RESUMO
The mechanical properties of the anterior cruciate ligament (ACL) in the knee have been highlighted, but its role in the regulation of the joint microenvironment remains unclear, especially in the progression of Knee Osteoarthritis (KOA). Here, single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) data were integrated to reveal the transcriptional and epigenomic landscape of ACL in normal and OA states. We identified a novel subpopulation of fibroblasts in ACL, which provides new insights into the role of the ACL in knee homeostasis and disease. Degeneration of the ACL during OA mechanically alters the knee joint homeostasis and influences the microenvironment by regulating inflammatory- and osteogenic-related factors, thereby contributing to the progression of KOA. Additionally, the specific mechanism by which these Inflammation-associated Fibroblasts (IAFs) regulate KOA progression was uncovered, providing new foundation for the development of targeted treatments for KOA.
Assuntos
Lesões do Ligamento Cruzado Anterior , Osteoartrite do Joelho , Humanos , Ligamento Cruzado Anterior , Articulação do Joelho , Fibroblastos , Análise de Célula ÚnicaRESUMO
Osteoarthritis, characterized by articular cartilage degradation, is the leading cause of chronic disability in older adults. Studies have indicated that circular RNAs are crucial regulators of chondrocyte development and are involved in the progression of osteoarthritis. In this study, we investigated the function and mechanism of a circular RNA and its potential for osteoarthritis therapy. The expression levels of circCREBBP, screened by circular RNA sequencing during chondrogenic differentiation in adipose tissue-derived stem cells, and TGFß2 were significantly increased in the cartilage of patients with osteoarthritis and IL-1ß-induced chondrocytes. circCREBBP knockdown increased anabolism in the extracellular matrix and inhibited chondrocyte degeneration, whereas circCREBBP overexpression led to the opposite effects. Luciferase reporter assays, rescue experiments, RNA immunoprecipitation, and RNA pulldown assays confirmed that circCREBBP upregulated TGFß2 expression by sponging miR-1208, resulting in significantly enhanced phosphorylation of Smad1/5 in chondrocytes. Moreover, intra-articular injection of adeno-associated virus-sh-circCrebbp alleviated osteoarthritis in a mouse model of destabilization of the medial meniscus. Our findings reveal a critical role for circCREBBP in the progression of osteoarthritis and provide a potential target for osteoarthritis therapy.
Assuntos
Cartilagem Articular , MicroRNAs , Osteoartrite , Animais , Camundongos , Apoptose , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Proteína de Ligação a CREB/metabolismo , Interleucina-1beta/metabolismo , MicroRNAs/genética , Osteoartrite/genética , Osteoartrite/metabolismo , RNA Circular/genética , Proteína Smad1/metabolismo , Proteína Smad1/farmacologiaRESUMO
tRNA-derived fragments (tRFs) have been reported to have critical regulatory roles in osteoarthritis (OA). Recent studies have suggested that autophagy promotes the homeostasis of the extracellular matrix of chondrocytes in OA. However, the role of tRFs in posttranscriptional gene regulation during autophagy in OA is unknown. Therefore, we explored the role of tRF-5009A in the posttranscriptional gene regulation of autophagy and cartilage degeneration in OA. Using RNA sequencing, we identified tRF-5009A, the tRNAValCAC-derived fragment, in OA tissues and explored its expression by quantitative reverse transcription PCR and fluorescence in situ hybridization. We further investigated the relationship between the expression of tRF-5009A and clinical factors in OA. Chondrocytes were transfected with a tRF-5009A inhibitor or mimic to determine their functions, including in relation to autophagy and the cartilage phenotype. A rescue experiment and dual-luciferase reporter assay were conducted to determine whether the 3'-untranslated region (UTR) of mTOR contains a tRF-5009A-binding site. tRF-5009A was downregulated in the cartilage of OA knees, especially in damaged areas. mTOR was highly expressed in damaged cartilage and negatively correlated with the expression of tRF-5009A; transfection with a tRF-5009A inhibitor promoted the expression of mTOR and suppressed autophagy, whereas transfection with a tRF-5009A mimic had the opposite effect. A dual-luciferase reporter assay showed that tRF-5009A silenced the expression of mTOR by binding to its 3'-UTR. Thus, tRF-5009A regulates autophagy and cartilage degeneration in OA by targeting mTOR. In summary, these findings provide an additional tool for the clinical diagnosis and novel targeted therapy of OA.
