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Back to Journal »International Journal of Nanomedicine» Volume 16

The therapeutic potential of exosomal circRNA from synovial mesenchymal cells by targeting circEDIL3/miR-485-3p/PIAS3/STAT3/VEGF functional module in rheumatoid arthritis

Authors: Zhang Jie, Zhang Yan, Ma Ying, Luo Li, Chu Ming, Zhang Zhen

Published on December 3, 2021 2021 Volume: 16 pages 7977-7994

DOI https://doi.org/10.2147/IJN.S333465

Single anonymous peer review

Editor who approved for publication: Dr. Mian Wang

Zhang Juan, 1 Zhang Yue, 1 Ma Yeye, 1 Luo Lili, 1 Chu Maolin, 2 Zhang Zhiyi 1 1 Department of Rheumatology, The First Affiliated Hospital of Harbin Medical University, Harbin, People's Republic of China; 2 Department of Urology, The Second Affiliated Hospital of Harbin Medical University , Harbin, People’s Republic of China Correspondent: Zhang Zhiyi; Zhu Maolin Tel +86 13303608999; +86 451 86605579 Fax +86 451 85556421 Email [Email protection]; [Email protection] Background: Synovial inflammation and related angiogenesis activation is in Plays a key role in rheumatoid arthritis (RA). As a carrier of genetic information, exosomes, including circular RNA (circRNA), have been explored as a delivery vehicle for therapeutic molecules. However, the role of exosomal circRNAs derived from synovial mesenchymal stem cells (SMSCs) and its mechanism in the progression of RA remain unclear. Methods: SMSCs-derived exosomes (SMSCs-Exos) were used for in vitro co-culture of RA fibroblast-like synovial cells (RA-FLS) and human dermal microvascular endothelial cells (HDMECs) and collagen-induced arthritis. CIA) In vivo mouse model. Their effects on VEGF expression and angiogenesis activity in vitro and their therapeutic effects in vivo were evaluated. Exosomes from SMSCs that overexpress circEDIL3 (Ad-circEDIL3-SMSCs-Exos) were used to further determine the role of circEDIL3 in SMSCs-Exo-based therapies. Results: Both SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos significantly down-regulated the IL-6/sIL-6R complex-induced RA-FLS and HDMECs co-culture supernatant and cell lysate VEGF expression co-cultured RA -The reduction of FLS, the degree of reduction of the latter is more obvious. Subsequent experiments showed that due to the decreased VEGF expression, SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos significantly down-regulated angiogenic activity. CircEDIL3 acts as a sponge for miR-485-3p, which targets PIAS3. PIAS3 is known to inhibit STAT3 activity and reduce downstream VEGF. In the CIA mouse model, the injection of SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos reduced synovial VEGF, thereby improving the severity of arthritis. Conclusion: The intracellular transfer of circEDIL3 via SMSCs-Exos may be a potential new treatment strategy for RA. Keywords: circEDIL3, exosomes, PIAS3, STAT3, VEGF

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by angiogenesis accompanied by inflammation leading to the formation of synovial pannus, leading to the destruction of cartilage and bone. 1 The inflammatory state of RA is through the enhancement of angiogenesis through inflammatory cells and oxygen. Therefore, angiogenesis has been explored as a promising therapeutic target for the treatment of RA. The synovium of RA patients contains fibroblast-like synovial cells (FLS), which produce large amounts of angiogenic factors, such as vascular endothelial growth factor (VEGF). Wang et al. and Li et al. confirmed that dysregulated autophagy 2 and immune dysregulation 3 are involved in the progression of RA synovial pannus. In particular, we found that the angiogenesis function module with VEGF as a key member plays a crucial role in angiogenesis, which indicates that drugs that inhibit VEGF production can be used as anti-rheumatic drugs. 4 However, current monoclonal antibody therapy has limited clinical application of targeting VEGF due to adverse effects such as hypertension and thromboembolism. Therefore, there is a need for more specific therapeutic targets than VEGF. 5

Circular RNA (circRNA) is a kind of non-coding RNA, which represents a new type of abundant, widely distributed and tissue-specific transcripts. They form a covalently closed continuous loop. 6 Circular RNA mainly regulates gene expression by acting as an effective sponge for microRNA (miRNA). It can bind to the complementary sequence in the 3ʹ untranslated region (3ʹ-UTR) of target mRNA to inhibit translation or degrade the target. 7 It is reported that CircRNAs are involved in the pathogenesis of diseases including tumors; 8 However, the role of tissue circRNAs in RA is unclear, and there are no reports on circRNAs that may play a key role in angiogenesis and inflammation. . In fact, in various diseases, inflammation and angiogenesis often follow and promote each other, and play a key role in the occurrence and development of the disease. 9 A systematic study of circRNA and its influence on inflammation-related neovascularization is necessary to develop them for clinical applications. In addition, despite the potential of circRNA as a therapeutic agent, there is still a lack of effective methods to deliver it to target tissues.

Exosomes (vesicles with a size of 30-200 nm) have attracted more and more attention as non-coding RNA transfer vectors. 10,11 A study showed that exosomes released by mesenchymal stem cells (MSC) can protect T lymphocytes from inflammation. 12 MSC-derived exosomes can reduce joint destruction by inhibiting synovial cell proliferation and angiogenesis. 13 Although circRNA is enriched and stable in exosomes, 11 the biological functions of circRNA secreted from MSC-derived exosomes in inflammation and angiogenesis are still unclear. In order to fill this knowledge gap, we designed the current study to study the regulatory function of MSC-derived exosomal circRNA in RA inflammation-induced angiogenesis.

In this study, one of the circRNAs (hsa_circ_0073244, circEDIL3) was found to be significantly expressed in normal synovial mesenchymal stem cells (SMSCs). Under IL-6-induced inflammation, SMSCs-derived exosomes circEDIL3 reduced the level of VEGF secreted by RA-FLS in a dose-dependent manner. Using online tools to predict miRNA-mRNA interactions, we found that circEDIL3 targets miR-485-3p and predicts its targeted activation of STAT3 protein inhibitors (signal transducer and activator of transcription 3) [PIAS3]. PIAS3 belongs to the small Rho GTPase family and is the main cell inhibitor of STAT3. Strikingly, our experiments showed that in the CIA model, the exosomes circEDIL3 derived from SMSC can target the VEGF functional module through the miR-485-3p/PIAS3/STAT3 axis. Therefore, circRNAs may be a new class of RA therapeutics, and the secreted circEDIL3 can be transferred to RA-FLS with complete functional activity through the new bioengineering carrier SMSCs-Exos. This new system may help alleviate disease progression by reducing inflammation-induced angiogenesis. It has great therapeutic potential for RA and possibly other diseases.

The isolation and expansion of synovial mesenchymal stem cells (SMSCs) are as previously reported. 14 In short, synovial tissues from human donors of different ages (average 44 years old, range 30-66 years old) are based on the ethics committee of the First Affiliated Hospital of Harbin Medical University, and signed consent from the donor. The synovium was digested with collagenase (Worthington Biochemical, Lakewood, NJ, USA) overnight at 37°C, and the cells were collected and cultured in high glucose DMEM supplemented with 10% FBS (HyClone, Logan, UT, USA). The cells obtained after 14 days were designated as passage 0. After another 14 days, single-cell-derived colonies were obtained and merged together. The cells were identified as SMSCs by flow cytometry (FCM) and antibodies against CD105, CD44, CD90, CD73, CD11b, CD14 and CD34 (BD Biosciences). We used a specific differentiation medium (CTCC, Wuxi, China) to further confirm the MSC through adipogenesis, osteogenic and chondrogenic analysis. In this study, cells from passage 4-7 were used.

According to previous reports, the exosomes in the SMSC medium were separated by differential centrifugation. 15 In short, after culturing SMSC in exosomal cell growth medium (SBI System Biosciences) for 48 hours, centrifuge the medium at 3000×g for 30 minutes to remove cell debris, and collect the supernatant, 16,500 × g Centrifuge for 30 minutes. Finally, the supernatant was centrifuged at 120,000 × g for 120 minutes, and the exosomes were harvested and stored at -80 °C.

For a traditional transmission electron microscope (TEM), the exosomal particles are placed in a carbon-coated copper grid, stained with 2% uranyl acetate, and observed with a transmission electron microscope (Libra 200 FE, Zeiss, Germany). Exosomes were also identified by Nanoparticle Tracking Analysis (NTA) and Western Blot analysis using Zetasizer Nano ZS (Malvern, UK).

