Mesenchymal Stem Cells Inhibit the Proliferation and Migration of Fibroblast-like Synoviocytes in Rheumatoid Arthritis via Exosome-mediated Delivery of miRNAs

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by aggressive and symmetrical polyarthritis. Fibroblast-Like Synoviocytes(FLSs)play a central role in the pathogenesis of RA. The abnormal expression of miRNAs in RA FLSs mediates RA joint inammation(cid:0)synovial hyperplasia(cid:0)and tissue destruction; MSC-Exos-derived miRNAs are a potential RA treatment strategy. This study aimed to investigate the hUCMSC-Exos can deliver miRNA to RA FLSs and affect their biological properties.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disease characterized by the abnormal synovial hyperplasia of joints and the destruction of cartilage and bone. In recent years the treatment strategies for RA have developed rapidly but there is still a long way to go before the complete cure of RA. Traditional NSAIDs are restricted by a variety of safety issues while traditional DMARDs have a slow effect and serious side effects. Although the glucocorticoids can quickly eliminate joint swelling and reduce pain it cannot prevent joint damage and the development of lesions moreover it also has potentially severe side effects after long-term use [1]. Although biological agents and molecular targeted drugs have improved remission rates in RA these drugs are expensive and target a particular step in the progression of RA. Mesenchymal stem cells MSCs have been reported as an interesting therapeutic cell candidate for the treatment of RA due to their immunomodulatory and differentiation effects. Unfortunately it also has been found to cause abnormal differentiation and tumor formation [2]. Patients with RA have a low treatment compliance rate and are prone to relapse after drug withdrawal. In short none of the existing treatment methods for RA can enable patients to achieve long-term remission without drugs and may even progress to persistent in ammation and progressive disability. Fibroblast-like synoviocytes (FLSs) are an important part of the synovial membrane which play a signi cant role in the process of local synovial hyperplasia in ammation and cartilage degradation in RA patients [3]. MicroRNAs (miRNAs) composed of 19-23 nucleotides are evolutionarily conserved single-stranded non-coding RNAs which could negatively regulate protein expression by degrading target RNA or blocking its translation [4]. Existing studies have shown that miRNA in RA FLSs may participate in the pathological process of RA by regulating Wnt signaling pathway NF-κB pathway and DNA methylation [5]. Mesenchymal stem cellderived exosomes (MSC-Exos) are common membrane-bound nanovesicles with a size of about 30-150nm derived from mesenchymal stem cells through exocytosis which could mimic the function of parental MSCs by transferring bioactive substances such as DNA proteins/peptides mRNA miRNA lipids and organelles. MSC-Exos are more effective less toxic and more stable than the parental cells.
Studies have shown that MSC-Exos can mediate intercellular communication and regulate different signaling pathways through miRNA transmission [6]. Thus we have reason to believe the cell-free therapy based on MSC-Exos may be more effective in repairing the joint damage of RA breaking through the limitations of existing treatments and becoming a potential method for the treatment of RA. Excitingly the miRNAs encapsulated in MSC-Exos transfer information to recipient cells [7]. Some studies characterized the mechanisms by which Exos particularly through miRNAs are involved in RA. The human MSC-Exos that overexpress miRNA-124a could inhibit cell proliferation migration and promote apoptosis in the FLSs line [8]. The bone marrow mesenchymal stem cell BMMSC -secreted exosomal miR-192-5p can delay the event of the in ammatory response in RA [9]. BMMSC-derived Exos participate in the intercellular transfer of miR-320a and subsequently inhibit the progression of RA [10]. The DBA/1J mice BMMSC-derived miR-150-5p Exos decreased migration and invasion in RA FLSs as well as reduced hind paw thickness and the clinical arthritic scores in collagen-induced arthritis (CIA) mice [11]. Exos were extracted from normal MSCs with over-expressed miR-146a/miR-155 increased forkhead box P3 (Fox-P3) TGFβ IL-10 RORγt IL-17 and IL-6 gene expression in the CIA mice [12]. These researches con rmed the value of MSC-Exos to mediate the direct intracellular transfer of miRNA and showed the e cacy of this strategy in RA.
The miRNAs have been found extracellularly being encapsulated within Exos or associated with Argonaute (Ago) [13].The mature miRNA is loaded into the RNA-induced silencing complex (RISC) which mediates the relationship between the interacting miRNA and its target mRNA molecule. The Ago protein is part of RISC and mediates the cleavage of target mRNA. Particularly Ago2 has splicing activity and can cut mRNA. Post-transcriptional Ago2 regulates miRNA expression abundance. Ago2 binds with miRNAs to form the RISC which can associate with multivesicular bodies which produce Exos after fusion with the plasma membrane [14]. In short Ago2 promotes miRNA maturation prevents miRNA degradation and controls the sorting of miRNA. Regulation of Ago2 could affect miRNA maturation.
In the current study We tested the uptake of hUCMSC-Exos by RA FLSs and knocked down Ago2 in hUCMSC to reduce the content of Ago2 in hUCMSC-Exos which is in order to the expression of miRNA in hUCMSC-Exos then analyzed the effect of miRNA in hUCMSC-Exos on RA FLSs. In summary we clari ed that miRNA delivered by hUCMSC-Exos may affect the proliferation and migration of RA FLSs. Ultimately the results provide a theoretical foundation for the application of hUCMSC-secreted exosomal miRNA in RA treatment.