Assuntos
Cartilagem Articular , MicroRNAs , Osteoartrite , Autofagia , Condrócitos , Humanos , Hibridização in Situ Fluorescente , RNA de Transferência , Serina-Treonina Quinases TORRESUMO
OBJECTIVES: Osteoarthritis (OA) is a degenerative disease causing the progressive destruction of articular cartilage; however, the aetiology has not yet been elucidated. Circular RNAs (circRNAs) are reportedly involved in cartilage degeneration and OA development. In the present study, we identified that circNFIX regulates chondrogenesis and cartilage homeostasis. MATERIALS AND METHODS: Microarray analysis was performed to explore circRNA expression during the chondrogenic differentiation of human adipose-drived stem cells (hADSCs). CircNFIX expression was determined using quantitative reverse transcription-polymerase chain reaction and in situ hybridization. Gain- and loss-of-function assays were performed to validate the role of circNFIX in cartilage homeostasis. RNA pull-down, Argonaute2-RNA immunoprecipitation and luciferase reporter assays were performed to evaluate the interactions among circNFIX, miR758-3p and KDM6A. RESULTS: CircNFIX expression was upregulated in the early and middle stages, whereas downregulated in the late stage of hADSC chondrogenesis. CircNFIX inhibition attenuated hADSC chondrogenesis. CircNFIX was remarkably downregulated in OA samples, circNFIX overexpression protected against chondrocyte degradation and alleviated OA progression in the destabilization of the medial meniscus OA model. Mechanistically, circNFIX acted as a sponge of miR758-3p and played a role in the chondrogenesis and chondrocyte degeneration by targeting the miR-758-3p/KDM6A axis. CONCLUSIONS: Our results revealed a key role of circNFIX in chondrogenesis and cartilage homeostasis, which may provide a potential therapeutic strategy for OA treatment.
Assuntos
Cartilagem Articular , MicroRNAs , Osteoartrite , RNA Circular , Humanos , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Condrogênese/genética , Histona Desmetilases/metabolismo , Homeostase/genética , MicroRNAs/genética , Osteoartrite/genética , Osteoartrite/metabolismo , RNA Circular/genéticaRESUMO
Mitochondrial dysfunction is related to the pathogenesis of osteoarthritis (OA); however, there are no effective drugs to treat OA for maintaining mitochondrial homeostasis. Studies have shown that mitochonic acid-5 (MA-5) has a protective effect against mitochondrial damage and plays a role in mitophagy. However, it is not clear whether MA-5 has a beneficial effect on inflammatory articular cartilage. Here, human OA cartilage was obtained from patients undergoing total joint replacement. Interleukin-1ß (IL-1ß) was used to stimulate chondrocytes and induce inflammatory injury. Cell Counting Kit-8, TUNEL, and flow cytometry assays were used to assess apoptosis. Gene expression was examined using quantitative reverse transcription-polymerase chain reaction. Mitochondrial function was evaluated using immunoblotting, mitochondrial membrane potential assay, JC-1 staining, and immunofluorescence analysis. Mitophagy was detected using immunoblotting and immunofluorescence. 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP), a specific inhibitor of Sirtuin 3 (SIRT3), was used to block the SIRT3/Parkin pathway. Mitophagy in the cartilage sections was evaluated via immunohistochemistry. IL-1ß was found to induce chondrocyte apoptosis by inhibiting SIRT3 expression and mitophagy. In addition, inflammatory damage reduced the mitochondrial membrane potential and promoted the production of intracellular reactive oxygen species (ROS), leading to increased mitochondrial division, mitochondrial fusion inhibition, and the consequent mitochondrial damage. In contrast, the MA-5 treatment inhibited excessive ROS production by upregulating mitophagy, maintaining the mitochondrial membrane potential, and reducing mitochondrial apoptosis. After chemically blocking SIRT3 with 3-TYP, Parkin-related mitophagy was also inhibited, an effect that was prevented by pretreatment of the chondrocytes with MA-5, thereby suggesting that SIRT3 is upstream of Parkin. Overall, MA-5 was found to enhance the activity of SIRT3, promote Parkin-dependent mitophagy, eliminate depolarized/damaged mitochondria in chondrocytes, and protect cartilage cells. In conclusion, MA-5 inhibits IL-1ß-induced oxidative stress and protects chondrocytes by upregulating the SIRT3/Parkin-related autophagy signaling pathway.