RA fibroblast-like synovial cells (RA-FLS) were purchased from Cell Applications (San Diego, CA, USA) and stored in 10% FBS DMEM. Human dermal microvascular endothelial cells (HDMEC) were obtained from ScienCell Research Laboratories (6076 Corte Del Cedro, Carlsbad, CA, USA) and were cultured in Endothelial Cell Growth Medium (EGM)-2 from the same company. Cells from passage 3-6 were used in the experiment. The co-cultivation system of RA-FLS and HDMECs was established as described above. 4 In short, RA-FLS (4×105 cells/mL) and HDMECs (ranging from 2×105 cells/mL to 3×105 cells/mL) were respectively seeded in the lower chamber of the Transwell device (Costar) And the upper chamber, and cultured in cell growth medium (SBI) without exosomes. As mentioned earlier, the culture was stimulated with IL-6/sIL-6R complex (IL-6 100 ng/mL, sIL-6R 100 ng/mL). 16 Then the exosomes from SMSCs (SMSCs-Exos, 100 µg) were added to the co-culture. After 48 hours of incubation in 1% FBS DMEM, the supernatant was collected and centrifuged at 1000 rpm to remove cell debris, and further centrifuged at 120,000 × g for 120 minutes to remove exosomes. A part of the culture supernatant was removed for subsequent Transwell test, tube formation test, and isolated aortic ring angiogenesis test. The remaining supernatant is frozen at -20°C until VEGF analysis with a commercial ELISA kit (R&D Systems). The mRNA and protein expression in the co-cultured RA-FLS were determined by quantitative real-time PCR (qRT-PCR) and Western blotting, respectively.

The Transwell measurement and the aortic ring measurement were performed as described above. 4 In short, for the Transwell assay, 600 µL of the supernatant from the co-culture is placed in the lower chamber. Add HDMEC (3×104 cells) to the upper chamber and incubate for 6 hours. The cells that migrated to the bottom of the Transwell membrane were stained and quantified (LEICA DMi8). Aortic rings isolated from 8-week-old C57BL/6 mice (SPF, Experimental Animal Center, Second Affiliated Hospital of Harbin Medical University) were incubated in 1% FBS EGM-2 on Matrigel for 3 days. After verifying the budding of the aortic ring, EGM-2 was replaced with 500 μL of fresh supernatant from the co-culture of RA-FLS and HDMEC, and the aortic ring was incubated for another 3 days. Check the microvessel growth with a microscope (LEICA DMi8) and analyze it with ImageJ software. In the tube formation test, a μ slide (ibidi, GmbH, Munich, Germany) was used to study angiogenesis. Each well was coated with 10 μL Matrigel (Corning, New York, USA), and 70 μL of cell suspension containing 1×104 cells was added to each well. After 6 hours of incubation, a phase-contrast microscope (LEICA DMi8) was used to evaluate the formation of endothelial cell tubes.

PKH67 Green Fluorescent Cell Linker Kit (Sigma) is used to stain exosomes. Suspend the exocrine body in 100 μL diluent C, mix with 100 μL PKH67 dye solution (4×10-6 M), and incubate for 1-5 minutes. Add 200 μL of exosome-free serum to stop the reaction. The labeled exosomes were then washed twice with PBS by centrifugation (100,000 xg, 1 hour) and incubated with the receptor RA-FLS for 6 hours, and then imaged under a confocal laser microscope (LEICA TCS SP8).

As mentioned earlier, TRIzol reagent (Invitrogen) was used to extract total RNA from cultured cells. 17 To isolate exosomes-RNA, after transferring the upper aqueous phase to a new EP tube, add Acryl Carrier (Solarbio, Beijing, China) and isopropanol. For circRNA detection, total RNA was incubated with RNase R (Geneseed Biotech, Guangzhou, China). PrimeScript RT Master Mix (Takara Biotechnology, Dalian, China) was used to synthesize cDNA for mRNA and circRNA analysis, and iTaq Universal SYBR Green Supermix (Bio-Rad) was used for qRT-PCR amplification. For miRNA detection, riboSCRIPTTM Reverse Transcription Kit (RiboBio, Guangzhou, China) was used for cDNA synthesis, and Applied Biosystems TaqMan MicroRNA Assay Kit was used for qRT-PCR. Use the 2-ΔΔCt method to normalize relative circRNA, mRNA, or miRNA expression to GAPDH or U6 snRNA levels. Each sample was tested in triplicate. The sequence of each primer is listed in Supplementary Table 1.

Fluorescence in situ hybridization (FISH) was performed using a fluorescence in situ hybridization kit (RiboBio, Guangzhou, China). In short, after treatment in the pre-hybridization buffer, the RA-FLS grown on the coverslip is combined with the CY3-labeled circRNA probe (RiboBio, Guangzhou, China, Supplementary Table 1) in the hybridization buffer in a humid and dark environment. Incubate in liquid for 12-16 hours. Cell nuclei were stained with DAPI, and the intracellular localization of circRNA was observed using TCS SP8 X laser confocal microscope (LEICA).

The miRNA targets of circEDIL3 are predicted by the bioinformatics database TargetScan (http://www.targetscan.org/) and CircInteractome (https://circinteractome.nia.nih.gov/). The mRNA target of miR-485-3p is predicted by TargetScan (http://www.targetscan.org/) and Starbase (http://starbase.sysu.edu.cn/).

Ad-Vectors encoding circEDIL3 (Ad-circEDIL3), PIAS3 (Ad-PIAS3), STAT3 (Ad-STAT3) and GFP control were prepared by Sino Biotech (Shanghai, China). In short, the circEDIL3, PIAS3 and STAT3 fragments were amplified and connected to the linearized vector, and the amplified sequence was verified by Sanger sequencing. Use Lipofectamine 3000 Transfection Reagent (Thermo Fisher) to co-transfect the packaging plasmid and viral vector into HEK-293T cells. Forty-eight hours after transfection, the medium was centrifuged and supplemented with polybrene (SolarBio). Finally, the medium mixed with polybrene is added to the target cells for infection. CircEDIL3 overexpression was established in SMSC and RA-FLS. PIAS3 and STAT3 overexpression were established in RA-FLS.

The pre-designed siRNA targeting human circEDIL3 (si-circEDIL3) was produced by GenePharma (Shanghai, China, Supplementary Table S1) and transfected into RA-FLS using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The cells were grown for 48 hours after transfection and harvested. Vectors (miRNA mimics and miRNA inhibitors) that up-regulate or down-regulate relative miRNA expression were designed and constructed by RiboBio (Guangzhou, China).

For the biotin-conjugated circRNA pull-down assay, the probe is designed to bind to the junction region of circEDIL3 and is synthesized by GenePharma (Shanghai, China). RA-FLS was washed in ice-cold PBS, lysed in lysis buffer, and incubated with 3 µg biotinylated probe against circEDIL3 sequence or negative control. Incubate the cell lysate with biotinylated probe with 50 µL streptavidin-coupled magnetic beads (Invitrogen, USA), and add TRIzol reagent (Invitrogen, Thermo, USA) to the washed magnetic beads To extract RNA. RNA is reverse transcribed and analyzed by qRT-PCR analysis.

For biotin-coupled miRNA capture, 3'-end biotinylated miR-485-3p mimic or control RNA (RiboBio, Guangzhou, China) was transfected into RA-FLS at a final concentration of 50 nM. After 48 hours, the cells were harvested and the cell pellet was lysed in a lysis buffer containing a complete protease inhibitor cocktail. The abundance of circEDIL3 and PIAS3 mRNA in the binding part pulled down by streptavidin-coated magnetic beads was evaluated by qRT-PCR analysis.

The expression plasmid of circEDIL3 was generated by inserting the entire human circEDIL3 sequence into pcDNA3.1 circRNA Mini Vector (Addgene). Part of the GFP fragment was amplified by PCR and cloned into pcDNA3.1 circRNA Mini Vector as a control plasmid. RA-FLS was transfected with miR-485-3p mimic (RiboBio, China) or a control mimic in combination with a luciferase reporter gene or an empty vector. The cells were also transfected with pcDNA3.1-circEDIL3 and its mutants. Insert the PIAS3 gene 3ʹ-UTR containing the miR-485-3p target site or its mutant sequence into the pGL3 promoter vector. According to the manufacturer's instructions, use riboFECTTM CP kit (RiboBio, China) to transfect MiR-485-3p mimics/inhibitors and their respective negative controls into RA-FLS at a final concentration of 100 nM. 24 hours after transfection, luciferase activity was measured using Dual-Glo Luciferase Assay System (Promega, Madison, Wisconsin) and Cytation 5 cell imaging multi-mode reader (BioTek). Each sample was prepared three times, and the entire experiment was repeated three times.

Protein extraction and Western blot analysis were performed as described previously. 17 Antibodies against specific proteins, including VEGF (Abcam, ab214424), PIAS3 (Cell Signaling Technology, 9042), p-STAT3 (Cell Signaling Technology, 9145), STAT3 (Cell Signaling) using Technology, 12640), CD63 (Abcam , ab134045) and TSG101 (Abcam, ab125011).

All animal experiments were performed in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines, and were approved by the Animal Care and Use Institutional Committee of the First Affiliated Hospital of Harbin Medical University. As mentioned earlier, male DBA/1J mice (8 weeks old, weight 19 g ± 2 g, SPF, SLAC, Shanghai, China) were used to establish the CIA model. 4 In short, bovine type II collagen (CII, Chondrex) is emulsified in an equal volume of complete Freund's adjuvant (CFA) (Chondrex). Each DBA/1J mouse was injected with 100 μL emulsion containing 100 μg CII at the base of the tail by intradermal injection. On the 21st day after the initial immunization, the mice received a booster injection of 100 μg CII, which was emulsified with an equal volume of incomplete Freund's adjuvant (Chondrex). After inducing arthritis (day 21), the mice were divided into the following groups (n = 5 for each experimental group): CIA control, SMSCs-Exo, Ad-circEDIL3-SMSCs-Exo, Ad-Vector-SMSCs-Exo ( CIA mice treated with designated exosomes at a dose of 100 μg suspended in 10 μL PBS twice a week), methotrexate (MTX, CIA treated with MTX at a dose of 1.5 mg/kg/week) Mice, as a treatment control) and a normal control group (not immunized). After the first immunization, intra-articular treatment continued from day 26 to day 39. Normal and CIA model control mice were given equal volumes of PBS. From day 24 to day 39, the weight of the mice was carefully monitored every three days.

From the 21st day after the initial immunization, the clinical arthritis will be evaluated every day, and the arthritis score will be evaluated every 3 days according to the standard scoring system until the end of the experimental period. 4 At the end of this study (day 40 after the initial injection of collagen), micro-CT imaging (Quantum GX, Perkin Elmer, Waltham, USA) was performed to study the effect of exosomes on three-dimensional (3D) bone structure. Influence. Caliper Analyze software (Analyze Direct, Kansas, USA) was used to calculate the average CT value of the hind paw and knee joint to assess bone loss.

After euthanasia, the knee joints of the mice were collected and stained with hematoxylin-eosin (H&E). In order to quantitatively assess the severity of arthritis, a scoring system was adopted according to the reported protocol. 4 The immunohistochemical analysis was performed as described earlier, with some modifications. 4 Knee joint sections on glass slides with anti-PIAS3, p-STAT3, STAT3 or VEGF (Wan Lei Biological, Shenyang, China). Subsequently, the sections were stained with the polymer HRP detection system (PV9001, ZSGB-BIO, Beijing, China) and visualized with the DAB peroxidase substrate kit (ZLI-9017, ZSGB-BIO, Beijing, China). After immunostaining, the joint synovial area of ​​each section was evaluated in three randomly selected areas under a microscope (LEICA DMi8, Germany) at 100x magnification. Image-Pro Plus 6 (Media Cybernetics, Inc.) is used to analyze the average integrated optical density (IOD) according to the previously described protocol. 2

Data are expressed as mean ± SEM. Statistical analysis was performed by using SPSS version 17.0 software using Student's t test or one-way analysis of variance (ANOVA) as appropriate. A value of p <0.05 is considered statistically significant.

We isolated synovial MSCs (SMSC) from humans and confirmed the MSC characteristics of the selected clones through differentiation assays (Supplementary Figure 1A-C). SMSC is also identified by flow cytometry (FCM) analysis and specific cell surface markers (Supplementary Figure 2). Extract exosomes from SMSCs (SMSCs-Exos) from conditioned cell culture medium supplemented with FBS without exosomes. The morphology and size were checked by transmission electron microscopy (TEM) (Figure 1A) and Nanoparticle Tracking Analysis (NTA) (Figure 1B). Exosomes were further confirmed by the presence of protein markers (such as CD63 and TSG101) and the absence of the negative marker GAPDH (Figure 1C). The fusion of PKH67-labeled SMSCs-Exos and RA-FLS was confirmed by fluorescence microscopy (Figure 1D). Then we studied the effect of SMSCs-Exos on RA-FLS-induced angiogenesis. In order to simulate the microenvironment of synovial tissue inflammation, IL-6 and its soluble receptor (sIL-6R) were added to the co-culture of RA-FLS and HDMECs, and the effect of SMSCs-Exos on RA-FLS-induced angiogenesis Were studied. After 48 hours of incubation with IL-6+sIL-6R, VEGF mRNA (Figure 1E) and protein expression (Figure 1F) in the co-cultured RA-FLS were significantly up-regulated, and this up-regulation was significantly inhibited by SMSCs-Exos. However, in the absence of IL-6+sIL-6R treatment, SMSCs-Exos did not significantly alter the expression of VEGF. The expression of VEGF protein in the supernatant of the co-cultured RA-FLS and HDMECs showed a similar trend to that of the cell lysate of the co-cultured RA-FLS (Figure 1G). Next, we asked whether the VEGF secreted from human RA-FLS into the co-culture supernatant would promote the angiogenic activity of HDMECs. HDMECs were treated with the supernatant from the co-culture, and the vascular structure formed by the treated cells was carefully examined after the exosomes were removed. HDMECs showed significant changes in migration by Transwell test (Figure 1H), tube formation test showed capillary structure formation (Figure 1I), and in vitro aortic ring angiogenesis test showed capillary sprouting (Figure 1J). These observations are consistent with the changes in VEGF expression in the supernatant from the co-culture of RA-FLS and HDMEC. Figure 1 SMSCs-Exos inhibited the up-regulated angiogenesis induced by RA-FLS under inflammatory conditions. (A) SMSCs-derived exosomes (SMSCs-Exos) were confirmed to be 150 nm or smaller in diameter by TEM and showed a characteristic lipid bilayer. (B) The average diameter of SMSCs-Exos is about 100 nm, as determined by the nanoparticle tracker. (C) It was confirmed by Western blot analysis that the specific markers of traditional exosomes CD63 and TSG101 were positive in SMSCs-Exos, and the levels of these markers in SMSCs-Exso were higher than in SMSCs. GAPDH in SMSCs-Exos is negative. (D) SMSCs-Exos were labeled with PKH67 (green) and co-cultured with RA-FLS for 6 hours. Fluorescence microscopy analysis showed that the stained exosomes could be endocytosed into cells. (E and F) After incubating RA-FLS and SMSCs-Exos for 48 hours under the induction of IL-6+sIL-6R, the expression of VEGF mRNA (E) and protein (F) in the co-cultured RA-FLS was passed through qRT- These are PCR and Western blotting. After IL-6+sIL-6R induction, RA-FLS co-cultured VEGF mRNA and protein expression levels were significantly up-regulated, and SMSCs-Exos significantly rescued these up-regulations. (G) Analyze the VEGF concentration of the supernatant by ELISA. It was observed that the change trend of VEGF secretion in the supernatant of the co-culture of RA-FLS and HDMECs was similar to the expression of VEGF in the co-culture of RA-FLS. (H, I and J) The 6-hour Transwell test (H) and tube formation test (I) and the isolated aortic ring angiogenesis test (J) showed that the migration of HDMEC, capillary-like structure formation and microvessel sprouting occurred. The changes were consistent with the expression of VEGF in the supernatants from the co-cultures of RA-FLS and HDMECs, respectively. The results are expressed as mean±SEM n = 3, each group; *p <0.05, **p <0.01, ***p <0.001. CTL=PBS control. IL-6+sIL-6R = IL-6100 ng/mL+ sIL-6R 100 ng/mL. Abbreviation: Veh, vehicle control.