Ethics Statement
Informed consent agreement forms were signed and collected from all participants before enrollment into our study. The research followed the tenets of the Declaration of Helsinki and was approved by the Ethical Committee of Shanxi Bethune Hospital (ethical approval code:2018LL007).

Sample Collection
The specimens of this study were obtained from patients in the Department of Orthopedics at Shanxi Bethune Hospital and the Second Hospital of Shanxi Medical University from March 2015 to March 2021. The synovial tissues taken from arthroplasty or synovectomy were from patients with RA. All patients ful lled the diagnosis of the American College of Rheumatology for RA.

Isolation and Culture of RA FLSs
The synovial tissues were collected cut under sterile conditions detached with trypsinethylenediaminetetraacetic acid (0. 25%EDTA Solarbio Beijing China) at 37°C for 1 h and centrifuged.
The cells were then collected and cultured in Dulbecco's modi ed Eagle's medium (DMEM)(Gibco USA) containing 10% fetal bovine serum (FBS)(Gibco USA). Cell passages were conducted every 3-4 days followed by 24-h adherent growth of cells at 37°C with 5% CO2. The cells were detached with 0. 25% EDTA and passaged after reached 85% con uence. The RA-FLSs at passage 3 to 5 were utilized for the following experiments.

Isolation and Culture of hUCMSCs
We stripped the umbilical vein and artery of the umbilical cord (the Department of Obstetrics and Gynecology of Shanxi Bethune Hospital supplied) of the healthy term neonates. The remaining tissues were cut to 0. 5mm3 then inoculated in petri dishes. After the tissues were attached to the wall we added serum-free medium (Excell Bio Chain). The cells were fused to 80% they were digested by trypsin passed 1:3. The p3-p5 generation hUCMSCs in a good growth state were used in the experiment.

Culturing of Hela cells
Hela cells supplied by the Chinese Academy of Sciences as Western blot positive reference cells were cultured at 37 ℃ in the incubator containing CO2 with a volume fraction of 5% and saturated humidity. The liquid was changed once every two days. When cells fused to 80% they were passed at a ratio of 1:5. to the manufacturer's instructions and RNA was precipitated using isopropanol. The RNA quality determined by the spectrophotometer was OD260/OD280: 1. 8-2. 0 and the RNA concentration was 1 000-2 000μg/mL. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control whose primers were synthesized by Guangzhou RiboBio Co. Ltd.Moreover their sequences were as follows: GAPDH-F5'-TGACTTCAACAGCGACACCCA-3' R5'-CACCCTGTTGCTGTAGCCAAA-3' Ago2- With GAPDH used as the normalizing controls ΔCt indicates the gene expression level in cells and 2−ΔΔCt re ects relative quanti cation.

