Cartilage Of Osteoarthritis Patients Research Articles

Osteoclast-derived exosomal miR-212-3p suppressed the anabolism and accelerated the catabolism of chondrocytes in osteoarthritis by targeting TGF-β1/Smad2 signaling

Osteoarthritis (OA) is a common aging-related disease affecting entire joint structures, encompassing articular cartilage and subchondral bone. Although senescence and dysfunction of chondrocytes are considered crucial factors in the occurrence of OA, the exact pathogenesis remains to be investigated. In our study, chondrocytes were incubated with a conditioned medium obtained from osteoclasts at different differentiation stages, suggesting that osteoclasts and osteoclast precursors suppressed anabolism and promoted the catabolism of chondrocytes in vitro. In contrast, the function of osteoclasts was more significant than osteoclast precursors. Further blocking of osteoclast exosome secretion by using GW4869 abolished the effect of osteoclasts on chondrocytes. Functionally, exosomal transfer of osteoclast-derived miR-212-3p inhibited Smad2 to mediate chondrocyte dysfunction, thus accelerating cartilage matrix degradation in OA via TGF-β1/Smad2 signaling. The mechanism was also confirmed within the articular cartilage in OA patients and surgery-induced OA mice. Our study provides new information on intercellular interactions in the bone microenvironment within articular cartilage and subchondral bone during OA progression. The miR-212-3p/Smad2 axis is a potential target for the prevention and therapy of OA.

APOD acts on fibroblast-like synoviocyte and chondrocyte to alleviate the process of osteoarthritis in vitro.

The pathogenesis of osteoarthritis (OA) is still unclear, leading to the lack of targeted treatment. We aimed to probe into the effect of apolipoprotein D (APOD), the key gene from our previous study through bioinformatics analysis, on fibroblast-like synoviocyte (FLS) and chondrocytes in vitro to confirm its potential roles on the delay of OA progression. Primary FLS and chondrocytes were extracted from synovium and cartilage of OA patients and stimulated with interleukin 1β (IL-1β) in vitro. After APOD intervention, viability and proliferation of FLS and chondrocytes were detected. Subsequently, the inflammatory factors of the two cells were detected by quantitative reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and western blot, and the apoptosis and autophagy-related substances were determined at the same time. Finally, the oxidation level in FLS and chondrocytes were detected. APOD reversed the change of gene expression stimulated by IL-1β in FLS and chondrocytes. APOD alleviated the proliferation of FLS while promoted proliferation of chondrocytes, and reduced the expression of inflammatory factors. Moreover, APOD promoted apoptosis of FLS and autography of chondrocytes, while reduced apoptosis of chondrocytes. Finally, decrease level of reactive oxygen species (ROS) in both cells were observed after APOD intervention, as well as the increased expression of antioxidant-related genes. APOD had effects on the proliferation of FLS and chondrocytes through apoptosis and autography as well as the reduction of oxidative stress, delaying the progress of OA.

Oral Administration of Bovine Milk-Derived Extracellular Vesicles Attenuates Cartilage Degeneration via Modulating Gut Microbiota in DMM-Induced Mice.

Osteoarthritis (OA) is the most common joint disease primarily characterized by cartilage degeneration. Milk-derived extracellular vesicles (mEVs) were reported to inhibit catabolic and inflammatory processes in the cartilage of OA patients. However, the current therapies target the advanced symptoms of OA, and it is significant to develop a novel strategy to inhibit the processes driving OA pathology. In this study, we investigated the therapeutic potential of mEVs in alleviating OA in vivo. The results revealed that mEVs ameliorated cartilage degeneration by increasing hyaline cartilage thickness, decreasing histological Osteoarthritis Research Society International (OARSI) scores, enhancing matrix synthesis, and reducing the expression of cartilage destructive enzymes in the destabilization of medial meniscus (DMM) mice. In addition, the disturbed gut microbiota in DMM mice was partially improved upon treatment with mEVs. It was observed that the pro-inflammatory bacteria (Proteobacteria) were reduced and the potential beneficial bacteria (Firmicutes, Ruminococcaceae, Akkermansiaceae) were increased. mEVs could alleviate the progression of OA by restoring matrix homeostasis and reshaping the gut microbiota. These findings suggested that mEVs might be a potential therapeutic dietary supplement for the treatment of OA.

