To the Editor: Osteoarthritis (OA) is one of the most common age-related diseases and is characterized by synovial inflammation and progressive degradation of cartilage. It was reported that one-third of 65-year-olds and 303 million people globally were affected by OA in 2017.[1,2] Although there is extensive research published on OA, its pathogenesis remains unclear and controversial. Cartilage degeneration is considered a key pathological factor that leads to disease progression. However, an increasing number of studies have revealed that synovial inflammation may be the main driving force for the development of OA. Wang et al suggested that synovial inflammation can accelerate cartilage degeneration by activating the toll-like receptors/myeloid differentiation factor 88/nuclear factor-κB (TLRs/MyD88/NF-κB) signaling pathway. Fibroblast-like synoviocytes (FLSs) can acquire an aggressive proliferative phenotype, driving cartilage and bone destruction. Regardless of which driving factor plays a leading role in OA, communication between FLSs and chondrocytes plays a crucial role. Exosomes (EXs) are vesicles with a diameter of approximately 50 to 150 nm and a lipid bilayer membrane structure. EXs contain many biologically active substances such as non-coding RNAs (ncRNAs), proteins, and messenger RNAs (mRNAs), which play important roles in the cross-talk of OA cells and the intra-articular microenvironment. MicroRNAs (miRNAs) are a group of small ncRNAs approximately 22 nucleotides in length that play key roles in the epigenetics of human diseases by regulating gene expression at the post-transcriptional level. However, few studies have examined differentially expressed miRNAs (DEMs) in synovial fluid (SF)-derived EXs.
Herein, we identified DEMs in SF-derived EXs through DEM analysis of an available dataset, followed by pathway analysis of the results and subsequent quantitative reverse transcription-polymerase chain reaction (RT-qPCR) validation of selected DEMs using an independent cohort, which provides novel insights into OA development and potential OA target molecules for therapeutic interventions. This study was approved by the Ethics Committee of Beijing University of Chinese Medicine Third Affiliated Hospital.
The dataset GSE126677 was downloaded from the GEO database, and the expression of miRNAs in SF-derived EXs was detected using the sequencing platform GPL16791 (Illumina HiSeq 2500 ,Illumina, San Diego, CA, USA). Through analysis using the NetworkAnalyst database, nine DEMs were identified, including four upregulated and five downregulated DEMs. The heatmap is shown in Figure 1A. Subsequently, we searched target genes of DEMs using the miRWalk database, and the targets verified by the miRTarBase database were used to construct the miRNA-mRNA network. A total of 349 targets for these nine DEMs were identified. Next, the miRNA-mRNA regulatory network was constructed and visualized in Cytoscape, as shown in Supplementary Figure 1, https://links.lww.com/CM9/B218. To investigate the functions of the targets, gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery online tool. The top 10 GO and KEGG items, including the biological processes (BP), cellular components (CC), and molecular functions (MF), with KEGG pathways that were significantly enriched, are listed in Supplementary Tables 1–4, https://links.lww.com/CM9/B218. KEGG pathway enrichment analysis results suggested that genes were mainly clustered in signaling pathways such as the “tumor protein P53 (p53) signaling pathway,” “mammalian target of rapamycin (mTOR) signaling pathway,” and “proteoglycans in cancer.” The details of the GO annotation and KEGG pathway analyses are listed in Supplementary Tables 1–4, https://links.lww.com/CM9/B218.
To obtain higher-purity EXs, we used the size exclusion chromatography (SECF) method to enrich EXs in the SF, which is recommended by the International Society for Extracellular Vesicles. SF samples were collected from patients undergoing total knee replacement (TKR) or arthroscopy. Twelve samples of SF were obtained, including six from patients with early-stage Knee OA (KOA) (Kellgren-Lawrence I–II) and six patients with late-stage KOA (Kellgren-Lawrence III–IV). Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting (WB) were used to detect the morphology, particle size, and biomarker proteins (CD9, CD63, and Flotillin-1) in OA SF-derived EXs. As shown in Figure 1D, SECF can extract circular or elliptical vesicles with round or oval structures from SF, with a diameter of approximately 30 to 150 nm, which is consistent with the morphological features of EXs.[4,5] NTA revealed that EXs isolated from SF were 100 to 120 nm in size, as shown in Figure 1E. WB analysis showed the presence of EX markers CD9, CD63, and Flotillin-1, as shown in Figure 1F. These data show that SECF can successfully isolate EXs from the SF.
To confirm the results of the bioinformatic analysis, we performed RT-qPCR in mild KOA and severe KOA SF-derived EXs. As shown in Figure 1G and Supplementary Figure 2, https://links.lww.com/CM9/B218, there were significant differences in the expression of four DEMs in early- and late-stage KOA SF-derived EXs, including two upregulated miRNAs (miR-130b-3p and miR-1271–5p) and two downregulated miRNAs (miR-3126-5p and miR-3976). These results suggest that SF EXs derived from miR-130-3p and miR-1271–5p may play a key role in the progression of OA.
OA is one of the most common degenerative joint diseases, which results in pain, stiffness, swelling, disability, and diminished quality of life. To date, there is no cure for KOA. TKR is considered the best option for treating end-stage KOA. However, there remains a series of problems after TKR, including infection, repair, stiffness, and unsatisfactory results. Therefore, preventing early- and mid-stage KOA from developing prematurely into end-stage KOA and slowing down the progression of KOA is the focus of treatment. In the development of radiographic OA, Atukorala et al suggested that synovial inflammation precedes other articular tissue damage rather than being a consequence of joint failure, and synovial inflammation and effusion increases the risk of cartilage loss. Meanwhile, synovial inflammation can also affect inflammation of the intra-articular fat pad and exacerbate cartilage damage.
EXs contain many active substances, and there are currently 9769 proteins, 3408 mRNAs, 2838 miRNAs, and 1116 lipids identified in EXs (www.exocarta.org). Kato et al extracted EXs from interleukin-1β (IL-1β)-stimulated FLSs, and then used them to stimulate articular chondrocytes. They found that the expression of matrix metallopeptidase 13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5), collagen type II alpha-1, and aggrecan was significantly upregulated as a result. The FLS-derived EX long ncRNA H19 promoted chondrocyte proliferation and migration and inhibited extracellular matrix degradation in OA through the miR-106b-5p/tissue inhibitor of metalloproteinases 2 axis. In contrast, chondrocyte-derived EXs enhanced the expression of IL-1β in macrophages, stimulated the activation of synovial macrophages, and aggravated synovial inflammation. Zhou et al observed the expression changes of differential miRNAs in rat OA SF and found that miR-126-3p was significantly reduced in OA SF, and overexpression of miR-126-3p could significantly inhibit chondrocyte apoptosis and inflammation. Therefore, communication between the synovium, cartilage, and fat pad plays an important role in the progression of OA. Identifying the key molecules involved in the communication between OA cells may be the key to delay the progression of OA.
In this study, based on bioinformatic analysis, nine DEMs were identified in SF-derived EXs from healthy and OA patients. Combined with the targets of these DEMs, we found 349 genes that were further used for enrichment analyses. The results indicated that these targets were mainly located in the “extracellular exosome,” which is consistent with our study of SF-derived EXs, and enriched in functions such as the “G1/S transition of mitotic cell cycle,” “regulation of ossification, ” “cellular response to DNA damage stimulus,” and enriched in pathways such as the “p53 signaling pathway” and “mTOR signaling pathway.” Several studies have demonstrated that the cell cycle, cartilage ossification, and DNA damage are closely related to the course of OA. Age is a major risk factor for OA, and the p53 signaling pathway is crucial in aging. It has been shown that p53 is highly expressed in OA chondrocytes, and the inhibition of p53 expression can delay cartilage damage. mTOR is a key regulator of cell growth, metabolism, survival, and lifespan. Silencing mTOR promotes autophagy and delays cartilage damage in mice. The mTOR signaling pathway is closely related to the phosphoinositide 3-kinases/protein kinase B, mitogen-activated protein kinase, and 5’ AMP-activated protein kinase signaling pathways, which play an important role in the course of OA.
To verify the expression of DEMs derived from SF EXs in clinical samples, we extracted and identified SF-derived EXs. Our previous research has shown that SECF can extract pure EXs; therefore, we used SECF to enrich the EXs in SF, which is also consistent with the method recommended by the 2018 International Society for Extracellular Vesicles Guidelines. The TEM and NTA results show that the morphological characteristics and particle size of the extract were consistent with those of EXs, and WB results show that the extract was positive for biological markers of EXs, namely CD9, CD81, and Flotillin-1, proving that SECF can successfully extract EXs secreted by SF. We then verified the expression of DEMs in SF-derived EXs from early- and late-stage KOA patients. Four miRNAs were validated, including two upregulated miRNAs (miR-130b-3p and miR-1271-5p) and two downregulated miRNAs (miR-3126-5p and miR-3976). Several studies have demonstrated that miR-130–3p is involved in regulating many BP, including cell proliferation, invasion, macrophage polarization, and inflammatory responses, all of which are involved in the progression of OA. miR-130–3p derived from M2 macrophage EXs promotes gastric cancer progression and mediates communication in the tumor microenvironment. miR-130b was also found to be specific for osteogenic differentiation. miR-1271–5p is highly expressed in OA while silencing miR-1271–5p can inhibit OA cartilage damage by targeting EGR. These data suggest that these two miRNAs may play a key role in the OA microenvironment and promote the progression of OA.
To conclude, we identified nine DEMs in SF-derived EXs from healthy and OA patients based on miRNA expression profiles from the GEO database. The significant upregu-lation of miR-130b-3p and miR-1271–5p was verified using RT-qPCR. These two miRNAs may play a critical role in the communication between OA cells. In future research, we will conduct a large number of sample verifications and functional experiments to confirm our results and explore its potential molecular mechanism.
This study was supported by the General Program of National Natural Science Foundation of China (Nos. 81373662 and 81874475), the Capacity building project of Chinese and Western Medicine Clinical Collaboration on major difficult disease in 2019, the National Natural Science Foundation of China (Nos. 82004383 and 81873314), and the Science and Technology Program of Guangzhou (No. 202102010273).
Conflicts of interest
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