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tRNA-derived fragments (tRFs) are new noncoding RNAs, and recent studies have shown that tRNAs and tRFs have important functions in cell metabolism via posttranscriptional regulation of gene expression. However, whether tRFs regulate cellular metabolism of the anterior cruciate ligament (ACL) remains elusive. The aim of this study was to investigate the role and action mechanism of tRFs in ACL cell metabolism. A tRF array was used to determine tRF expression profiles in different human ACL cells, and quantitative real-time polymerase chain reaction and fluorescence in situ hybridisation were used to determine TRF365 expression. ACL cells were transfected with a TRF365 mimic or a TRF365 inhibitor to determine whether TRF365 regulates IKBKB expression. A rescue experiment and dual-luciferase reporter assay were conducted to determine whether the 3'-untranslated region (UTR) of IKBKB has a TRF365-binding site. TRF365 was weakly expressed in osteoarthritis (OA) ACL and interleukin-1ß-treated ACL cells. IKBKB was highly expressed in OA ACL and interleukin-1ß-treated ACL cells; transfection with the TRF365 mimic suppressed IKBKB expression, whereas transfection with the TRF365 inhibitor had the opposite effect. A dual-luciferase reporter assay showed that TRF365 silenced the expression of IKBKB by binding to its 3'-UTR. Thus, TRF365 regulates the metabolism of ACL cells by targeting IKBKB. In summary, TRF365 may provide a new direction for the study of ACL degeneration and on the pathophysiological process of OA.
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Hyaluronan (HA) provides a favorable environment for chondrogenesis of bone marrow mesenchymal stem cells (BMSCs). A previous report from our group indicated that addition of HA increases the chondro-inductive capacity of scaffolds. Therefore, this study aimed to investigate whether the Mw of the HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. Human BMSCs (hBMSCs) and rabbit BMSCs (rBMSCs) were isolated and expanded. TCP-COL scaffolds and TCP-COL-HA scaffolds with two different HA Mws were assessed for their capacity to induce cartilage regeneration from hBMSCs in vitro and in vivo. The results showed that about 96.96% of hBMSCs expressed CD44. Moreover, Hyal-1 and chondrogenic marker genes expressions were increased in hMSCs seeded on TCP-COL-HA scaffolds, and blocking the HA-CD44 interaction with an anti-CD44 antibody reduced the expression levels of Hyal-1 and chondrogenic marker genes. Additionally, TCP-COL-HA scaffolds with 2000 kDa Mw showed greater induction of BMSC chondrogenesis induction compared with those with 80 kDa Mw. Similar results were observed in an ectopic implantation nude mouse model. In a rabbit osteochondral defect repair model, rBMSCs seeded on TCP-COL-HA scaffolds with 2000 kDa Mw showed greater cartilage regeneration than those seeded with 80 kDa Mw. In addition, hBMSC-seeded TCP-COL-HA scaffolds with 2000 kDa Mw showed a significantly higher mechanical strength than those with 80 kDa Mw. Collectively, these results indicate that the Mw of HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. The TCP-COL-HA scaffolds might be used as allogenic off the shelf products in cartilage tissue engineering in future.
Assuntos
Condrogênese , Células-Tronco Mesenquimais , Animais , Fosfatos de Cálcio , Diferenciação Celular , Células Cultivadas , Colágeno/farmacologia , Humanos , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Coelhos , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
Long non-coding RNAs (lncRNAs) play pivotal roles in mesenchymal stem cell differentiation. However, the mechanisms by which non-coding RNA (ncRNA) networks regulate osteogenic differentiation remain unclear. Therefore, our aim was to identify RNA-associated gene and transcript expression profiles during osteogenesis in bone marrow mesenchymal stem cells (BMSCs). Using transcriptome sequencing for differentially expressed ncRNAs and mRNAs between days 0 and 21 of osteogenic differentiation of BMSCs, we found that the microRNA (miRNA) miR-503-5p was significantly downregulated. However, the putative miR-503-5p target, sorbin and SH3 domain containing 1 (SORBS1), was significantly upregulated in osteogenesis. Moreover, through lncRNA-miRNA-mRNA interaction analyses and loss- and gain-of-function experiments, we discovered that the lncRNAs LOC100126784 and POM121L9P were abundant in the cytoplasm and enhanced BMSC osteogenesis by promoting SORBS1 expression. In contrast, miR-503-5p reversed this effect. Ago2 RNA-binding protein immunoprecipitation and dual-luciferase reporter assays further validated the direct binding of miR-503-5p to LOC100126784 and POM121L9P. Furthermore, SORBS1 knockdown suppressed early osteogenic differentiation in BMSCs, and co-transfection with SORBS1 small interfering RNAs counteracted the BMSCs' osteogenic capacity promoted by LOC100126784- and POM121L9P-overexpressing lentivirus plasmids. Thus, the present study demonstrated that the lncRNAs LOC100126784 and POM121L9P facilitate the osteogenic differentiation of BMSCs via the miR-503-5p/SORBS1 axis, providing potential therapeutic targets for treating osteoporosis and bone defects.