Kartogenin Promotes the BMSCs Chondrogenic Differentiation in Osteoarthritis by Down-Regulation of miR-145-5p Targeting Smad4 Pathway.

Transplantation of mesenchymal stem cells (MSCs) is a potential therapeutic strategy for cartilage degeneration of osteoarthritis (OA). But controlling chondrogenic differentiation of the implanted MSCs in the joints remains a challenge. The role of kartogenin (KGN) for chondrogenesis of MSCs has been widely reported, however, the mechanism of chondrogenesis has not been elucidated in OA. In this study, we investigated the miR-145-5p, TGF-β, Samd4, and p-stat3/stat3 expression in cartilage of OA patients and bone marrow mesenchymal stem cells (BMSCs) treated with KGN or miR-145-5p inhibitor. In addition, the cell proliferation and chondrogenic differentiation in vitro and in vivo of BMSCs treated with KGN was also detected. In OA patients, the expression of miR-145-5p was up-regulated, and the expression of TGF-β, Samd4, and p-stat3/stat3 was inhibited. When the BMSCs treated with miR-145-5p inhibitor, the expression of TGF-β, Samd4, and p-stat3/stat3 was also significantly up-regulated. KGN-treated BMSCs had better proliferation and chondrogenic differentiation by up-regulating the expression of Sox 9, Col-2a1, aggrecan in vitro and in OA by down-regulation of miR-145-5p targeting Smad4 pathway. Moreover, intra-articular injection of KGN-treated BMSCs had a better pain relief effect in OA. The double effect on cartilage protection and pain relief indicates a great potential of intra-articular injection of KGN-treated BMSCs for the treatment of OA.

Hsa_circ_0134111 promotes osteoarthritis progression by regulating miR-224-5p/CCL1 interaction.

Mechanical, metabolic, inflammatory, and immune factors contribute to the development of osteoarthritis (OA), a joint disease characterized by cartilage destruction. The circular RNA (circRNA) hsa_circ_0134111 is upregulated in the cartilage of OA patients; however, its potential role in OA pathogenesis and progression remains unexplored. In this study, the effects of hsa_circ_0134111 knockdown were evaluated in primary human chondrocytes treated with IL-1β to simulate OA, as well as in a rat model of OA. Hsa_circ_0134111 expression was upregulated in IL-1β-stimulated chondrocytes. CCK-8 and flow cytometry assays showed that hsa_circ_0134111 knockdown reversed IL-1β-induced cell decline by inhibiting apoptosis. Following prediction analysis of circRNA and miRNA targets, dual-luciferase reporter and silencing/overexpression assays suggested that a regulatory network composed of hsa_circ_0134111, miR-224-5p, and CCL1 modulates IL-1β-mediated OA-like effects in chondrocytes. Accordingly, CCL1 overexpression abrogated the prosurvival effects of hsa_circ_0134111 knockdown in vitro. Moreover, hsa_circ_0134111 silencing in vivo alleviated cartilage destruction in an OA rat model, decreased IL-6 and TNF-α levels in synovial fluid, and downregulated CCL1 expression in the affected joints. These results suggest that hsa_circ_0134111 contributes to OA development by binding to miR-224-5p, thereby releasing the inhibition that miR-224-5p exerts over CCL1.

MiR-485-3p promotes proliferation of osteoarthritis chondrocytes and inhibits apoptosis via Notch2 and the NF-κB pathway

