diversity of mechanisms [5]. LncRNAs have been corroborated to have significant roles in complex human pathologies through regulating cell biological processes, such as proliferation, apoptosis, metastasis, metabolism, and inflammation [6][7][8], and the dysregulation of lncRNAs is associated with the physiological status and pathological progression of many diseases, including RA [9,10]. LncRNA heart and neural crest derivatives expressed 2antisense RNA 1 (HAND2-AS1) is a well-recognized tumor suppressor in different types of cancer [11][12][13]. Previous study found that the level of HAND2-AS1 was low in RA-FLSs, suggesting that abnormal HAND2-AS1 expression might be involved in synovial aggression and joint destruction in RA [14].
Recently, the use of exosomes as biological vehicles for lncRNAs transfer has attracted great research interest.
Exosomes are small (30-150 nm) membranous spherical vesicles, which are actively released by almost all living cells [15]. They actually are natural information carriers and act as mediators in intercellular communications via delivering biofunctional cargoes, like proteins, lipids, DNA, and lncRNA, between cells, thereby impacting the behavior of recipient cells [16][17][18]. Mesenchymal stem cells (MSCs) are multipotent stem cells, which have been reported may be applied for the treatment of RA [19,20]. MSCs produce abundant amounts of exosomes [20], and MSC-derived exosomes are more immunosuppressive in inflammatory arthritis [21]. Besides, emerging evidence has revealed that exosomes originated from MSCs play substantial roles in bone remodeling processes [22,23]. However, whether HAND2-AS1 functions as an exosomal lncRNA related to MSCs in RA progression is unclear.
Herein, the objective of this study was to explore the effects and mechanism of HAND2-AS1 on the development of RA, evaluating the potential therapeutic ability of MSCs and related exosomes on RA treatment.

Clinical samples
The synovial specimens of RA (RA) were collected from 28 RA patients who underwent knee joint replacement surgery or knee synovial debridement at the Affiliated Hospital of Weifang Medical University. All patients were diagnosed as RA according to the American College of Rheumatology classification. The normal synovial specimens were collected to serve as controls from 19 patients with severe joint trauma, and all enrolled subjects had no other joint abnormalities or systemic diseases. The samples were removed from discarded tissues and instantly kept in − 80°C until used. The protocol of this study was permitted by the Ethics Committee of Affiliated Hospital of Weifang Medical University. Written informed consent was obtained from each patient.

Cell culture
Human synovial cell line MH7A was purchased from Beijing Institute for Cancer Research Collection (Beijing, China) and cultured in the Dulbecco's modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS). Human bone marrowderived MSCs were obtained from ATCC (PCS-500-012; Manassas, VA, USA) and grown in the α-modified Eagle's medium (MEM) containing 10% FBS and 1% penicillin-streptomycin at 37°C. All cultures were maintained in a humidified atmosphere of 5% CO 2 at 37°C. Passages 3 and 5 of MH7A and hMSCs were used for further experiments.

Transwell assay
Transwell chamber (8-mm pore size) coated with Matrigel (BD Bioscience) was employed to evaluate the invasion capacity of cells. Following different treatments, MH7A cells (1 × 10 5 ) with 200 μL serum-free media were seeded into the upper chamber of transwell. Five hundred microliter media containing serum was added into the lower chambers. Following 24-h incubation, cells invaded to the lower surface were fixed with 4% paraformaldehyde and stained 0.1% with crystal violet (Sigma-Aldrich, St. Louis, MO, USA). Finally, the average numbers of invaded cells in five random fields were assessed by a microscope.

Enzyme-linked immunosorbent assay (ELISA)
After appropriate treatment, the culture supernatants from MH7A cells culture were collected, then the concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were assayed using commercial the ELISA kit (R&D Systems, Minneapolis, MN, USA) referring to the producer's guidance.

RNA immunoprecipitation (RIP) assay
MH7A cells were lysed using RIP lysis buffer. The IgG antibody or Argonaute 2 (Ago2) antibody was coupled to magnetic beads and incubated with cell lysates for 4 h at 4°C, followed by incubation with protease K buffer to remove the protein. After that, the immunoprecipitated RNA was eluted and analyzed by qRT-PCR assay.

Statistical analysis
All experiments were performed in triplicate. The data was displayed as the mean ± standard deviation (SD) and processed by GraphPad Prism 7 software. Student's t test or one-way analysis of variance (ANOVA) was utilized to analyze significant differences. The P < 0.05 indicated statistically significant.

HAND2-AS1 upregulation inhibits tumor-like biologic behaviors of RA-FLSs via NF-κB pathway
Initially, the expression profile of HAND2-AS1 was investigated in synovial tissues. In comparison with normal synovial specimens, HAND2-AS1 was lowly expressed in synovial tissues with RA (Fig. 1a). Next, the role of HAND2-AS1 in RA progression was studied, we transfected HAND2-AS1 plasmid into MH7A cells. qRT-PCR analysis indicated that HAND2-AS1 level in MH7A cells was significantly upregulated after cells were transfected with HAND2-AS1 (Fig. 1b). After that, a CCK-8 assay suggested that the proliferation of MH7A cells was markedly reduced by HAND2-AS1 upregulation (Fig.  1c). On the contrary, HAND2-AS1 overexpression significantly promoted apoptosis in MH7A cells (Fig. 1d). Moreover, wound healing and transwell assays were performed; we found that the migration and invasion abilities of HAND2-AS1-increased MH7A cells were markedly suppressed (Fig. 1e, f). Results of ELISA indicated HAND2-AS1 upregulation repressed inflammation of MH7A cells, reflected by the decrease of IL-6 and TNF-α levels (Fig. 1g). Besides that, the level of phosphorylated p65 (p-p65) was found to be reduced by HAND2-AS1 overexpression in MH7A cells, suggesting that HAND2-AS1 could inactivate NF-κB pathway (Fig.  1h). Taken together, HAND2-AS1 upregulation inhibited the proliferation, motility, and inflammation and induced apoptosis in RA-FLSs via the inactivation of NF-κB pathway.

TNFAIP3 is a target of miR-143-3p and could be indirectly regulated by HAND2-AS1
Through searching the starBase database, it was found that there was a potential base-complementary binding site between miR-143-3p and the TNFAIP3 3'UTR (Fig. 4a). The Fig. 2 MiR-143-3p is a target of HAND2-AS1. a The potential binding sites of miR-143-3p on HAND2-AS1. b-d The interaction between miR-143-3p and HAND2-AS1 was confirmed using dual-luciferase reporter assay and RIP assay. e qRT-PCR analysis of miR-143-3p expression in MH7A cells transfected with vector or HAND2-AS1. f qRT-PCR analysis of miR-143-3p expression in RA synovial tissues and normal synovial tissues. g Pearson correlation analysis of miR-143-3p and HAND2-AS1 expression in 28 RA tissues. ***P < 0.0001 dual-luciferase reporter assay showed a remarkable reduction in the luciferase activity following co-transfection of MH7A cells with miR-143-3p mimics and wild-type TNFA IP3 3'UTR vector, but not with the mutant-type TNFAIP3 3′ UTR vector (Fig. 4b). RIP assay further demonstrated that TNFAIP3 and miR-143-3p were enriched by anti-Ago2 compared to control IgG (Fig. 4c). Furthermore, miR-143-3p overexpression repressed the expression of TNFAIP3 in MH7A cells (Fig. 4d). Therefore, we confirmed that miR-143-3p directly targeted TNFAIP3 and negatively regulate its expression.

Discussion
FLSs are the major cell types that make up the synovial intima structure, and RA-FLSs exhibit "tumor-like" properties, including aggressive proliferation, apoptosis arrest, and increased invasiveness, which are critical in the formation of synovial pannus; besides that, activated RA-FLSs release abundant inflammatory cytokines, chemokines, and metalloproteinases, mediating inflammation and further exacerbating joint damage, and ultimately exacerbating the progression of RA [3,25,26]. Therefore, an investigation of the aggressive phenotype of RA-FLSs is required to develop novel therapy regimens for RA patients.
In the current study, a low expression of HAND2-AS1 was observed in synovial tissues with RA. Then, we upregulated the level of HAND2-AS1 in RA-FLSs in vitro, and it was proved that HAND2-AS1 re-expression impaired the proliferation, invasion, migration, and inflammation and contributed to the apoptosis in RA-FLSs; thus, we knew that HAND2-AS1 was of great importance in the pathogenesis of RA. NF-κB is a ubiquitously expressed pleiotropic transcription factor present in almost all cell types; aberrant regulation of NF-κB and its downstream targets often result in inflammation, cell growth, drug resistance, and cancer metastasis [27]. p65, as one of the five components that form the NF-κB transcription factor family, is typically involved in the body's inflammatory response [28]. In this study, we also observed that HAND2-AS1 reduced the level of p-p65 in RA-FLSs. Therefore, we concluded that HAND2-AS1 might repress RA progression via the inactivation of NF-κB pathway. MSCs possess the ability to differentiate into multiple cell lines, including osteoblasts, chondrocytes, and adipocytes; form bone and cartilage; and exert immunosuppressive functions [29][30][31]; besides owing to their capacity to abolish the exacerbated pathogenic immune response observed in these patients, MSCs have been considered as interesting therapeutic cell candidates for the treatment of RA [32]. Exosomes are small spherical packages, which possess the physical properties of artificial nanoparticles and additional advantages such as excellent biocompatibility, biodegradability, and sequence programmability, and are ideal drug delivery carriers [33]. In the current study, we generated hMSC-HAND2-AS1-Exos (exosomes derived from HAND2-AS1-overexpressing hMSCs); after the co-incubation of RA-FLSs with hMSC-HAND2-AS1-Exos, HAND2-AS1 Previous studies have shown that HAND2-AS1 can perform a suppressive role in tumor progression by serving as a sponge of miRNAs [11,12]. Emerging evidence has revealed that the novel regulatory network of lncRNAs-miRNAs-mRNAs plays a significant role in the pathophysiological processes of many diseases, including RA [34,35]. Therefore, the HAND2-AS1-miRNAs-mRNAs network in RA was investigated in this study. It was confirmed that HAND2-AS1 served as a sponge for miR-143-3p and positively regulated TNFAIP3 expression, which was verified to be a direct target of miR-143-3p. Subsequent analysis suggested that the effects of HAND2-AS1 on RA-FLSs and the inactivation of NF-κB pathway were abolished by miR-143-3p overexpression or TNFAIP3 downregulation. Moreover, when the level of HAND2-AS1 was elevated in RA-FLSs through the co-culture with hMSC-HAND2-AS1-Exos, we found miR-143-3p expression was inhibited and HAND2-AS1 expression was increased; importantly, hMSC-derived exosomal HAND2-AS1 also suppressed tumor-like biologic behaviors of RA-FLSs via NF-κB pathway.
In conclusion, this study demonstrated that overexpression of HAND2-AS1 in exosomes derived from MSCs suppressed the proliferation, invasion, migration, and inflammation and induced apoptosis in RA-FLSs through the inactivation of NF-κB pathway via miR-143-3p/ TNFAIP3 axis. MSCs are easily isolated and amenable to culture expansion in vitro, MSC transplantation is undergoing extensive evaluation as a cellular therapy in human clinical trials [36]; however, rejection by the host and tumorigenicity have limited its clinical application [37]. Exosomes are one of extracellular vesicles that stably present in body fluids due to their phospholipid bilayer; besides that, owing to their RNA transport capacity and ability crossing the blood brain barrier, exosomes are considered candidate drug delivery vehicles [38]. MSCs produce abundant amounts of exosomes, and MSC-derived exosomes have a content that includes cytokines and growth factors, signaling lipids, mRNAs, and regulatory miRNAs [36]. Therefore, loading specific lncRNA into MSC-derived exosomes may provide a new therapeutic paradigm for cellfree MSC-based therapies in clinic. Our study suggests a potential therapeutic strategy for the treatment of RA.