Figure 1 SMSCs-Exos inhibits RA-FLS-induced up-regulation of angiogenesis under inflammatory conditions. (A) SMSCs-derived exosomes (SMSCs-Exos) were confirmed to be 150 nm or smaller in diameter by TEM and showed a characteristic lipid bilayer. (B) The average diameter of SMSCs-Exos is about 100 nm, as determined by the nanoparticle tracker. (C) It was confirmed by Western blot analysis that the specific markers of traditional exosomes CD63 and TSG101 were positive in SMSCs-Exos, and the levels of these markers in SMSCs-Exso were higher than in SMSCs. GAPDH in SMSCs-Exos is negative. (D) SMSCs-Exos were labeled with PKH67 (green) and co-cultured with RA-FLS for 6 hours. Fluorescence microscopy analysis showed that the stained exosomes could be endocytosed into cells. (E and F) After incubating RA-FLS and SMSCs-Exos for 48 hours under the induction of IL-6+sIL-6R, the expression of VEGF mRNA (E) and protein (F) in the co-cultured RA-FLS was passed through qRT- These are PCR and Western blotting. After IL-6+sIL-6R induction, RA-FLS co-cultured VEGF mRNA and protein expression levels were significantly up-regulated, and SMSCs-Exos significantly rescued these up-regulations. (G) Analyze the VEGF concentration of the supernatant by ELISA. It was observed that the change trend of VEGF secretion in the supernatant of the co-culture of RA-FLS and HDMECs was similar to the expression of VEGF in the co-culture of RA-FLS. (H, I and J) The 6-hour Transwell test (H) and tube formation test (I) and the isolated aortic ring angiogenesis test (J) showed that the migration of HDMEC, capillary-like structure formation and microvessel sprouting occurred. The changes were consistent with the expression of VEGF in the supernatants from the co-cultures of RA-FLS and HDMECs, respectively. The results are expressed as mean±SEM n = 3, each group; *p <0.05, **p <0.01, ***p <0.001. CTL=PBS control. IL-6+sIL-6R = IL-6100 ng/mL+ sIL-6R 100 ng/mL.

Compared with the differentiated stromal cell counterparts, the 10 most up-regulated circRNAs in MSCs have been previously reported. 18 Using qRT-PCR, it was confirmed that the expression levels of 4 circRNA in SMSC were significantly higher than RA-FLS (Supplementary Figure 3A). All 4 are different from linear RNA or actin. CircRNA is resistant to RNase R digestion (Supplementary Figure 3B). We further confirmed that hsa_circ_0073244 (circEDIL3) is the most highly expressed circRNA among the above four circRNAs in SMSCs-Exos (Supplementary Figure 4A). The characterization of hsa_circ_0073244 is shown in Supplementary Figure 4B. Therefore, we suspect that circRNA can be transferred to RA-FLS as cargo in SMSCs-Exos, and may explain the biological functions of SMSCs-Exos.

In order to explore whether circRNA derived from SMSCs can affect the secretion of VEGF from synovial cells, SMSCs (Ad-circEDIL3-SMSCs) overexpressing circEDIL3 were constructed through adenovirus infection, and SMSCs-Exos were extracted from the culture supernatant. qRT-PCR confirmed the overexpression of circEDIL3 (Supplementary Figure 4C). Carefully extract exosomes from the culture supernatant of Ad-circEDIL3 transfected SMSCs (Ad-circEDIL3-SMSCs-Exos). The expression of circEDIL3 was significantly increased in Ad-circEDIL3-SMSCs-Exos (Supplementary Figure 4D). Ad-circEDIL3-SMSCs-Exos and its corresponding vector control (Ad-Vector-SMSCs-Exos) were added to the co-culture of RA-FLS and HDMECs, with/without IL-6+sIL-6R. We found that compared with Ad-Vector-SMSCs-Exo treatment, Ad-circEDIL3-SMSCs-Exo treatment significantly reduced the levels of VEGF mRNA and protein in RA-FLS and the VEGF concentration in the supernatant of the co-culture. It was induced with IL-6+sIL-6R; however, in the absence of IL-6+sIL-6R, there was no significant change in VEGF levels after Ad-circEDIL3-SMSCs-Exo treatment (Figure 2A-C). In summary, we examined circEDIL3 in SMSCs-Exos and proved that circEDIL3 is abundant and stable in SMSCs-Exos, and this circular RNA may be transferred to RA-FLS by SMSCs-Exos to further inhibit the IL-6-induced RA-FLS Inflammatory environment. Figure 2 SMSC-derived exosomes circEDIL3 inhibited the secretion of VEGF produced by RA-FLS, and the interaction of circEDIL3 with miR-485-3p targeting PIAS3. Transfect Ad-circEDIL3 or its corresponding vector control (Ad-Vector) into SMSCs, and transfer circEDIL3 overexpression cells (Ad-circEDIL3-SMSCs-Exos) or vector control cells (Ad-Vector-SMSCs-Exos) outside Extrinsic culture media. Under the induction of IL-6/sIL-6R complex, Ad-circEDIL3-SMSCs-Exos and Ad-Vector-SMSCs-Exos can be endocytosed into RA-FLS after 6 hours of co-cultivation. The expression of CircEDIL3 in exosomes was detected by qRT-PCR. (AC) The levels of VEGF mRNA (A) and protein (B) in the RA-FLS co-cultured with Ad-circEDIL3-SMSCs-Exo and the V EGF concentration in the supernatant (C) were significantly lower than those of the control group. Ad-Vector-SMSCs-Exo group induced by IL-6+sIL-6R. (D) The qRT-PCR test showed that compared with the control groups, circEDIL3 knockdown significantly increased the expression of miR-485-3p, while overexpression of circEDIL3 significantly reduced the expression of miR-485-3p. (E) Compared with each control group, miR-485-3p mimic/inhibitor can significantly reduce/enhance the level of circEDIL3. (F) RA-FLS is co-transfected with the firefly luciferase reporter gene containing wild type (WT) or mutant (Mut) circEDIL3, with miR-485-3p mimic and corresponding control. Compared with the control reporter gene or mutant luciferase reporter gene, when co-transfected with wild circEDIL3 and miR-485-3p mimics, the relative luciferase activity of circEDIL3 was significantly inhibited. (G) The pull-down analysis showed that, compared with the control probe, the biotin-labeled circEDIL3 specific probe produced a significant amount of miR-485-3p. (H) qRT-PCR analysis showed that compared with the control group, circEDIL3 and PIAS3 mRNA captured by biotin-miR-485-3p were significantly enriched. (I) The luciferase reporter gene test showed that compared with the control reporter gene or the mutant luciferase reporter gene, when transfected with miR-485-3p mimic, the luciferase activity of PIAS3 wild-type reporter gene was significant reduce. The results are expressed as mean±SEM n = 3, each group; *p <0.05, **p <0.01, ***p <0.001. CTL=PBS control. Veh = vehicle control. si-scrl=scramble siRNA. IL-6+sIL-6R = IL-6100 ng/mL + sIL-6R 100 ng/mL.

Figure 2 SMSC-derived exosomes circEDIL3 inhibited the secretion of VEGF produced by RA-FLS, and the interaction of circEDIL3 with miR-485-3p targeting PIAS3. Transfect Ad-circEDIL3 or its corresponding vector control (Ad-Vector) into SMSCs to isolate circEDIL3 overexpression cells (Ad-circEDIL3-SMSCs-Exos) or vector control cells (Ad-Vector-SMSCs-Exos) Exosomes culture media. Under the induction of IL-6/sIL-6R complex, Ad-circEDIL3-SMSCs-Exos and Ad-Vector-SMSCs-Exos can be endocytosed into RA-FLS after 6 hours of co-cultivation. The expression of CircEDIL3 in exosomes was detected by qRT-PCR. (AC) The levels of VEGF mRNA (A) and protein (B) in the RA-FLS co-cultured with Ad-circEDIL3-SMSCs-Exo and the V EGF concentration in the supernatant (C) were significantly lower than those of the control group. Ad-Vector-SMSCs-Exo group induced by IL-6+sIL-6R. (D) The qRT-PCR test showed that compared with the control groups, circEDIL3 knockdown significantly increased the expression of miR-485-3p, while overexpression of circEDIL3 significantly reduced the expression of miR-485-3p. (E) Compared with each control group, miR-485-3p mimic/inhibitor can significantly reduce/enhance the level of circEDIL3. (F) RA-FLS is co-transfected with the firefly luciferase reporter gene containing wild type (WT) or mutant (Mut) circEDIL3, with miR-485-3p mimic and corresponding control. Compared with the control reporter gene or mutant luciferase reporter gene, when co-transfected with wild circEDIL3 and miR-485-3p mimics, the relative luciferase activity of circEDIL3 was significantly inhibited. (G) The pull-down analysis showed that, compared with the control probe, the biotin-labeled circEDIL3 specific probe produced a significant amount of miR-485-3p. (H) qRT-PCR analysis showed that compared with the control group, circEDIL3 and PIAS3 mRNA captured by biotin-miR-485-3p were significantly enriched. (I) The luciferase reporter gene test showed that compared with the control reporter gene or the mutant luciferase reporter gene, when transfected with miR-485-3p mimic, the luciferase activity of PIAS3 wild-type reporter gene was significant reduce. The results are expressed as mean±SEM n = 3, each group; *p <0.05, **p <0.01, ***p <0.001. CTL=PBS control. Veh = vehicle control. si-scrl=scramble siRNA. IL-6+sIL-6R = IL-6100 ng/mL + sIL-6R 100 ng/mL.

Recent studies have shown that the main regulatory mechanism of circRNA is to act as a miRNA sponge, which mainly occurs in the cytoplasm. 7 Fluorescence in situ hybridization (FISH) results show that most of the circEDIL3 transcription signal is located in the cytoplasm. SMSCs have some hybridization signals in the nuclear region (Supplementary Figure 5). Since circEDIL3 has been confirmed to be located in the cytoplasm, we want to know whether circEDIL3 can also act as a miRNA sponge. Use Arraystar's proprietary algorithm based on public databases TargetScan and miRanda to search for microRNAs "sponged" by circEDIL3, and select the highest-ranked candidate miRNA. At least one miRNA binding site identified by Arraystar's proprietary algorithm was confirmed using the CircInteractome database (Supplementary Figure 6A). In order to verify whether the selected miRNA can interact with circEDIL3, a luciferase reporter gene test was performed. The sequence of circEDIL3 is inserted directly downstream of the Renilla luciferase reporter gene, and each candidate interacting miRNA is co-transfected with the luciferase reporter gene into RA-FLS. Compared with the negative control, miR-485-3p, miR-198 and miR-326 reduced Renilla luciferase reporter gene activity (Supplementary Figure 6B). Next, the level of circEDIL3 was manipulated in RA-FLS, and the efficiency of knockdown or overexpression was confirmed (Supplementary Figure 6C and D). qRT-PCR analysis showed the opposite trend of miR-485-3p and circEDIL3 expression (Figure 2D); other candidate miRNAs also had similar effects, but miR-485-3p showed the most significant effect (Supplementary Figure 6E). The interaction of circEDIL3 with its potential target miR-485-3p has been further confirmed. Compared with the respective controls, miR-485-3p mimics/inhibitors significantly reduced/enhanced the expression level of circEDIL3 (Figure 2E). Next, the miRNA target site in the circEDIL3 sequence was mutated (Supplementary Figure 7A), and the Renilla luciferase assay was repeated. No significant changes in luciferase levels were observed, demonstrating the specificity of the interaction between circEDIL3 and miR-485-3p target sites (Figure 2F). The pull-down analysis of biotin-labeled oligonucleotides complementary to the reverse splicing connection of circEDIL3 verified the direct interaction between miR-485-3p and circEDIL3 in its natural loop structure. Consistent with the Renilla luciferase reporter gene detection, the circEDIL3 specific probe instead of the control probe can reduce a large amount of miR-485-3p (Figure 2G). We also used miR-485-3p labeled with 3ʹ end biotin to pull down circRNA, and confirmed the significant enrichment of circEDIL3 in the pulled down product (Figure 2H). Bioinformatics database StarBase predicts that the 3ʹ UTR of human PIAS3 contains a putative miR-485-3p binding site (Supplementary Figure 6A). The luciferase plasmids pGL3-PIAS3-WT and pGL3-PIAS3-Mut (Supplementary Figure 7B) were constructed and co-transfected with miR-485-3p mimics into RA-FLS. The luciferase activity of the wild-type PIAS3 reporter gene was inhibited, but the activity of the mutant luciferase reporter gene remained unchanged (Figure 2I). In addition, we used a biotinylated RNA pull-down assay to confirm the results. Compared with the control, the PIAS3 mRNA captured by biotin-miR-485-3p increased significantly (Figure 2H). The expression of PIAS3 mRNA in RA-FLS was determined by qRT-PCR. As shown in Figure 3A and B, the expression of PIAS3 mRNA and protein was strongly inhibited/promoted by miR-485-3p mimics/inhibitors in RA-FLS. In summary, PIAS3 may be a potential target gene of miR-485-3p, and circEDIL3 can regulate PIAS3 by adsorbing miR-485-3p. Figure 3 CircEDIL3 regulates p-STAT3/STAT3 activity through miR-485-3p/PIAS3 pathway. (A) MiR-485-3p mimics/inhibitors decrease/increase PIAS3 mRNA expression in RA-FLS. There was no significant change in STAT3 mRNA expression. (C) PIAS3 overexpression was applied on the basis of miR-485-3p mimic processing. The down-regulation of PIAS3 mRNA by miR-485-3p mimics can be partially restored by PIAS3 overexpression. In other words, PIAS3 overexpression significantly increases PIAS3 mRNA expression, and this up-regulation can be significantly suppressed by miR-485-3p mimics. However, STAT3 mRNA expression remained stable. (E) Apply CircEDIL3 knockdown based on the management of miR-485-3p inhibitors. The upregulation of PIAS3 mRNA by miR-485-3p inhibitors can be significantly rescued by knocking down circEDIL3. In other words, circEDIL3 knockdown significantly reduced PIAS3 mRNA expression, and this down-regulation could be significantly suppressed by miR-485-3p inhibitors, while STAT3 mRNA levels did not change significantly. (B, D, F) The change trend of PIAS3 protein expression is consistent with PIAS3 mRNA level, while the change trend of STAT3 activity is opposite, that is, the change trend of p-STAT3 protein level is opposite to the change trend of PIAS3 protein expression, and the level of STAT3 protein does not change significantly. The results are expressed as mean±SEM n = 3, each group; *p <0.05, **p <0.01, ***p <0.001. CTL=PBS control. si-scrl=scramble siRNA.

Figure 3 CircEDIL3 regulates p-STAT3/STAT3 activity through miR-485-3p/PIAS3 pathway. (A) MiR-485-3p mimics/inhibitors decrease/increase PIAS3 mRNA expression in RA-FLS. There was no significant change in STAT3 mRNA expression. (C) PIAS3 overexpression was applied on the basis of miR-485-3p mimic processing. The down-regulation of PIAS3 mRNA by miR-485-3p mimics can be partially restored by PIAS3 overexpression. In other words, PIAS3 overexpression significantly increases PIAS3 mRNA expression, and this upregulation can be significantly suppressed by miR-485-3p mimics. However, STAT3 mRNA expression remained stable. (E) Apply CircEDIL3 knockdown based on the management of miR-485-3p inhibitors. The upregulation of PIAS3 mRNA by miR-485-3p inhibitors can be significantly rescued by knocking down circEDIL3. In other words, circEDIL3 knockdown significantly reduced PIAS3 mRNA expression, and this down-regulation could be significantly suppressed by miR-485-3p inhibitors, while STAT3 mRNA levels did not change significantly. (B, D, F) The change trend of PIAS3 protein expression is consistent with PIAS3 mRNA level, but the change trend of STAT3 activity is opposite, that is, the change trend of p-STAT3 protein level is opposite to the change trend of PIAS3 protein expression, and there is no significant change in STAT3 protein level. The results are expressed as mean±SEM n = 3, each group; *p <0.05, **p <0.01, ***p <0.001. CTL=PBS control. si-scrl=scramble siRNA.

PIAS3 can bind to p-STAT3 and antagonize the function of STAT3, which is a key transcription factor for VEGF. Therefore, we speculate that circEDIL3 may regulate STAT3 activity through the miR-485-3p/PIAS3 axis. To test this hypothesis, the protein level of PIAS3 and the p-STAT3/STAT3 ratio were checked by Western blot analysis when miR-485-3p was overexpressed or knocked down. As shown in Figure 3B, when miR-485-3p mimics were added, PIAS3 protein expression was significantly reduced, while the opposite effect was observed on p-STAT3 expression; however, the protein level of STAT3 did not change significantly, indicating STAT3 activity Rise, not protein levels. In addition, miR-485-3p inhibitors have opposite effects on PIAS3 protein expression and STAT3 activity.

Next, PIAS3 was overexpressed in cells treated with miR-485-3p mimics. PIAS3 was successfully overexpressed at the mRNA and protein levels (Figure 3C and D), despite being inhibited by miR-485-3p mimics, PIAS3 protein expression was still up-regulated by PIAS3 overexpression. The increase in p-STAT3 induced by miR-485-3p mimics was blocked by PIAS3 overexpression, and there was no significant change in STAT3 protein levels.

Finally, the cells were treated with a combination of miR-485-3p inhibitor treatment and circEDIL3 knockdown. Si-circEDIL3 inhibited the increase in PIAS3 protein expression and the decrease in p-STAT3 expression induced by miR-485-3p inhibitors, while the expression of STAT3 protein did not change significantly (Figure 3F), indicating that the down-regulation of circEDIL3 restored STAT3 activity.

PIAS3 and STAT3 mRNA expression levels were also determined by qRT-PCR. The results showed that PIAS3 protein expression changed in parallel with its mRNA expression; however, regardless of whether miR-485-3p mimics/inhibitors, PIAS3 overexpression, and circEDIL3 knockdown were administered alone or in combination, STAT3 mRNA levels did not change significantly (Figure 3A, C and E).

Transfect Ad-circEDIL3 or its corresponding negative control (Ad-Vector) into SMSCs, and isolate exosomes from the culture medium to deliver circEDIL3 (Ad-circEDIL3-SMSCs-Exos) or vector (Ad-Vector-SMSCs- Exos). The results showed that regardless of the induction of IL-6+sIL-6R, Ad-circEDIL3-SMSCs-Exos inhibited the expression of miR-485-3p, increased PIAS3 mRNA, and maintained stable STAT3 mRNA levels. A similar effect on miR-485-3p/PIAS3/STAT3 mRNA expression was observed in the SMSCs-Exos-treated samples, but not in the PBS-treated control (Figure 4A-C). Figure 4 SMSCs-Exos circEDIL3 directly regulates the expression of VEGF through the miR-485-3p/PIAS3/STAT3 axis. (AC) qRT-PCR showed that, regardless of IL-6+sIL-6R induction, Ad-circEDIL3-SMSCs-Exos inhibited miR-485-3p (A) and promoted PIAS3 mRNA with stable STAT3 mRNA (B). C) Compared with the level of SMSCs-Exo or Ad-Vector-SMSCs-Exo in RA-FLS. Compared with the PBS group, the SMSCs-Exo treatment group showed similar changes in miR-485-3p/PIAS3/STAT3 mRNA expression. (D) Western blot analysis showed that compared with the SMSCs-Exos or Ad-Vector-SMSCs-Exos group under IL-6+, Ad-circEDIL3-SMSCs-Exos increased the expression of PIAS3 protein and decreased the expression of p-STAT3 and STAT3 protein. Stable sIL-6R induction. Compared with the PBS group, the SMSCs-Exo treatment group showed similar changes in PIAS3/p-STAT3/STAT3 protein expression. (E, F) Overexpression of STAT3 significantly increased the expression of VEGF mRNA (E) and protein (F). And when IL-6+sIL-6R is induced, the increase in VEGF expression is more obvious. (G, H) Under the induction of IL-6+sIL-6R, under the treatment of Ad-Vector-SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos, the overexpression of STAT3 significantly upregulated VEGF mRNA (G) and protein expression ( H). The results are expressed as mean±SEM n = 3, each group; *p <0.05, **p <0.01, ***p <0.001. CTL=PBS control. Veh = vehicle control. IL-6+sIL-6R = IL-6100 ng/mL + sIL-6R 100 ng/mL.

Figure 4 SMSCs-Exos circEDIL3 directly regulates the expression of VEGF through the miR-485-3p/PIAS3/STAT3 axis. (AC) qRT-PCR showed that, regardless of IL-6+sIL-6R induction, Ad-circEDIL3-SMSCs-Exos inhibited miR-485-3p (A) and promoted PIAS3 mRNA with stable STAT3 mRNA (B). C) Compared with the level of SMSCs-Exo or Ad-Vector-SMSCs-Exo in RA-FLS. Compared with the PBS group, the SMSCs-Exo treatment group showed similar changes in miR-485-3p/PIAS3/STAT3 mRNA expression. (D) Western blot analysis showed that compared with the SMSCs-Exos or Ad-Vector-SMSCs-Exos group under IL-6+, Ad-circEDIL3-SMSCs-Exos increased the expression of PIAS3 protein and decreased the expression of p-STAT3 and STAT3 protein. Stable sIL-6R induction. Compared with the PBS group, the SMSCs-Exo treatment group showed similar changes in PIAS3/p-STAT3/STAT3 protein expression. (E, F) Overexpression of STAT3 significantly increased the expression of VEGF mRNA (E) and protein (F). And when IL-6+sIL-6R is induced, the increase in VEGF expression is more obvious. (G, H) Under the induction of IL-6+sIL-6R, under the treatment of Ad-Vector-SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos, the overexpression of STAT3 significantly upregulated VEGF mRNA (G) and protein expression ( H). The results are expressed as mean±SEM n = 3, each group; *p <0.05, **p <0.01, ***p <0.001. CTL=PBS control. Veh = vehicle control. IL-6+sIL-6R = IL-6100 ng/mL + sIL-6R 100 ng/mL.

Protein detection was performed by Western blotting. Interestingly, regardless of IL-6+sIL-6R induction, PIAS3 protein expression paralleled PIAS3 mRNA levels. After IL-6+sIL-6R induction, opposite effects on STAT3 activity and PIAS3 protein levels were observed (Figure 4D). However, in the absence of IL-6+sIL-6R induction, p-STAT3 protein levels remained stable, STAT3 activity, that is, the ratio of p-STAT3/STAT3 protein did not change significantly, while PIAS3 protein levels changed significantly (Supplementary Figure 8).

In order to explore the actual role of STAT3 in the regulation of VEGF expression, STAT3 was transfected into RA-FLS using adenoviral vectors to increase intracellular STAT3. In this case, we found that p-STAT3 (Supplementary Figure 9) and VEGF mRNA and protein expression increased significantly, regardless of IL-6+sIL-6R induction (Figure 4E and F). In addition, under the induction of IL-6+sIL-6R, Ad-STAT3 significantly rescued the down-regulation of VEGF expression induced by SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos (Figure 4G and H). These data further indicate that circEDIL3 can transfer across cells through SMSCs-Exos, and then promote PIAS3 as a miR-485-3p sponge, thereby up-regulating STAT3 activity, accelerating VEGF expression and subsequent angiogenesis induced by RA-FLS.

In order to further study the effect of exosomes circEDIL3 on arthritis in vivo, CIA mice were injected intra-articularly with Ad-circEDIL3-SMSCs-Exos, Ad-Vector-SMSCs-Exos or SMSCs-Exos. Exosomal treatment lasted from the 26th day to the 39th day after the first (initial) immunization. We evaluated the severity of arthritis according to the standard arthritis scoring system 4 from the 24th day after the initial immunization, and recorded the scores of CIA mice every 3 days until the end of the experiment. The micro-CT images were taken and evaluated on the 40th day. As shown in Figure 5A and B, after mice were immunized with collagen II, CIA developed rapidly in the paws, with obvious clinical symptoms and joint destruction. Methotrexate (MTX) treatment (1.5 mg/kg/week) as a positive control has a strong inhibitory effect on the development of arthritis (Figure 5A-C). Compared with CIA control mice, CIA mice treated with SMSCs-Exos continued to show significantly reduced signs of arthritis, lower clinical arthritis scores, and less joint destruction (Figure 5A-C). In addition, compared with SMSCs-Exos (Figure 5A-C) or Ad-Vector-SMSCs, Ad-circEDIL3-SMSCs-Exos treated CIA mice had significantly reduced hind paw thickness, clinical arthritis score and bone erosion-Exo Treatment group (Supplementary Figure 10A and B). Figures 5A and B show representative clinical and micro-CT pictures of mouse paws in different treatment groups. The arthritis score and the evaluation of the experimental plan are shown in Figure 5C. The average microCT value was evaluated and shown in Supplementary Figure 10B. From day 24 to day 39, the average body weight of the CIA control group was significantly lower than that of normal mice. However, the average body weight of the Ad-circEDIL3-SMSCs-Exos and Ad-Vector-SMSCs-Exos treatment groups, SMSCs-Exos and SMSCs-Exos were significantly higher than those treated with PBS, and there was no significant difference compared with normal mice (Supplementary Figure 10C). Figure 5 Injection of Ad-circEDIL3-SMSCs-Exos reduced arthritis in CIA mice. (A) Representative pictures of mouse paws after the first immunization on day 27 (I), day 33 (II), and day 39 (III). Obvious clinical symptoms were observed in the CIA control group. Compared with mice injected with PBS, the joints of CIA mice injected with exosomes showed a significant improvement in arthritis symptoms. The improvement of joint symptoms in Ad-circEDIL3-SMSCs-Exo group was more obvious than that in SMSCs-Exo group. Methotrexate (MTX) treatment (1.5 mg/kg/week) as a positive control has a strong inhibitory effect on the development of arthritis. (B) Representative micro-CT photograph. The articular surface of the normal control group was smooth and complete. Obvious joint structural changes and disability were observed in CIA control mice. CIA mice treated with exosomes and MTX showed obvious mild cartilage and bone destruction, and the improvement was more significant in the Ad-circEDIL3-SMSCs-Exo treatment group. In short, the joint damage shown by micro-CT is consistent with clinical symptoms. (C) Evaluation and display of clinical scores and process diagrams. Data are expressed as mean ± SEM * p <0.05, ** p <0.01, *** p <0.001 relative to the PBS-treated group. NC = normal control mice.

Figure 5 Injection of Ad-circEDIL3-SMSCs-Exos reduced arthritis in CIA mice. (A) Representative pictures of mouse paws after the first immunization on day 27 (I), day 33 (II), and day 39 (III). Obvious clinical symptoms were observed in the CIA control group. Compared with mice injected with PBS, the joints of CIA mice injected with exosomes showed a significant improvement in arthritis symptoms. The improvement of joint symptoms in Ad-circEDIL3-SMSCs-Exo group was more obvious than that in SMSCs-Exo group. Methotrexate (MTX) treatment (1.5 mg/kg/week) as a positive control has a strong inhibitory effect on the development of arthritis. (B) Representative micro-CT photograph. The articular surface of the normal control group was smooth and complete. Obvious joint structural changes and disability were observed in CIA control mice. CIA mice treated with exosomes and MTX showed obvious mild cartilage and bone destruction, and the improvement was more significant in the Ad-circEDIL3-SMSCs-Exo treatment group. In short, the joint damage shown by micro-CT is consistent with clinical symptoms. (C) Evaluation and display of clinical scores and process diagrams. Data are expressed as mean ± SEM * p <0.05, ** p <0.01, *** p <0.001 relative to the PBS-treated group. NC = normal control mice.

On day 40, the knee joints of CIA mice were evaluated by histopathological evaluation of micro-CT and HE stained sections. Micro-CT results showed that the joint structure and rough bone surface of the mice in the CIA group were damaged. Compared with the PBS treatment group, the joint damage in the SMSCs-Exo group was significantly reduced. It is worth noting that compared with the SMSCs-Exo and Ad-Vector-SMSCs-Exo groups, the knee joints of Ad-circEDIL3-SMSCs-Exo injected mice showed significant improvement in arthritis (Figure 6A and Supplementary Figure 10D) ). HE staining analysis showed that after CIA induction, the synovial tissue of mice showed a large amount of inflammation infiltration, synovial hyperplasia and pannus formation; compared with the CIA control group, the leukocyte infiltration in the SMSCs-Exo and Ad-circEDIL3-SMSCs-Exo groups And the destruction of bone and cartilage is significantly improved. The improvement of joint pathological changes in the Ad-circEDIL3-SMSCs-Exo group was more obvious than that in the SMSCs-Exo group (Figure 6B and D). Overall, the joint damage demonstrated by micro-CT and histological findings is consistent with the clinical symptoms and severity of arthritis scores in CIA mice. In addition, the immunohistochemical staining and average integrated optical density (IOD) value of joint tissue sections showed that compared with the normal control group, the PIAS3 staining intensity of mice in the CIA control group decreased, the p-STAT3/STAT3 ratio increased, and the VEGF staining intensity increased mouse. Compared with CIA control mice, SMSCs-Exo treatment increased the staining intensity of PIAS3 and VEGF, and decreased the ratio of p-STAT3/STAT3. In addition, compared with the mice treated with SMSCs-Exos, the immunohistochemical staining of PIAS3 in the Ad-circEDIL3-SMSCs-Exo group was significantly stronger, the p-STAT3/STAT3 ratio was lower, and the VEGF was stronger. The trend of the staining intensity of PIAS3/p-STAT3/STAT3/VEGF in the MTX-treated group was similar to that of the Ad-circEDIL3-SMSCs-Exos-treated mice (Figure 6C, E, and F). Figure 6 Histological analysis of the synovial tissue of CIA mice treated with Ad-circEDIL3-SMSCs-Exos or SMSCs-Exos. (A) Representative micro-CT images of mouse knee joints under different administrations. (B) HE staining analysis showed that after induction of CIA, mice showed a large amount of inflammatory infiltration, synovial hyperplasia and pannus formation in the synovial tissue. Compared with the CIA control group, the joint inflammation, cartilage and bone erosion of the SMSCs-Exo treatment group were significantly reduced, and the leukocyte infiltration and synovial hyperplasia of the Ad-circEDIL3-SMSCs-Exo group was even better than that of the SMSCs-Exo group. (C) The immunostaining of PIAS3 (I), p-STAT3 (II), STAT3 (III) and VEGF (IV) was demonstrated respectively. We observed that compared with NC mice, PIAS3 in synovial tissue of CIA control mice was reduced, p-STAT3/STAT3 intensity ratio increased, and VEGF staining increased. In addition, compared with CIA control mice, CIA mice treated with Ad-circEDIL3-SMSCs-Exos or SMSCs-Exos showed increased PIAS3, decreased p-STAT3/STAT3 ratio, decreased VEGF staining intensity in synovial tissue, and a more trendy change. obvious. The Ad-circEDIL3-SMSCs-Exo group was significantly higher than the SMSCs-Exo group. The trend of the staining intensity of PIAS3/p-STAT3/STAT3/VEGF in the MTX-treated group was similar to that of the Ad-circEDIL3-SMSCs-Exos-treated mice. The histological evaluation score (D), the average integrated optical density (IOD) value (E), and the STAT3 activity value (p-STAT3/STAT3) (F) are shown. Data are expressed as mean ± SEM *p <0.05, **p <0.01 compared with PBS treatment group, #p <0.05, ##p <0.01, ###p <0.001 compared with NC. NC = normal control. MTX = methotrexate.

Figure 6 Histological analysis of the synovial tissue of CIA mice treated with Ad-circEDIL3-SMSCs-Exos or SMSCs-Exos. (A) Representative micro-CT images of mouse knee joints under different administrations. (B) HE staining analysis showed that after induction of CIA, mice showed a large amount of inflammatory infiltration, synovial hyperplasia and pannus formation in the synovial tissue. Compared with the CIA control group, the joint inflammation, cartilage and bone erosion of the SMSCs-Exo treatment group were significantly reduced, and the leukocyte infiltration and synovial hyperplasia of the Ad-circEDIL3-SMSCs-Exo group was even better than that of the SMSCs-Exo group. (C) The immunostaining of PIAS3 (I), p-STAT3 (II), STAT3 (III) and VEGF (IV) was demonstrated respectively. We observed that compared with NC mice, PIAS3 in synovial tissue of CIA control mice was reduced, p-STAT3/STAT3 intensity ratio increased, and VEGF staining increased. In addition, compared with CIA control mice, CIA mice treated with Ad-circEDIL3-SMSCs-Exos or SMSCs-Exos showed increased PIAS3, decreased p-STAT3/STAT3 ratio, decreased VEGF staining intensity in synovial tissue, and a more trendy change. obvious. The Ad-circEDIL3-SMSCs-Exo group was significantly higher than the SMSCs-Exo group. The trend of the staining intensity of PIAS3/p-STAT3/STAT3/VEGF in the MTX-treated group was similar to that of the Ad-circEDIL3-SMSCs-Exos-treated mice. The histological evaluation score (D), the average integrated optical density (IOD) value (E), and the STAT3 activity value (p-STAT3/STAT3) (F) are shown. Data are expressed as mean ± SEM *p <0.05, **p <0.01 compared with PBS treatment group, #p <0.05, ##p <0.01, ###p <0.001 compared with NC. NC = normal control. MTX = methotrexate.

It has been previously reported that circRNA is involved in the pro-inflammatory state of RA patients. 19 However, the relationship between circRNA and inflammation-induced angiogenesis has not yet been determined. STAT3 signaling has recently become a major regulator of inflammation, and the early suppression of STAT3 is closely related to the long-term clinical benefits of RA. 20 In our research, a circRNA (hsa_circ_0073244, circEDIL3) was found to target the core molecule STAT3 and then inhibit downstream VEGF, thereby playing a key role in inflammation and angiogenesis events. This is in contrast to other circRNA previously reported because it does not simply directly target and promote VEGF molecules to induce angiogenesis. 21,22 VEGF-induced angiogenesis in the inflammatory microenvironment is still a considerable challenge for the treatment of RA and even many other diseases related to inflammation 23,24 As far as we know, we found that this key circRNA is the first simultaneous Involved in inflammation and angiogenesis. Therefore, this discovery will provide more specific treatment targets and benefit precision treatment.

More interestingly, circEDIL3 is one of the most highly expressed circRNAs in SMSCs, which can be delivered to the recipient RA-FLS via exosomes derived from SMSCs (SMSCs-Exos), which also inhibits co-cultured RA-FLS cell lysates For example, the expression of VEGF in the co-culture supernatant of RA-FLS and HDMECs reduces the distribution of blood vessels. In addition, after adding SMSCs-Exos overexpressing circEDIL3, the expression of VEGF in the co-cultured RA-FLS lysate was observed to decrease, which indicates the feasibility of a new exosome therapy for circRNA. The discovery of the therapeutic potential of SMSCs-Exos in inhibiting abnormal angiogenesis is consistent with previous reports; 13,25 However, circRNA involved in the anti-angiogenic effects of SMSCs-Exos during inflammation has not been reported. Due to the stable loop structure, 7 circRNA delivery will be more promising than other non-coding RNAs for treatment, and SMSCs-Exos overexpressing circRNA may further protect RNA cargo from degradation and is expected to become a new treatment method. This method may have greater advantages over miRNA-expressing MSC-Exos that has been reported for treatment. 13

In order to verify in detail the interdependent regulatory interactions between circEDIL3 and other molecules, we examined the expression levels after overexpression or knockdown in vitro. We observed that the level of miR-485-3p significantly increased/decreased after circEDIL3 knockdown/overexpression, and miR-485-3p mimics/inhibitors could significantly reduce/enhance the level of circEDIL3. The expression of PIAS3 was significantly reduced/increased by miR-485-3p mimics/inhibitors, respectively. The down-regulation/up-regulation of PIAS3 expression by miR-485-3p mimics/inhibitors can be rescued by PIAS3 overexpression/circEDIL3 knockdown. Interestingly, the biological activity of STAT3, that is, the p-STAT3/STAT3 ratio, showed a change opposite to the PIAS3 level, while there was no discernible change in STAT3 mRNA expression. These results indicate the existence of the circEDIL3/miR-485-3p/PIAS3/STAT3 functional module (Figure 7) and confirm that PIAS3 is a cytostatic inhibitor of STAT3, which is consistent with earlier observations in different cell models. 26,27 Although recent developments have targeted the JAK/STAT3 signaling pathway as therapeutic targets, such as JAK2 inhibitors, which have been shown to be effective in treating RA, 28 due to the heterogeneity of patients, many patients still respond poorly to treatment. In this study, we focused on PIAS3, a STAT3 inhibitor, and modulated this Rho GTPase family member instead of blocking the JAK pathway. Our research provides an alternative to the currently available treatment options, and of course, it will be better than previous options. Since different people are more sensitive to different drugs, precision treatment will produce better results. Figure 7 Schematic diagram of the circEDIL3 mechanism from SMSCs-Exos in RA. CircEDIL3 derived from SMSCs-Exos (exosomes of synovial mesenchymal stem cells) may act as a sponge for miR-485-3p targeting PIAS3, and then inhibit inflammatory factors such as IL-6-induced p-STAT3 activity and reduce The downstream VEGF combined with its angiogenesis will ultimately improve RA disease. Exosomes derived from stable circEDIL3 overexpressing SMSCs through adenovirus infection (Ad-circEDIL3-SMSCs-Exos) showed better efficacy than SMSCs-Exos treatment. Therefore, the intracellular transfer of circEDIL3 via SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos indicates a potential new treatment strategy for RA.

Figure 7 Schematic diagram of the circEDIL3 mechanism from SMSCs-Exos in RA. CircEDIL3 derived from SMSCs-Exos (exosomes of synovial mesenchymal stem cells) may act as a sponge for miR-485-3p targeting PIAS3, and then inhibit inflammatory factors such as IL-6-induced p-STAT3 activity and reduce The downstream VEGF combined with its angiogenesis will ultimately improve RA disease. Exosomes derived from stable circEDIL3 overexpressing SMSCs through adenovirus infection (Ad-circEDIL3-SMSCs-Exos) showed better efficacy than SMSCs-Exos treatment. Therefore, the intracellular transfer of circEDIL3 via SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos indicates a potential new treatment strategy for RA.

Overexpression of CircEDIL3 up-regulates the expression of PIAS3 protein, which in turn inactivates STAT3 to alleviate the progression of RA disease. This is the first report on circRNAs related to PIAS3, thereby inhibiting STAT3 in the treatment of arthritis. The benefit of increasing PIAS3 expression on RA is consistent with the results of Samarpita et al. 29, 30 but different from the results of Laos et al. 31 may be due to different disease activities or different tissue backgrounds leading to different states of STAT3 activity. In addition, we found that the expression of VEGF may be increased by overexpression of STAT3, which indicates that VEGF is a downstream mediator of the action of STAT3, according to previous studies. 32,33 Since STAT3 is a key transcription factor for inflammation and other diseases such as pathological events such as angiogenesis, the discovery of 34,35 circEDIL3/miR-485-3p/PIAS3/STAT3 functional modules may also provide treatment for other diseases where STAT3 plays a central role New target.

Surprisingly, in this study, we also found that in the absence of inflammation induced by the IL-6/sIL-6R complex, the cell lysate of co-cultured RA-FLS and the supernatant of RA co-cultured The expression of VEGF-FLS and HDMECs in the SMSCs-Exo treatment did not change significantly, although the levels of circEDIL3, miR-485-3p and PIAS3 changed consistently after the SMSCs-Exo treatment, and compared with IL-6+sIL The addition of -6R is irrelevant. Interestingly, STAT3 activity was significantly up-regulated after IL-6+sIL-6R induction, which is consistent with previous reports.36 And without IL-6+sIL-6R induction, STAT3 activity did not change significantly, despite the fact that circEDIL3 , MiR -485-3p and PIAS3 have been changed. The above results indicate that the induction of IL-6 is a significant promoter of STAT3 activity, and the direct inhibitory effect of PIAS3 on STAT3 depends on the activity of STAT3. Perhaps this could explain why SMSCs-Exo treatment stimulated VEGF-induced angiogenesis in regenerated tissues, in which IL-6 expression did not increase significantly. Therefore, the effect of SMSCs-Exos on angiogenesis may be closely dependent on the activity of STAT3 in different tissue environments. The findings of this study will undoubtedly give us a more comprehensive understanding of the impact of SMSCs-Exos on angiogenesis, and will be of great help to the rational application of SMSCs-Exos in future treatments.

Another significance of our current research is that we have proved the clinical potential of SMSCs-Exos produced by bioengineering as a drug carrier38, which can "kill two birds with one stone." In this study, SMSCs-Exos has the advantage of exerting anti-inflammatory effects and blocking its related angiogenesis. The positive effects of targeting STAT3 may make them more suitable as STAT3 antagonists (including JAK inhibitors, miRNAs targeting STAT3 mRNA, carriers of 39 and other chemical drugs (including arsenic trioxide) 4, 17, 40) to treat RA or other diseases (Such as cancer) -42, has a similar anti-angiogenesis effect. SMSCs-Exos may act synergistically with loaded drugs, thereby reducing drug resistance and reducing drug loading, achieving equivalent or even superior therapeutic effects, and significantly reducing the toxic and side effects of loaded drugs. We will continue to study the optimal dose ratio of SMSCs-Exos and loaded drugs, and further study how to increase the yield of exosomes for large-scale production.

In summary, it was found that circEDIL3 derived from SMSCs-Exos inhibited inflammation-induced angiogenesis through miR-485-3p/PIAS3/STAT3/VEGF functional modules in vitro and in vivo, promoted the progression of pannus, and improved RA (Figure 7). In addition, SMSCs-Exos overexpressing circEDIL3 may be a valuable drug for the development of new precision treatment strategies for RA, whether as a monotherapy or as a combination therapy with a new drug carrier.

RA, rheumatoid arthritis; circular RNA, circular RNA; miRNA, microRNA; MSCs, mesenchymal stem cells; SMSCs, synovial mesenchymal stem cells; SMSCs-Exos, SMSCs-derived exosomes; Ad- circEDIL3-SMSCs, adenovirus expressing circEDIL3 transfected SMSCs; Ad-circEDIL3-SMSCs-Exos, exosomes secreted by Ad-circEDIL3-SMSCs; RA-FLS, fibroblast-like synovial cells from RA patients; HDMECs, Human dermal microvascular endothelial cells; CII, bovine type II collagen; CIA, collagen-induced arthritis; VEGF, vascular endothelial growth factor; STAT3, signal transducer and activator of transcription 3; PIAS3, a protein inhibitor that activates STAT3; TEM , Transmission electron microscope; NTA, nanoparticle tracking analysis; sIL-6R, IL-6 and its soluble receptors; FISH, fluorescence in situ hybridization; MTX, methotrexate; IOD, integrated optical density; FCM, flow cytometry Surgery; CFA, complete Freund's adjuvant; micro-CT, micro-computed tomography; H&E, hematoxylin-eosin.

Member of the Ethics Committee of the First Affiliated Hospital of Harbin Medical University.

This work was supported by the National Natural Science Foundation of China (NSFC 81901638), China Postdoctoral Science Foundation (approval number: 2019M651309), Heilongjiang Province Postdoctoral Science Foundation (approval number: LBH-Z18226) and the First Affiliated Hospital Scientific Research and Innovation Fund. Harbin Medical The university (approval number: 2019B18) awarded Zhang Juan, and the young and middle-aged scientific research and innovation fund of the Second Affiliated Hospital of Harbin Medical University (approval number: KYCX2019-10) awarded Zhu Maolin.

The authors report no conflicts of interest in this work.

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