Context Osteoarthritis (OA) is one of the leading causes of disability worldwide. microRNAs (miRs) has been shown to be involved in multiple pathological processes during OA. But the possible mechanism of miR-485-3p in OA remains unclear. Objective This study was designed to identify the effect of miR-485-3p on OA. Methods miR-485-3p expression in the cartilage of OA patients and healthy controls was detected. OA cell model was established by lipopolysaccharide (LPS). miR-485-3p expression in SW1353 and CHON-001 chondrocytes treated with LPS was detected. After overexpressing miR-485-3p in chondrocytes, cell proliferation, and apoptosis were detected. Apoptosis-, extracellular matrix (ECM)-, inflammatory-, and oxidative stress-related factors were detected. The target gene of miR-485-3p was predicted by online software and verified by dual luciferase reporter gene assay. Notch2 was intervened in CHON-001 chondrocytes to detect proliferation and apoptosis. Finally, the phosphorylation of NF-κB pathway-related proteins was detected. Results miR-485-3p expression was low in OA patients and LPS-treated chondrocytes. After LPS treatment, the proliferation of SW1353 and CHON-001 chondrocytes was decreased, and apoptosis was increased. The above outcomes were reversed after overexpressing miR-485-3p. Overexpressing miR-485-3p also reduced ECM degradation, inflammation and oxidative stress in chondrocytes. miR-485-3p could target Notch2. After LPS treatment, the NF-κB pathway was activated, but miR-485-3p overexpression inhibited the pathway. Notch2 inhibition promoted proliferation and inhibited apoptosis of LPS-treated CHON-001 chondrocytes, and inhibited the NF-κB pathway. Conclusion Overexpression of miR-485-3p inhibited Notch2 and the NF-κB pathway, and promoted proliferation of OA chondrocytes and inhibited apoptosis.

Downregulation of IRAK3 by miR-33b-3p relieves chondrocyte inflammation and apoptosis in an in vitro osteoarthritis model.

Interleukin-1 receptor-associated kinase-3 (IRAK3) has a distinctive role in regulating inflammation. However, the functional role of IRAK3 and regulatory mechanism underlying the pathogenesis of osteoarthritis (OA) remain unclear. Here, we first found that IRAK3 was upregulated, while miR-33b-3p was downregulated in the cartilage of OA patients and IL-1β-induced CHON-001 cells. IRAK3 was confirmed as the direct target of miR-33b-3p and negatively regulated by miR-33b-3p. There was an inverse correlation between IRAK3 mRNA expression and miR-33b-3p expression in OA cartilage tissues. The in vitro functional experiments showed that miR-33b-3p overexpression caused a remarkable increase in viability, a significant decrease in inflammatory mediators (IL-1β and TNF-α), and apoptosis in IL-1β-induced CHON-001 cells. Importantly, IRAK3 knockdown imitated, while overexpression reversed the effects of miR-33b-3p on IL-1β-induced inflammation and apoptosis in CHON-001 cells. Collectively, miR-33b-3p significantly alleviated IL-1β-induced inflammation and apoptosis by downregulating IRAK3, which may serve as a promising target for OA.

CCAL1 enhances osteoarthritis through the NF-κB/AMPK signaling pathway.

Osteoarthritis (OA) is a chronic joint disease characterized by articular cartilage degeneration and secondary osteogenesis. It has been previously demonstrated that the CCAL1 locus is the gene encoding tumor necrosis factor receptor superfamily member 11B (TNFRSF11B). The purpose of this study was to demonstrate the role of CCAL1 in OA progression and to elucidate its molecular mechanisms. We report that CCAL1 is highly expressed in the cartilage of OA patients and its expression level is positively correlated with the severity of OA. We found that CCAL1 causes a switch to the fibrosis-prone phenotype of Human Chondrocyte-Osteoarthritis (HC-OA) cells. In addition, CCAL1 enhances cell viability and promotes the proliferation of HC-OA cells. Finally, the detection of proteins associated with the NF-κB/AMPK signaling pathway by western blot suggested that CCAL1 exerts its role on HC-OA cells by activating the NF-κB signaling pathway and inhibiting the AMPK signaling pathway, which was verified through the addition of NF-κB inhibitor caffeic acid phenethyl ester (CAPE) and AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR). In summary, we report that CCAL1 may promote OA through the NF-κB and AMPK signaling pathways.

CircTMBIM6 promotes osteoarthritis-induced chondrocyte extracellular matrix degradation via miR-27a/MMP13 axis.

Osteoarthritis is a degenerative disease characterized by degeneration of articular cartilage, but the current mechanism is unclear. Circular RNA (circRNA) plays a significant role in a series of biological processes related to osteoarthritis, but its mechanism remains unclear. The purpose of this study was to investigate the role of the circTMBIM6/miR-27a/MMP13 axis in osteoarthritis. The expression levels of circTMBIM6, miR-27a and MMP13 in cartilage of osteoarthritis patients and normal human cartilage were detected by reverse transcription polymerase chain reaction (RT-PCR). Osteoarthritis cell model was induced by IL-1β and TNF-α, and the expression changes of circTMBIM6, miR-27a and MMP13 in the in vitro model were detected. In addition, the in vitro regulation of circTMBIM6 and miR-27a in osteoarthritis was verified by transfection of circTMBIM6 and miR-27a plasmids, and the regulation of miR-27a on MMP13 was also verified. The dimethylmethylene blue (DMMB) method was used to analyze the secretion and formation of soluble glycosaminoglycan sulfate (sGAG), and the effects of circTMBIM6 and miR-27a chondrocytes were evaluated. The expression levels of circTMBIM6 and MMP13 in cartilage tissue of patients with osteoarthritis were higher than that of normal group, while the expression level of miR-195 in cartilage tissue of patients with osteoarthritis was lower. After IL-1β and TNF-α treatment, the expression of circTMBIM6 and MMP13 in chondrocytes increased, while the expression of miR-27a decreased. CircTMBIM6 overexpression reduced miR-27a expression but increased MMP13 expression. The circTMBIM6 gene knockout showed the opposite effect. CircTMBIM6 promotes osteoarthritis-induced chondrocyte extracellular matrix degradation via miR-27a/MMP13 axis.

Knee Osteoarthritis: A Review of Pathogenesis and State-Of-The-Art Non-Operative Therapeutic Considerations.

Being the most common musculoskeletal progressive condition, osteoarthritis is an interesting target for research. It is estimated that the prevalence of knee osteoarthritis (OA) among adults 60 years of age or older is approximately 10% in men and 13% in women, making knee OA one of the leading causes of disability in elderly population. Today, we know that osteoarthritis is not a disease characterized by loss of cartilage due to mechanical loading only, but a condition that affects all of the tissues in the joint, causing detectable changes in tissue architecture, its metabolism and function. All of these changes are mediated by a complex and not yet fully researched interplay of proinflammatory and anti-inflammatory cytokines, chemokines, growth factors and adipokines, all of which can be measured in the serum, synovium and histological samples, potentially serving as biomarkers of disease stage and progression. Another key aspect of disease progression is the epigenome that regulates all the genetic expression through DNA methylation, histone modifications, and mRNA interference. A lot of work has been put into developing non-surgical treatment options to slow down the natural course of osteoarthritis to postpone, or maybe even replace extensive surgeries such as total knee arthroplasty. At the moment, biological treatments such as platelet-rich plasma, bone marrow mesenchymal stem cells and autologous microfragmented adipose tissue containing stromal vascular fraction are ordinarily used. Furthermore, the latter two mentioned cell-based treatment options seem to be the only methods so far that increase the quality of cartilage in osteoarthritis patients. Yet, in the future, gene therapy could potentially become an option for orthopedic patients. In the following review, we summarized all of the latest and most important research in basic sciences, pathogenesis, and non-operative treatment.

Quercitrin alleviates cartilage extracellular matrix degradation and delays ACLT rat osteoarthritis development: An in vivo and in vitro study

Introduction: Disruptions of extracellular matrix (ECM) degradation homeostasis play a significant role in the pathogenesis of osteoarthritis (OA). Matrix metalloproteinase 13 (MMP13) and collagen Ⅱ are important components of ECM. Earlier we found that quercitrin could significantly decrease MMP13 gene expression and increase collagen Ⅱ gene expression in IL-1β-induced rat chondrocytes and human chondrosarcoma (SW1353) cells.Objectives: The effects and mechanism of quercitrin on OA were explored.Methods: Molecular mechanisms of quercitrin on OA were studied in vitro in primary chondrocytes and SW1353 cells. An anterior cruciate ligament transection (ACLT) rat model of OA was used to investigate the effect of quercitrin in vivo. Micro-CT analysis and Safranin O-Fast Green Staining of knee joint samples were performed to observe the damage degree of tibial subchondral bone. Immunohistochemistry of knee joint samples were conducted to observe the protein level of MMP13, collagen Ⅱ and p110α in articular cartilage.Results: In vitro, quercitrin promoted cell proliferation and delayed ECM degradation by regulating MMP13 and collagen II gene and protein expressions. Moreover, quercitrin activated the Phosphatidylinositol 3-kinase p110α (p110α)/AKT/mTOR signaling pathway by targeting p110α. We also firstly showed that the gene expression level of p110α was remarkably decreased in cartilage of OA patients. The results showed that intra-articular injection of quercitrin increased bone volume/tissue volume of tibial subchondral bone and cartilage thickness and reduced the Osteoarthritis Research Society International scores in OA rats. Meanwhile, immunohistochemical results showed that quercitrin exerted anti-OA effect by delaying ECM degradation.Conclusion: These findings suggested that quercitrin may be a prospective disease-modifying OA drug for prevention and treatment of early stage OA.

MiRNA-103 promotes chondrocyte apoptosis by down-regulation of Sphingosine kinase-1 and ameliorates PI3K/AKT pathway in osteoarthritis.

Objectives: The aim of the present study was to determine the effects of miRNA-103 on chondrocyte apoptosis and molecular mechanisms in osteoarthritis (OA) progression. Methods: The cell proliferation, apoptosis, and recovery ability were measured by cell counting kit-8 (CCK-8), flow cytometry, and wound healing assays. The interaction of miRNA-103 and Sphingosine kinase-1 (SPHK1) were determined by using luciferase reporter assay. The expression of mRNA and proteins were measured by qRT-PCR and Western blot. OA rat model was established by surgery stimulation. Results: miRNA-103 expression was significantly increased in the cartilage of OA patients and surgery-induced OA rat models. miRNA-103 transfection into primary rat chondrocytes reduced SPHK1 expression, induced apoptosis, inhibited cell proliferation, and impeded scratch assay wound closure. Moreover, expression of total AKT, and p-AKT were significantly reduced in miRNA-103-overexpressing chondrocytes while SPHK1 up-regulation increased the expression of phosphatidylinsitol-3-kinase (PI3K) and p-AKT, and reversed the proliferation suppression induced by the miRNA-103 mimic. Conclusions: Our studies suggest that miRNA-103 contributes to chondrocyte apoptosis, promoting OA progression by down-regulation of PI3K/AKT pathway through the reduction in SPHK1 activity.

Transient receptor potential ankyrin 1 (TRPA1) as a factor and drug target in osteoarthritis: TRPA1 mediates fibroblast growth factor 2 expression in chondrocytes

Purpose: Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, which is known to be involved in nociception and neurogenic inflammation. TRPA1 is widely expressed in neurons, and according to more recent findings, also in non-neuronal cells, including synoviocytes and keratinocytes. TRPA1 acts as a chemosensor of pungent environmental compounds, but it is also activated by agents formed endogenously in inflammatory and hypoxic conditions such as those found in osteoarthritic joints. We have recently shown in monosodium iodoacetate-induced experimental osteoarthritis that TRPA1 activation mediates inflammation, cartilage degradation and pain (Moilanen et al. Osteoarthritis Cartilage 2015). We have also shown that TRPA1 is functionally expressed in human chondrocytes, where it mediates inflammatory effects (Nummenmaa et al. Arthritis Res Ther 2016). Further, we found that anti-inflammatory drugs dexamethasone and aurothiomalate downregulate the expression of TRPA1 in human chondrocytes (Nummenmaa et al. RMD Open 2017), revealing TRPA1 as a potential mediator and drug target in osteoarthritis. Fibroblast growth factor 2 (FGF-2) belongs to the fibroblast growth factor family, which is a large group of molecules involved in the regulation of connective tissue development and metabolism. FGF-2 is found in the synovial fluid and cartilage of osteoarthritis patients, and has previously been associated with the pathogenesis of osteoarthritis. In the present study, we investigated the functional consequences of TRPA1 activation in chondrocytes focusing in particularly on the expression of FGF-2. Methods: Chondrocytes were isolated from cartilage tissue from wild type (WT) and TRPA1 deficient (knock-out, KO) mice and cultured in the absence or presence of IL-1β. Total RNA was isolated and genome-wide expression analysis was carried out with next generation RNA sequencing (NGS) method with Illumina HiSeq2500 at the Institute for Molecular Medicine Finland. Results from the RNA sequencing analysis were verified by quantitative RT-PCR. Primary human OA chondrocytes were isolated from cartilage samples obtained from patients undergoing knee replacement surgery. Chondrocytes were cultured in the presence of the TRPA1-inducing cytokine IL-1β alone and together with the selective TRPA1 antagonist HC-030031. For experiments investigating the effects of FGF-2 in chondrocytes, the cells were cultured in the presence of FGF-2. Expression of FGF-2 subsequent to TRPA1 activation was measured by quantitative RT-PCR, and aggrecan, collagen II and MMP-enzymes by quantitative RT-PCR/immunoassay. Results: The expression of FGF-2 was significantly downregulated in chondrocytes from TRPA KO mice compared to WT mice. The downregulation of FGF-2 was verified by performing quantitative RT-PCR analysis on the sequenced samples, and on a larger set of corresponding samples. The results were confirmed using primary human OA chondrocytes. Pharmacological inhibition of TRPA1 with the selective antagonist HC-030031 downregulated FGF-2 expression in these cells. We further investigated the effects of FGF-2 in human OA chondrocytes, and found that FGF-2 upregulated the production of cartilage matrix degrading enzymes MMP-1 and MMP-13, and downregulated the expression of cartilage matrix components aggrecan and collagen II. Conclusions: The results revealed a hitherto unknown role for TRPA1 in the regulation of FGF-2 expression in chondrocytes. Further, FGF-2 was confirmed to have catabolic and anti-anabolic effects in human OA chondrocytes. Inhibition of TRPA1 and subsequent downregulation of FGF-2 could therefore have beneficial effects on the balance of catabolic and anabolic mediators within OA cartilage. These results together with our recent data discussed above support the concept of TRPA1 as a potential mediator and drug target in osteoarthritis.

Clinical utility and potential of ultrasound in osteoarthritis.

Osteoarthritis (OA) is the most common disorder of human joints. Imaging is necessary in daily practice for OA patients, and conventional radiography remains the gold standard. However, conventional radiography is not sensitive in the early stage of OA and cannot clearly detect inflammatory condition. Recently, ultrasonography (US) is widely used in musculoskeletal field and US performs better or at least equally well for identification of osteophytes and morphologic degeneration of cartilage in OA patients. US provides relevant additional diagnostic information on pathologic changes in soft tissue (e.g., synovitis, meniscal injuries and Baker's cysts) not depicted by conventional radiography. The advantage of US is its ability to visualize even in small and hidden morphologic change even in small joints. Thus, US may be a useful imaging technique for not only knee OA but also hand OA. This review article explains relevant pathologic findings in OA and clinical usefulness in daily practice with US images.

Long non-coding RNA PVT1, a molecular sponge for miR-149, contributes aberrant metabolic dysfunction and inflammation in IL-1β-simulated osteoarthritic chondrocytes.

Osteoarthritis (OA), a common form of degenerative joint disease, is typified by inflammatory response and the loss of cartilage matrix. Long non-coding RNAs (lncRNAs) are emerging as a new player in gene regulation and exert critical roles in diverse physiologic and pathogenic processes including OA. The lncRNA plasmacytoma variant translocation 1 (PVT1) has been implicated in cancer, diabetes and septic acute kidney injury. Recent research confirmed the elevation of PVT1 in patients with OA. However, its role in the development of OA remains poorly elucidated. In the present study, high expression of PVT1 was observed in cartilage of OA patients and IL-1β-stimulated chondrocytes. Moreover, cessation of PVT1 expression dramatically reversed the inhibition of IL-1β on collagen II and aggrecan expression, but suppressed IL-1β-induced elevation of matrix metalloproteinases (MMPs), including MMP-3, MMP-9 and MMP-13. Simultaneously, PVT1 inhibition also antagonized the production of inflammatory cytokines upon IL-1β stimulation, including prostaglandin E2 (PGE2), NO, IL-6, IL-8 and TNF-α. Further molecular mechanism analysis identified PVT1 as an endogenous sponge RNA that could directly bind to miR-149 and repress its expression and activity. More importantly, miR-149 inhibition reversed the protective roles of PVT1 cessation in attenuating IL-1β-evoked matrix aberrant catabolism and inflammation. Together, this research confirms that lowering PVT1 expression may ameliorate the progression of OA by alleviating cartilage imbalance toward catabolism and inflammatory response, thus supporting a promising therapeutic strategy against OA.

Expression of dishevelled-2 in cartilage of rheumatoid arthritis and its effect on cartilage destruction

Objective: To study the expression of dishevelled-2 (DVL2) in rheumatoid arthritis cartilage and its effect on cartilage destruction. Methods: Cartilage DVL2 expression in rat models of rheumatoid arthritis (RA), osteoarthritis(OA) and collagen-induced arthritis(CIA) were tested by Western blotting. DVL2 overexpressed lentivirus was transfected into the knee of CIA rats. Primary chondrocytes were extracted from RA patients by knee arthroplasty and transfected with DVL2 overexpressed lentivirus. Gene expression of related inflammation related cytokines was detected by real-time polymerase chain reaction (PCR) . Results: Compared with knee articular cartilage in OA patients and normal rats, DVL2 protein was highly expressed in knee cartilage of RA patients and CIA rats (P values 0.041 and 0.032, respectively). DVL2 did not significantly affect the destruction of knee cartilage in CIA rats (P=0.885). DVL2 overexpression in chondrocytes enhanced gene expression of cyclo-oxygenase-2 (COX-2), inducible nitric oxide synthase (NOS), matrix metalloproteinase (MMP) 2, MMP-3, and MMP-9, which could be more pronounced when tumor necrosis factor alpha was added. Conclusions: DVL2 is highly expressed in RA articular cartilage and promotes the expression of inflammatory cytokines and MMP gene in chondrocytes by activating Wnt/β-catenin pathway, which involves in the destruction of articular cartilage in RA.

MiR-34a Enhances Chondrocyte Apoptosis, Senescence and Facilitates Development of Osteoarthritis by Targeting DLL1 and Regulating PI3K/AKT Pathway

Background/Aims: Osteoarthritis (OA) is the prevalent degenerative disease caused by various factors. MicroRNAs are important regulators in the inflammation and immune response. The aim of this study was to investigate the effect of microRNA-34a (MiR-34a) on the death of chondrocytes, senescence, as well as its role in OA progression. Methods: A series of experiments involving CCK-8, flow cytometry, β-galactosidase staining and wound healing assays were conducted to determine the cellular capabilities of proliferation, cell apoptosis, senescence and the ability of cells to recover from injury, respectively. Binding sites between miR-34a and delta-like protein 1 (DLL1) were identified using a luciferase reporter system, whereas mRNA and protein expression of target genes was determined by RT-PCR and immunoblot, respectively. OA model was generated via surgery. Results: We found that miR-34a expression was increased in the cartilage of OA patients. In rat chondrocytes and chondrosarcoma cells, miR-34a transfections noticeably inhibited the expression of DLL1, triggered cell death and senescence, suppressed proliferation, and prevented scratch assay wound closure. However, transfection of a miR-34a inhibitor displayed adverse effects. Additionally, secretion and expression of factors associated with cartilage degeneration were altered via miR-34a. Moreover, miR-34a directly inhibits DLL1 mRNA. Furthermore, concentrations of DLL1, total PI3K, and p-AKT declined in chondrocytes that overexpress miR-34a. DLL1 overexpression elevated PI3K and p-AKT levels, and eliminated cell death triggered by a miR-34a mimic. In vivo, miR-34a remarkably inhibited miR-34a up-regulation, while enhanced the level of DLL1 expression. In the knee joints of surgery-induced OA rats, articular chondrocyte death and loss of cartilage were attenuated via miR-34a antagomir injection. Conclusions: These findings indicate that miR-34a contributes to chondrocyte death, causing OA progression through DLL1 and modulation of the PI3K/AKT pathway.