Isolation and identi cation of Exos
We cultured hUCMSCs hUCMSC KD3-Ago2 and hUCMSC NC with serum-free medium(Excell China) and separately collected supernatant then we used ultra ltration combined with differential centrifugation to obtain the corresponding exosomes. The process is shown in Figure 1.
Exos were visualized using transmission electron microscopy (JEM-2010 F JEOL Japan). The particle size of Exos was determined by highly sensitive nanoparticle size analysis and Nanoparticle Tracking Analysis (NTA) using a Zetasizer NanoZS (Malvern Instruments Malvern UK). Exos protein concentrations were detected using a BCA Protein Assay kit (Solarbio Beijing China).

Western blot analysis
The RIPA lysate was used to extract proteins from Hela cells hUCMSCs hUCMSC KD-Ago2 and hUCMSC NC . 40 μg samples were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene uoride(PVDF) membrane Millipore USA) after electrophoresis. After incubation with primary antibodies Anti-Argonaute-2 antibody Abcam Cambridge MA USA 1:1000 dilution in TBST with 5% BSA the membranes were washed then incubated with goat anti-rabbit IgG-Horseradish peroxide SantaCruz USA 1:2000 dilution secondary antibodies. The signals were visualized with ECL chemiluminescent solution (Thermo Fisher USA) We used western blot to analyze the Exos surface antigen phenotype and Ago2 level. UCMSC-Exos hUCMSC KD3-Ago2 -Exos and hUCMSC NC -Exos proteins were separated on 10 % sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels (Solarbio China) and transferred to PVDF membrane after electrophoresis. PVDF membrane was blocked in TBST with 5% skim milk for 1h. After incubation with primary antibodies against CD9(Abcam Cambridge MA USA 1:1000 dilution) CD63(Abcam Cambridge MA USA 1:500 dilution) TSG101 (sino biological China 1:500 dilution) and Argonaute-2 (Abcam Cambridge MA USA 1:1000 dilution) in TBST with 5% BSA the membranes were washed four times with TBST (8 min each time) then incubated with horseradish peroxidase-conjugated goat anti-rabbit (CST USA 1:10000 dilution) secondary antibodies. After the ECL method combined with X-ray imaging and we analyzed the Ago2 level.
Veri cation of the uptake of RNA in hUCMSC-Exos by RA FLSs According to the instructions we have con gured RNA Select™ Green Fluorescent Cell Stain (Thermo Fisher USA)working uid then the working uid was added to the prepared hUCMSC-Exos suspension to label RNA. Exosome Spin Columns(Thermo Fisher USA)were used to remove residual dye from hUCMSC-Exos.
We set up control groups and hUCMSC-Exos intervention groups. First 9mm cell climbing sheets(NEST China)were plated into 48-well plates and inoculated with RA FLSs(2×10 5 cells/ well) After the cells were adherent the medium was changed. Complete medium was still added into the control wells and the UCMSC-Exos intervention wells were added with complete medium with 200μg/mL RNA-labeled hUCMSC-Exos then cells were incubated at 37℃ in the dark for 24h. Next the cells were washed with PBS xed with the xative and the nucleus were stained with Hoechst 33342(Solarbio China);Finally we took out the cell climbing sheets and mounted them and uorescence image were visualized with wideeld high-content image analysis system(PerkinElmer USA).
Analysis of the effect of hUCMSC-Exos on the proliferation and migration of RA FLSs hUCMSC-Exos suspension was used to interfere with RA FLSs (0ug/mL 10ug/mL 30ug/mL 60ug/mL 90ug/mL 120ug/mL 150ug/mL 180ug/mL concentration);real-time CelI AnaIysis (RTCA)(ACEA USA) was performed to detect Cell Index every 1 h to re ect the effect of hUCMSC-Exos on the proliferation and migration of RA FLSs. The RA FLSs suspension was inoculated into wells of E-Plate We constructed the RA FLSs culture system for each group of cells according to Table 4.
Statistical analysis SAS 9. 4 is used for data processing and statistical analysis. Normally distributed data with homoscedasticity are expressed as mean ± standard deviation while the Non-normally distributed data are expressed as the interval between the median and quartile and the qualitative data rate was expressed by the ratio. Statistical analysis: repeated measure ANOVA was used for normality; Nonnormality was compared by non-parametric tests based on rank. P values less than 0. 05 were considered statistically signi cant.

Results
Isolation culturing and identi cation of RA FLSs RA FLSs grew by adhering to the substratum and displayed a broblast-like morphology (Figure 2a). Flow cytometry analysis of RA FLSs surface antigen phenotype revealed that PDPN CDH-11 were expressed while CD68 were not. (Figure 2b).
Isolation culturing and identi cation of hUCMSCs hUCMSCs grew by adhering to the substratum and displayed a long spindle-like morphology (Figure 3a). Flow cytometry analysis of hUCMSCs surface antigen phenotype revealed that CD105 CD73 CD90 were expressed while CD45 CD34 CD31 were not (Figure 3b).

Assessment of hUCMSCs transfected by GV493
After GV493 was used to transfect hUCMSC under different conditions for 72 hours the cells had normal growth morphology. GV493 is moderately di cult to transfect hUCMSCs and HiTransG P can signi cantly increase the transfection rate. The 80% uorescence rate is most suitable between the B and C groups Figure 4a . In other words the corresponding MOI is between 10-50. It follows that as few viruses as possible transfect cells without affecting the cell morphology we determined MOI=20. According to the most suitable transfection conditions that MOI=20 and add HiTransG P we used GV493 with or without different targets transfected hUCMSCs. It showed that they successfully transfected hUCMSCs (Figure 4b).
The All in all the results of real-time PCR and Western blot showed that the Ago2 knockdown rate was the highest in the hUCMSC KD3-Ago2 so the corresponding target LVPSC85386-11 was selected for the experiment.

The uptake of RNA in hUCMSC-Exos by RA FLSs
After hUCMSC-Exos with green uorescent-labeled RNA and RA FLSs were incubated for 24 hours we saw that the blue-stained nucleus of RA FLSs were accumulated by green uorescence. It indicates that RA FLSs can uptake the RNA in hUCMSC-Exos but compared with the control group the amount of cells in the experimental group is reduced (Figure 8a-b).

The effect of hUCMSC-Exos on the proliferation and migration of RA FLSs
After 6 hours of inoculation all the RA FLSs adhered to the wall. At the same time we added the complete medium containing hUCMSC-Exos which caused a sharp bulge at the 6th hour of the curve. The cell index proliferation curve showed attening after 25h. The 150ug/mL hUCMSC-Exos inhibited the proliferation of RA FLSs most strongly (Figure 9a).
The proliferation of RA FLSs without hUCMSC-Exos intervention was slow and we considered that it is related to fewer cells. In the subsequent experiments the number of cells were increased to 6×10 3 cells/well . The migration curve showed that 120ug/mL hUCMSC-Exos strongly inhibited the migration of RA FLSs (Figure 9b).

The effect of hUCMSC-Exos miRNA on the proliferation and migration of RA FLSs
The proliferation curves and migration curves of RA FLSs re ect that hUCMSC NC -Exos and hUCMSC KD3-Ago2 -Exos inhibited the proliferation and migration of RA FLSs compared with the blank control group at the same time(p<0. 01 p<0. 05) particularly the hUCMSC NC -Exos had the strongest inhibitory effect which. The proliferation and migration of RA FLSs in each group were statistically different in the Cell Index at different time points(p<0. 01 p<0. 05) and there were statistical differences between each group as a whole (p<0. 01 p<0. 05) (Figure 10a-b). The proliferation and migration of RA FLSs at different time points in each group were expressed by cell index (Table 5a-b). We knocked down the Ago2 and also reduced the expression of miRNA in hUCMSC KD3-Ago2 -Exos so the Ago2 of hUCMSC KD3-Ago2 -Exos is less than hUCMSC NC -Exos. Those results indicates that miRNA in hUCMSC-Exos could affect the proliferation and migration of RA FLSs. It combines with that RA FLSs could take up the RNA of hUCMSC-Exos which concludes hUCMSC-Exos may affect the proliferation and migration of RA FLSs by delivering miRNAs to RA FLSs.

Discussion
MSCs have high proliferation immune regulation and multidirectional differentiation capabilities. they could inhibit the proliferation and differentiation of various immune cells secretion of in ammatory factors and antibody production and promote the repair of damaged tissues. It has become a new method to explore the treatment of RA. We have proved that BMMSCs can inhibit The CXCL10/CXCR3 chemotactic axis adjusts the ratio of RANKL/OPG differentiates directly into chondrocytes and improves the bone destruction of ClA [15].
MSCs were initially isolated from bone marrow and later found to be widely distributed in various tissues such as umbilical cord fat synovium and gums. activation of the NF-κB pathway and expression of in ammatory factors to inhibit the pathogenesis of RA [33]. miR-10a participates in the NF-κB-YY1-miR 10a pathway to promote the production of in ammatory factors and the proliferation and migration of RA FLSs [34]; miR-203 depends on the activity of NF-κB to regulate the expression of IL-6 in RA FLS [35]; miR-10a is expressed in RA FLSs and inhibits the production of pro-in ammatory cytokines and matrix metalloproteinases by inhibiting NF-κB p65 induced by tumor necrosis factor-α; miR-18a directly acts on tumor necrosis factor-α-induced protein 3 to inhibit NF-κB pathway promotes the production of matrix-degrading enzymes and in ammatory mediators in RA FLS to promote the development of in ammation and cartilage destruction [36]; miR-19b positively regulates NF-κB signaling to promote in ammatory activation of RA FLSs [37]; DNA methylation and miRNAs in RA FLSs.The interaction between miR-124a is involved in the pathogenesis of RA. The methylation of miR-124a in RA FLSs promotes the proliferation of RA FLS and the expression of tumor necrosis factor-α[38]; ectopic expression of miR-152 down-regulates DNA methyltransferase DNMT1 and activates Wnt Pathway [29]; high expression of miR-34a * enhances the apoptosis of RA FLSs mediated by Fas-L and TRAIL [39].
The regulation of RA FLS by hUCMSC-Exos through miRNA will become a potential strategy for RA treatment. The amount of miRNA in Exos is extremely low sequence analysis showed that approximately 13% of the total RNA content of EVs from milk is miRNAs [40]. So we labeled the RNA of hUCMSC-Exos and then co-incubated it with RA FLSs nally found that RA FLSs could take up RNA in hUCMSC-Exos. The RNA in hUCMSC-Exos may enter the RA FLSs directly through the surface protein and lipid ligands to directly activate the RA FLSs membrane surface receptors to generate signal complexes and activate the intracellular signaling pathway.It can also fuse with the plasma membrane of the RA FLSs or be endocytosed directly into the RA FLSs. The tracer methods for Exos include uorescence imaging magnetic resonance imaging nuclear imaging CT imaging bioluminescence imaging and photoacoustic imaging [41] which help us to monitor Exos biological behavior in real-time and promote the development and delivery of targeted Exos in the eld of diagnosis and medicine.
In addition the co-incubation of hUCMSC-Exos and RA FLSs affected the proliferation and migration of RA FLSs. The 150ug/ml hUCMSC-Exos strongly inhibited the proliferation of RA FLSs and 120ug/ml hUCMSC-Exos strongly inhibited the migration of RA FLSs. MSC-Exos can transport various biologically active proteins and RNA (mRNA miRNA siRNA etc. ) to target cells to perform their biological functions. MSCs-derived exosomal miRNA-124a[8] miR-192-5p [9] miR-320a [10] miR-150-5p [11] and miR-146a/miR-155 [12] have been proven to be the treatments strategy for RA. In the current study we knocked down Ago2 in hUCMSC and then the content of Ago2 in hUCMSC-Exos decreased. Ago2 participates in the sorting of miRNA in Exos so the knockdown of Ago2 reduces the miRNAs expression exported by hUCMSC-Exos [14]. We used ago2 knockdown and non-knockdown hUCMSC-Exos to interfere with RA FLSs respectively both of which inhibited the proliferation and migration of RA FLSs especially the hUCMSC-Exos non-knockdown of ago2 had a stronger effect. The interfered miRNA in hUCMSC-Exos has a reduced effect on RA FLSs indicating that miRNA in hUCMSC-Exos affects the proliferation and migration of RA FLSs. Combining with that RA FLSs could uptake the RNA of hUCMSC-Exos we clari ed that miRNA delivered by hUCMSC-Exos might affect the proliferation and migration of RA FLSs.
In the future we will use live cells to dynamically track hUCMSC-Exos in real-time to visualize the dynamic cellular uptake of Exos to provide a scienti c basis for the interaction mechanism between Exos and cells. Not only that we will detect the changes of miRNAs in Exos and RA FLSs before and after Exos intervention in RA FLSs and select more differentially expressed miRNAs to nd more strategies for RA treatment. on reasonable request. Ethics approval and consent to participate The study was approved by the ethical committee of Shanxi Bethune Hospital. All experimental procedures were performed according to the Institutional.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was approved by the ethical committee of Shanxi Bethune Hospital. All experimental procedures were performed according to the Use Committee guidelines of Shanxi Medical University.

Consent for publication
Not applicable.   The supernatant of hUCMSCs was sequentially removed from dead cells and cell debris under different centrifugation conditions. After that the supernatant was concentrated with an ultra ltration tube and ltered with 0. 22μm lter membranes. Then the higher centrifugation conditions removed smaller debris.
Finally ultra-high-speed centrifugation to pellet and wash exosomes. Characteristics of RAFLSs. a The primary RAFLSs gradually adhere to the wall on the 3rd to 7th days and are fully grown and passable at about 18-25 days. They displayed a broblast-like morphology.
When the growth density is ≥70% they will appear "vortex". b RAFLSs express PDPN and CDH-11 positively on the surface and negatively express CD68  a The morphology and proliferation of the transfected hUCMSCs were monitored under bright-eld and dark eld microscopy and they are normal. Adding HiTransG P to each group can enhance the transfection e ciency. The dark-eld uorescence rate of group A group B group C and group D is about 100% 90% 60% 20% The best uorescence rate is 80% obviously The best MOI is between 10-50. We nally choose MOI=20 and HiTransG P to transfect hUCMSCs. Expression of the green uorescence protein GFP is observed as green colored hUCMSCs. b hUCMSCs morphology was monitored under bright-eld microscopy as well as dark eld microscopy . GV493 with or without siRNA targets could successfully transfected hUCMSCs Expression of the GFP is observed as green colored hUCMSCs.

Figure 5
RT-PCR results of Ago2 in hUCMSCs hUCMSCNC and hUCMSCKD-Ago2 a Ampli cation curves of GAPDH and Ago2 genes in hUCMSCs. b Dissolution curve of Ago2 in hUCMSCs. c Dissolution curve of GAPDH in hUCMSCs. d Ampli cation curves of GAPDH and Ago2 in hUCMSCNC and hUCMSCKD-Ago2. e Dissolution curve of Ago2 in hUCMSCNC and hUCMSCKD-Ago2. f Dissolution curve of GAPDH in hUCMSCNC and hUCMSCKD-Ago2. g Knockdown e ciency of Ago2 in hUCMSCNC and hUCMSCKD-Ago2.

Figure 6
Ago2 in hUCMSCs hUCMSCNC and hUCMSCKD-Ago2 were analyzed by Western blotting. a Expression of Ago2 in hUCMSCs. b Expression of Ago2 in hUCMSCNC and hUCMSCKD-Ago2. c Ago2 band gray value of hUCMSCNC and hUCMSCKD-Ago2.

Figure 8
The uptake of RNA in hUCMSC-Exos by RAFLSs. a The nucleus of RA FLS stained by Hoechst 33342. b The blue-stained nucleus of RA FLSs were accumulated by green uorescencet which is hUCMSC-Exos with green uorescent-labeled RNA.  The effect of hUCMSC-Exos miRNA on the proliferation and migration of RAFLSs. a The RA FLS proliferation curve showed that compared with the blank control both the hUCMSCKD3-Ago2-Exos and hUCMSCNC-Exos groups inhibited the proliferation of RAFLSs particularly hUCMSCNC-Exos had the strongest inhibitory effect. b The RA FLS migration curve showed that compared with the blank control both the hUCMSCKD3-Ago2-Exos and hUCMSCNC-Exos groups inhibited the migration of RAFLSs particularly hUCMSCNC-Exos had the strongest inhibitory effect.