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OBJECTIVES: Aberrations in exosomal circular RNA (circRNA) expression have been identified in various human diseases. In this study, we investigated whether exosomal circRNAs could act as competing endogenous RNAs (ceRNAs) to regulate the pathological process of osteoarthritis (OA). This study aimed to elucidate the specific MSC-derived exosomal circRNAs responsible for MSC-mediated chondrogenic differentiation using human bone marrow-derived MSCs (hMSCs) and a destabilization of the medial meniscus (DMM) mouse model of OA. METHODS: Exosomal circRNA deep sequencing was performed to evaluate the expression of circRNAs in human bone marrow-derived MSCs (hMSCs) induced to undergo chondrogenesis from day 0 to day 21. The regulatory and functional roles of exosomal circRNA_0001236 were examined on day 21 after inducing chondrogenesis in hMSCs and were validated in vitro and in vivo. The downstream target of circRNA_0001236 was also explored in vitro and in vivo using bioinformatics analyses. A luciferase reporter assay was used to evaluate the interaction between circRNA_0001236 and miR-3677-3p as well as the target gene sex-determining region Y-box 9 (Sox9). The function and mechanism of exosomal circRNA_0001236 in OA were explored in the DMM mouse model. RESULTS: Upregulation of exosomal circRNA_0001236 enhanced the expression of Col2a1 and Sox9 but inhibited that of MMP13 in hMSCs induced to undergo chondrogenesis. Moreover, circRNA_0001236 acted as an miR-3677-3p sponge and functioned in human chondrocytes via targeting miR-3677-3p and Sox9. Intra-articular injection of exosomal circRNA_0001236 attenuated OA in the DMM mouse model. CONCLUSIONS: Our results reveal an important role for a novel exosomal circRNA_0001236 in chondrogenic differentiation. Overexpression of exosomal circRNA_0001236 promoted cartilage-specific gene and protein expression through the miR-3677-3p/Sox9 axis. Thus, circRNA_0001236-overexpressing exosomes may alleviate cartilage degradation, suppressing OA progression and enhancing cartilage repair. Our findings provide a potentially effective therapeutic strategy for treating OA.
Assuntos
Cartilagem Articular , Exossomos , MicroRNAs , Condrócitos , Condrogênese/genética , Exossomos/genética , MicroRNAs/genética , RNA CircularRESUMO
Histone deacetylase 3 (HDAC3) plays a pivotal role in the repression of cartilage-specific gene expression in human chondrocytes. The aim of this study was to determine whether microRNA-193b-3p (miR-193b-3p) regulates the expression of HDAC3 during chondrogenesis and chondrocyte metabolism. Methods: miR-193b-3p expression was assessed in a human mesenchymal stem cell (hMSC) model of chondrogenesis, in interleukin-1ß (IL-1ß)-treated primary human chondrocytes (PHCs), and in non-degraded and degraded cartilage. hMSCs and PHCs were transfected with miR-193b-3p or its antisense inhibitor. A direct interaction between miR-193b-3p and its putative binding site in the 3'-untranslated region (3'-UTR) of HDAC3 mRNA was confirmed by performing luciferase reporter assays. Chondrocytes were transfected with miR-193b-3p before performing a chromatin immunoprecipitation assay with an anti-acetylated histone H3 antibody. To investigate miR-193b-3p-transfected PHCs in vivo, they were seeded in tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffolds, which were then implanted in nude mice. In addition, plasma exosomal miR-193b-3p in samples from normal controls and patients with osteoarthritis (OA) were measured. Results: miR-193b-3p expression was elevated in chondrogenic and hypertrophic hMSCs, while expression was significantly reduced in degraded cartilage compared to non-degraded cartilage. In addition, miR-193b-3p suppressed the activity of reporter constructs containing the 3'-UTR of HDAC3, inhibited HDAC3 expression, and promoted histone H3 acetylation in the COL2A1, AGGRECAN, COMP, and SOX9 promoters. Treatment with the HDAC inhibitor trichostatin A (TSA) increased cartilage-specific gene expression and enhanced hMSCs chondrogenesis. TSA also increased AGGRECAN expression and decreased MMP13 expression in IL-1ß-treated PHCs. Further, 8 weeks after implanting PHC-seeded TCP-COL-HA scaffolds subcutaneously in nude mice, we found that miR-193b overexpression strongly enhanced in vivo cartilage formation compared to that found under control conditions. We also found that patients with OA had lower plasma exosomal miR-193b levels than control subjects. Conclusions: These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs.