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

doi:10.21203/rs.3.rs-965123/v1

Background

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 inflammation，synovial hyperplasia，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.

Methods

Primary hUCMSCs were isolated and cultured by tissue adherence method，and surface markers were identified by flow cytometry. We used differential centrifugation combined with ultrafiltration to obtain hUCMSC-Exos and identify them；The primary RA FLSs were isolated and cultured by trypsin digestion，then surface markers were identified by flow cytometry. We labeled the RNA of hUCMSC-Exos. hUCMSC-Exos was co-cultured with RA FLSs then the dyed RA FLSs were observed with a fluorescence microscope.

GV493 with siRNA target sequences that could knock down Ago2 transfected hUCMSCs. Real-time PCR and Westen blot analysis Ago2 levels in hUCMSCs and transfected hUCMSCs to determine knockdown efficiency. Lentivirus GV493 with the target with the highest knockdown efficiency to transfect hUCMSCs，named hUCMSC^KD3-Ago2. Then hUCMSC-Exos，hUCMSC^KD3-Ago2-Exos，hUCMSC^NC-Exos were extracted by the differential centrifugal method combined ultrafiltration method，then were identified. The real-time cell analysis was used to detected the proliferation and migration of RA FLSs in different concentrations of hUCMSC-Exos. Finally，we used the optimal concentration of hUCMSC^KD3-Ago2-Exos and hUCMSC^NC-Exos to intervene in RA FLSs.

Results

After incubation with hUCMSC-Exos and RA FLSs，the hUCMSC-Exos accumulated in the blue nuclei of RA FLSs. hUCMSC^NC-Exos and hUCMSC^KD-Ago2-Exos inhibited the proliferation and migration of RA FLSs compared ，particularly the hUCMSCNC-Exos had the significant inhibitory effect.

Conclusions

hUCMSC-Exos RNA can be taken up by RA FLSs，and hUCMSC may affect the proliferation and migration of RA FLSs via exosome-mediated delivery of miRNA.

Stem Cell & Developmental Cell Biology

Rheumatoid arthritis

Fibroblast-like synoviocytes

Human umbilical cord mesenchymal stem cells-derived Exosomes

miRNA

Proliferation

Migration

hUCMSCs can deliver hUCMSC-Exos to RA FLSs.
Knockdown of Ago2 in hUCMSCs would reduce Ago2 in hUCMSC-Exos to affect miRNAs in hUCMSC-Exos.
hUCMSC-Exos inhibits the proliferation and migration of RA FLSs.
hUCMSC-Exos may affect RA FLSs by delivering miRNAs.

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 inflammation and progressive disability. Fibroblast-like synoviocytes (FLSs) are an important part of the synovial membrane，which play a significant role in the process of local synovial hyperplasia，inflammation，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 cell-derived 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 inflammatory 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 confirmed the value of MSC-Exos to mediate the direct intracellular transfer of miRNA and showed the efficacy 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 clarified 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 fulfilled 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 trypsin-ethylenediaminetetraacetic 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 modified 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% confluence. 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.

Flow cytometry analysis

RA FLSs cell suspension (1×10⁵ cells/mL) and hUCMSCs cell suspension (1×10⁵cells/mL)were used for flow cytometry（FCM）(ACEA，USA) analysis to detect cell surface antigen phenotype. RA FLSs were incubated on ice for 1h with anti-rat antibodies (BioLegend，USA) conjugated with fluorescein isothiocyanate(FITC)，Allophycocyanin(APC)，or phycoerythrin (PE). Then FCM evaluated and analyzed CDH 11、PDPN、CD68(Abcam，USA). The hUCMSCs were incubated with anti-human monoclonal antibodies (BioLegend，USA) conjugated with FITC or PE. The FCM evaluated and analyzed markers of hUCMSCs (CD105、CD73 and CD90)，endothelial cells (CD31 and CD34) and hematopoietic cells (CD45) (Biolengd，USA).

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.

Transfection of hUCMSCs with Lentivirus GV493

hUCMSCs suspension was inoculated in 96-well culture plates (5×10⁴ cells/well) to ensure that the cell fusion rate was about 20% after 24 hours. Experimental groupings，dosage of GV493 and transfection conditions are shown in Table 1. The cell culture medium was changed 16 hours after transfection. The fluorescence expression was observed under the fluorescence microscope (Olympus，Japan)after 72h.

Transfection of hUCMSCs with lentivirus GV493 with or without siRNA target

Because exosomal miRNA is associated with Ago2，we had designed three different siRNA targets that can knock down Ago2，then respectively loaded them on GW493 to transfect hUCMSCs to knock down hUCMSCs Ago2 and exosomal miRNA. The hUCMSCs suspension was inoculated in a 6-well plate (1×10⁵cells/well) whose fusion rate was about 20% after being cultured for 24 hours. hUCMSCs were transfected by GV493 according to the previously established transfection conditions. According to the different types of lentiviruses transfected，the experiment was divided into 4 groups，hUCMSC^KD1-Ago2 group: LVPSC85384-1 transfected hUCMSCs，hUCMSC^KD2-Ago2 group: LVPSC85385-11 transfected hUCMSCs，hUCMSC^KD3-Ago2 group: LVPSC85386-11 transfected hUCMSCs，NC group:the empty GV493 transfected hUCMSCs. The hUCMSCs in the KD group were named hUCMSC^KD-Ago2，which including hUCMSC^KD1-Ago2，hUCMSC^KD2-Ago2，and hUCMSC^KD3-Ago2. At the same time，those in the NC group were named hUCMSC^NC. The informations of group and virus are shown in Table 2. After 16 hours of transfection，the cells in each well were collected and transferred to T25 culture flasks for continued cultivation. The fluorescence rate was the positive transfection rate，which was observed under a fluorescence microscope 72 hours after transfection.

Detect the levels of Ago2 in hUCMSCs，hUCMSC^KD-Ago2 and hUCMSC^NC

Real-time PCR analysis

Total RNA was extracted and purified using Trizol reagent (Shanghai Pufei Biotech Co. ，Ltd. ) according 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—F5'-TCCACCTAGACCCGACTTT-3'，R5'-GTTCCACGATTTCCCTGTT-3'. Reverse transcription of the extracted RNA was carried out according to the protocols of the M-MLV kit（Promega，USA）. Quantitative real-time PCR was performed using PCR Kit（QIAGEN，USA）on Real-time PCR instrument (Roche，USA). With GAPDH used as the normalizing controls，ΔCt indicates the gene expression level in cells，and 2−ΔΔCt reflects relative quantification.

Isolation and identification of Exos

We cultured hUCMSCs，hUCMSC^KD3-Ago2 and hUCMSC^NC with serum-free medium(Excell，China) and separately collected supernatant，then we used ultrafiltration 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 fluoride(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 sulfate-polyacrylamide 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.

Verification of the uptake of RNA in hUCMSC-Exos by RA FLSs

According to the instructions，we have configured RNA Select™ Green Fluorescent Cell Stain (Thermo Fisher，USA)working fluid，then the working fluid 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(2×10⁵cells/ 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，fixed with the fixative，and the nucleus were stained with Hoechst 33342(Solarbio，China);Finally，we took out the cell climbing sheets and mounted them and fluorescence image were visualized with wide-field 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 reflect the effect of hUCMSC-Exos on the proliferation and migration of RA FLSs. The RA FLSs suspension was inoculated into wells of E-Plate 16(ACEA，USA)，(3×10³ cells/well). After the cells adhered，the cell supernatant was replaced with the complete medium containing hUCMSC-Exos at different concentrations，and then the cells were cultured for 30 hours. Similarly，RTCA was performed to detect the effect of hUCMSC-Exos on the migration of RA FLSs. The lower chambers of CIM-Plate 16(ACEA，USA)were filled with 15% FBS medium，and the upper chamber was added with serum-free RA FLSs suspension containing different concentrations of hUCMSC-Exos (serum-free RA FLSs suspension containing different concentrations of hUCMSC-Exos (2. 5×10⁴cells/well)，then the cells were cultured for 48 hours.

Assessment of hUCMSC-Exos miRNA on the proliferation and migration of RA FLSs

We determined that the intervention concentration is 150ug/mL，which is the concentration that hUCMSC-Exos has the strongest effect on the proliferation of RA FLSs. RA FLSs were inoculated on E-Plate 16 (6×10³cells/well) for 48 hours. After the cells of each group adhered，we changed the culture conditions according to Table 3. RTCA detected Cell Index every 0. 5h. RA FLSs were inoculated on CIM-Plate 16 (3×10⁴cells/well) for 46h hours，and RTCA detected Cell Index every 0. 5h to reflects cells migration. 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; Non - normality was compared by non-parametric tests based on rank. P values less than 0. 05 were considered statistically significant.

Isolation，culturing，and identification of RA FLSs

RA FLSs grew by adhering to the substratum and displayed a fibroblast-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 identification 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 difficult to transfect hUCMSCs，and HiTransG P can significantly increase the transfection rate. The 80% fluorescence 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 Ago2 levels in hUCMSCs，hUCMSC^KD-Ago2 and hUCMSC^NC

Real-time PCR analysis

The larger the cell ΔCt，the lower the target gene level. When the cell tΔCt≤12，the gene level of the cell is high; when ΔCt≥16，the gene level in the cell is low. ΔCt=Ct value^Ago2-Ct value^GAPDH. The average value of ΔCt in hUCMSCs is 8. 91，so Ago2 is highly expressed in hUCMSCs. Amplification curve and dissolution curve of GAPDH and Ago2 of hUCMSCs，hUCMSC^NC and hUCMSC^KD-Ago2 are shown in Figure 5a-f；Quantitative PCR showed that compared with hUCMSC^NC，the Ago2 gene knockdown efficiency of hUCMSCs^KD1-^Ago2 ，hUCMSCs^KD2-^Ago2 ，hUCMSCs^KD3-^Ago2 were 51. 4%，10. 1% and 70. 7% (Figure 5g).

Western blot analysis

Western blot showing Ago2 expression in Hela cells (positive reference cells)，which proves that the antibody is qualified. Ago2 also expressed in hUCMSCs(Figure 6a)，hUCMSC^KD-Ago2(Figure 6b) and hUCMSC^NC(Figure 6b). The gray band values of hUCMSC^NC are normalized，hUCMSC^KD1-Ago2hUCMSC^KD2-Ago2 and hUCMSC^KD3-Ago2 band gray value ratios respectively are 0. 848847139，0. 4890497，0. 373544889.

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.

Exos isolation and characterization

Transmission electron microscopy revealed that hUCMSC-Exos，hUCMSC^KD3-Ago2-Exos，hUCMSC^NC-Exos were oval phospholipid bilayer-bound structures with diameters of 30-150 nm(Figure 7a). NTA showed that the diameter distribution of hUCMSC-Exos was a single peak，the median particle diameter was 146nm，and the concentration was 1. 8×10¹¹ Particles/mL(Figure 7b). Highly sensitive nanoparticle size analysis showed that the particle size distributions of hUCMSC-Exos，hUCMSC^KD3-Ago2-Exos，and hUCMSC^NC-Exos were single peaks，and their peak values respectively were 142nm，106nm，122nm(Figure 7c). The protein concentrations of hUCMSC-Exos，hUCMSC^KD3-Ago2-Exos，and hUCMSC^NC-Exos respectively were 1. 384ug/ul，1. 133ug/ul，1. 038ug/ul. Western blot showing CD9，CD63 and TSG101 expression in hUCMSC-Exos，hUCMSC^KD3-Ago2-Exos，hUCMSC^NC-Exos (Figure 7d). The level of Ago2 in hUCMSC^KD3-Ago2-Exos is lower than that in hUCMSC^NC-Exoss and hUCMSC-Exos(Figure 7e-f).

The uptake of RNA in hUCMSC-Exos by RA FLSs

After hUCMSC-Exos with green fluorescent-labeled RNA and RA FLSs were incubated for 24 hours，we saw that the blue-stained nucleus of RA FLSs were accumulated by green fluorescence. 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 flattening 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 6×10³ 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 reflect 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.

MSCs have high proliferation，immune regulation and multidirectional differentiation capabilities. they could inhibit the proliferation and differentiation of various immune cells，secretion of inflammatory 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. Compared with other sources，UCMSCs have great differentiation potential，strong proliferation ability，low immunogenicity，wide sources，convenient materials，and no moral and ethical restrictions. In CIA rats，hUCMSCs inhibited the proliferation of T lymphocytes and promoted T lymphocyte apoptosis，down-regulated RORyt mRNA and protein expression，reduced the proportion of Th17 cells，up-regulated Foxp3 mRNA and protein expression，and increased the proportion of spleen Treg cells. In addition，hUCMSCs down-regulated the expression of RORyt and Foxp3 in the joints，inhibited IL-17 and promoted the expression of TGF-B in serum，thereby improving arthritis，delaying radiological progress，and inhibiting synovial hyperplasia[16]. UCMSCs are most likely to become pluripotent stem cells with clinical application prospects. However，UCMSCs have some problems in the treatment of RA. The survival and biological activity of UCMSCs after entering the body is not easy to control. Because MSCs treatment inhibits anti-tumor immune response，there is a potential carcinogenic risk[17]. At the same time，intravenous infusion of UCMSCs may cause capillary bed blockage，ectopic bone formation，cartilage formation，and tumor formation risks. Cell therapy of MSCs shows low implantation rate and poor survival. They play a role in animal models through paracrine[18][19]，in other words，the protective effects of MSCs secretome depended on the presence of MSC-Exos[20].

Extracellular vesicles (EVs) released by MSCs in resting or stress state can mimic the immune regulation and tissue repair effects of MSCs，including Exos，microparticles (MPs) and apoptotic bodies[21]. Our previous research shows hUCMSC-EVs can improve CIA by simulating the immunomodulatory effects of MSCs，regulating the proliferation，apoptosis and differentiation of T lymphocytes，and regulating the levels of inflammatory factors related to T lymphocytes[22]. As the most important component，Exos contains rich biological information delivery media such as mRNA and miRNA，which can regulate the function，survival and homing of immune cells. Its immunomodulatory effect is stronger than MPs[23]，and it has better properties as a non-cellular therapy than MSCs. hUCMSC-Exos are expected to become a new strategy for the treatment of RA.

Synovial hyperplasia can lead to irreversible cartilage and bone damage，which is the main pathological feature of RA and the difficulty in treatment. The Synovial lining layer is consisted of macrophage-like synovial cells and FLSs . The FLSs of RA patients have obstacles in growth regulation mechanism，resulting in excessive proliferation of FLSs cells and showing "tumor-like growth"，which plays a central role in joint inflammation and cartilage degradation during the progression of RA. FLSs express vimentin，CD45，CD55， CD90，CDH11，PDPN and other molecules on the surface. They can produce inflammatory factors and chemokines，chemoattract immune cells to promote immune response and delay the resolution of inflammation[24]. RA FLSs may be a potential target for RA treatment.

Varieties of studies have reported that miRNAs in FLS can participate in the pathogenesis of RA by regulating multiple signaling pathways. MiRNAs could participate in the occurrence and development of diseases by blocking mRNA translation and then inhibiting protein expression.The Wnt signaling pathway is the first pathway worth mentioning. In RA FLSs，miRNAs participate in the pathogenesis of RA by regulating the Wnt signaling pathway. miR-26 increases the production of pro-inflammatory cytokines and promotes the apoptosis of RA FLSs[25]; miR-375 inhibits the production of matrix metalloproteinases and fibronectin[26]; the upregulation of miR-663 promotes the proliferation of RA FLSs[27]; miR-323-3P positively regulates the Wnt pathway to affect RA FLSs [28]. MiR-152 ectopic expression indirectly induces negative regulators of the Wnt pathway to inhibit FLS proliferation.It can regulate the Wnt signaling pathway at the level of ligand-receptor and intracellular factors to regulate a variety of proteins that promote the migration and differentiation of RA FLSs[29]. In addition，the miRNA in RA FLSs can also participate in the pathological progress of RA by regulating the NF-κB pathway. The up-regulated MiR-155 and miR-146a in RA FLSs inhibit the NF-κB pathway to promote the activation of RA FLSs and promote osteoclastogenesis[30][31]，miR-27a reduces the levels of MMP2，MMP9，MMP13 and Rho family proteins in RA FLSs[31]，thereby inhibiting the migration and invasion of RA FLSs，and blocking the NF-κB pathway of RA FLSs.In RA FLSs，miR-21 acts as an activator of the NF-κB pathway to promote NF-κB nuclear translocation and promote the proliferation of RA FLSs [32]; miR-23b inhibits IL-17-induced activation of the NF-κB pathway and expression of inflammatory factors to inhibit the pathogenesis of RA [33]. miR-10a participates in the NF-κB-YY1-miR 10a pathway to promote the production of inflammatory 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-inflammatory 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 inflammatory mediators in RA FLS to promote the development of inflammation and cartilage destruction [36]; miR-19b positively regulates NF-κB signaling to promote inflammatory 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，finally，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 fluorescence 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 field 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 clarified 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 scientific 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 find more strategies for RA treatment.

hUCMSC-Exos may affect RA FLSs by delivering miRNAs. We traced that the RNA of hUCMSC-Exos was taken up by RA FLSs，and then knocked down the expression of hUCMSC-Exos Ago2，and confirmed that hUCMSC-Exos miRNA could inhibit the proliferation and migration of RA FLSs. Next，we will clarify the miRNA profile in RA FLSs，predict and anchor related therapeutic genes，and use MSC-Exos-derived miRNAs to open new strategies for RA targeted therapy.

RA:Rheumatoid arthritis; FLSs: Fibroblast-Like Synoviocytes; MSCs: Mesenchymal stem cells;miRNAs: MicroRNAs; MSC-Exos: Mesenchymal stem cell-derived exosomes; CIA:collagen-induced arthritis ; Fox- P3:forkhead box P3;Ago: Argonaute ;RISC: RNA-induced silencing complex ; DMEM:Dulbecco’s modified Eagle’s medium; FBS:fetal bovine serum ;FCM:flow cytometry; FITC:fluorescein isothiocyanate; APC:Allophycocyanin;PE:phycoerythrin; GAPDH:Glyceraldehyde-3-phosphate dehydrogenase ; NTA:Nanoparticle Tracking Analysis ;FGF:Fibroblast growth factor;PVDF:polyvinylidene fluoride;RTCA:real-time CelI AnaIysis .

Authors’ contributions

KX directed the research.GLZ，NZ， YL and LYM designed the research， LYM and NL performed the experiments， analyzed and interpreted the data， and drafted the manuscript. LYZ participated in the design and revised the manuscript. LYM，XYP，RXH and MMF performed the experiments. All authors have read and approved the final manuscript.

Acknowledgements

The authors thank the Translational Medicine Research Center of Shanxi Medical University for the excellent technical assistance.

Funding

This work was supported by grants from the National Natural Science Foundation (81871292) and the Key Research and Development Projects of Shanxi Province (201803D31136).

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 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.

Competing interests

The authors have no competing interests.

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Table 1 The groups，dosage of GV493 and infection conditions of hUCMSCs infected by GV493

Infection

Condition

GV493

mount

（TU）

A

B

C

Control

Group A

1×10⁸

MOI=100

Complete medium:90μL

GV493:10μL

Complete medium:86μL

HitransG A:4μL

GV493:10μL

Complete medium:86μL

HitransG P:4μL

GV493:10μL

Complete medium:100μL

Group B

5×10⁷

MOI=50

Complete medium:90μL

GV493:10μL

Complete medium:86μL

HitransG A:4μL

GV493:10μL

Complete medium:86μL

HitransG P:4μL

GV493:10μL

Group C

1×10⁷

MOI=10

Complete medium:90μL

GV493:10μL

Complete medium:86μL

HitransG A:4μL

GV493:10μL

Complete medium:86μL

HitransG P:4μL

GV493:10μL

Group D

1×10⁶

MOI=1

Complete medium:90μL

GV493:10μL

Complete medium:86μL

HitransG A:4μL

GV493:10μL

Complete medium:86μL

HitransG P:4μL

GV493:10μL

hUCMSC complete medium （Cyagen Biosciences，USA） was used to cultivate hUCMSC. HitransG P （Genechem，China） and HitransG A （Genechem，China） are lentiviral infection enhancer. GV493 was diluted with hUCMSC basal medium （Cyagen Biosciences，USA） to different titers，which corresponded to different multiplicity of infection （MOI）.

Table 2 The groups and virus information of hUCMSCs infected by GV493 vector
Group	virus name	Target sequence	Virus titer （TU/mL）	Virus dosage （μL）
KD1	LVPSC85384-1	gcACAGCCAGTAATCGAGTTT	2×10⁹	2. 00
KD2	LVPSC85385-11	cgGCAAGAAGAGATTAGCAAA	1×10⁹	4. 00
KD3	LVPSC85386-11	caATCAAATTACAGGCCAATT	1×10⁹	4. 00
NC	GW493		1×10⁹	4. 00

Table 3 The groups and culture conditions of RAFLSs in E-Plate 16 after RA FLSs adherence
Groups（n=3）	Culture condition （DMEM high sugar medium with 10% FBS）
Blank control group
hUCMSC^KD3-Ago2-Exos group	150ug/m hUCMSC^KD3-Ago2-Exos
hUCMSC^NC-Exos group	150ug/mL hUCMSC^NC-Exos

Table 4 The groups and culture system of RAFLSs in CIM-Plate 16
Group（n=3）	upper chamber （Serum-free DMEM high sugar medium）	lower chambers
Blank control group		DMEM high sugar medium with 10% FBS
hUCMSC^KD3-Ago2-Exos group	150ug/mL hUCMSC^KD3-Ago2-Exos
hUCMSC^NC-Exos group	150ug/mL hUCMSC^NC-Exos

Table 5a The proliferation of RA FLSs at different time points in each group
Group Time point	Blank control group（n=3）	hUCMSC^KD3-Ago2-Exos group （n=3）	hUCMSC^NC-Exos group （n=3）
10 h	1. 5394±0. 0724222	1. 37±0. 0753538	1. 3084333±0. 0346791
20 h	1. 5847667±0. 0686025	1. 3421667±0. 0810739	1. 2312333±0. 0228715
30 h	1. 5327±0. 0559024	1. 1817667±0. 0421836	1. 0848333±0. 0167804
40 h	1. 8286333±0. 0687184	1. 4050333±0. 0777613	1. 2751333±0. 0329749

Table 5b The migration of RA FLSs at different time points in each group
Group Time point	Blank control group（n=3）	hUCMSC^KD3-Ago2-Exos group （n=3）	hUCMSC^NC-Exos group （n=3）
10 h	1. 2837±0. 0145506	1. 2204667±0. 0461880	1. 0878000±0. 0775234
20 h	1. 098±0. 0401598	1. 0769±0. 0270217	0. 9862667±0. 0544596
30 h	1. 2325333±0. 0631646	1. 2112±0. 0409297	1. 1449667±0. 0442838
40 h	1. 3565±0. 0780123	1. 3427667±0. 041502	1. 2802333±0. 0581369

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

Status:

Version 1

Abstract

Figures

Highlights

Introduction

Materials And Methods

Results

The effect of hUCMSC-Exos on the proliferation and migration of RA FLSs

The effect of hUCMSC-Exos miRNA on the proliferation and migration of RA FLSs

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Status:

Version 1