MiR-30a-5p promotes the apoptosis of chondrocytes in patients with osteoarthritis by targeting protein kinase B

Objective: To investigate the expression of miR-30a-5p in cartilage of osteoarthritis patients, and to explore its mechanism of chondrocyte apoptosis. Methods: From May 2015 to December 2016, tissue specimen of 289 patients with osteoarthritis was collected in Department of Orthopedics, Changzhou traditional Chinese Medicine Hospital Affiliated to Nanjing University of Chinese Medicine.The expression of miR-30a-5p and protein kinase B(Akt) mRNA in cartilage of different patients was detected by qPCR.The apoptosis of chondrocytes was detected by Tunel method.The expression of related proteins in tissues and cells was detected by immunoblotting, and apoptosis and cell cycle were detected by flow cytometry. Results: The expression of miR-30a-5p in OA patients was significantly higher than control patients (P<0.05), but Aktwas positively related[(3.64±0.95)vs(1.03±0.31), P<0.05]. The expression of miR-30a-5p in cartilage of OA patients was negatively correlated with Akt mRNA expression (r=0.729 3, P<0.001), but it had a positive correlation with the apoptotic rate (r=0.847 5, P<0.001). miR-30a-5p targets negative regulation of Akt gene expression in SW1353 cells, and the expression of p-Akt, IkB-α, p-IkB-α, p65, p-p65 and mTOR and p-mTOR were significantly down-regulated by miR-30a-5p (P<0.05). Compared with normal SW1353 cells, the apoptosis rate of SW1353 cells which was transfected with miR-30a-5p-mimics increased by 9.65 times, G0/G1 phase cells increased by 1.37 times, S phase cells decreased by 60.94%, G2/M phase cells decreased 19.53%. Conclusion: miR-30a-5p is highly expressed in cartilage of osteoarthritis patients, and its high expression can block chondrocytes in G0/G1 phase by targeting Akt gene, and induce apoptosis of chondrocytes.

Peripheral Blood miR-181-5p Serves as a Marker for Screening Patients with Osteoarthritis by Targeting TNFα.

Increasing evidence has suggested the important role of miRNAs in the progression of osteoarthritis (OA). In the current study, we explored the role of miR-181-5p in the peripheral blood and bone tissues of OA patients for the first time. The level of miR-181-5p was evaluated in the peripheral blood and cartilage of OA patients using real time PCR analysis. ROC analysis was performed to explore whether peripheral blood and cartilage miR-181-5p could screen OA patients from normal controls. Western blot assay and dual luciferase reporter assay were carried out to validate the possible target gene of miR-181-5p in SW1353 cells. For the first time, we confirmed that the expression of miR-181-5p was significantly reduced in the peripheral blood and cartilage of OA patients, suggesting that miR-181-5p may be involved in the process of cartilage injury in OA degradation. Furthermore, ROC analysis showed that both peripheral blood and cartilage miR181-5p could screen OA patients from healthy controls. Moreover, we found that overexpression of miR-181-5p significantly suppressed the expression of TNFα, while inhibition of miR-181-5p enhanced the level of TNFα. Bioinformatic predictions identified a conserved binding site in the 3'UTR of TNFα by miR-181-5p. In addition, dual luciferase reporter assay indicated that miR-181-5p markedly suppressed the relative luciferase activity of TNFα. These data indicated that TNFα was a target gene of miR-181-5p. In summary, reduced miR-181-5p expression in the peripheral blood and cartilage tissues may serve as a potential biomarker to screen OA patients from healthy controls mainly by targeting TNFα.

Correlation of the osteoarthritis susceptibility variants that map to chromosome 20q13 with an expression quantitative trait locus operating on NCOA3 and with functional variation at the polymorphism rs116855380.

ObjectiveTo functionally characterize the osteoarthritis (OA) susceptibility variants that map to a region of high linkage disequilibrium (LD) on chromosome 20q13 marked by the single‐nucleotide polymorphism (SNP) rs6094710 and encompassing NCOA3 and SULF2.MethodsNucleic acids were extracted from the cartilage of OA patients. Overall and allelic expression of NCOA3 and SULF2 were measured by quantitative reverse transcription–polymerase chain reaction and pyrosequencing, respectively. The functional effect of SNPs within the 20q13 locus was assessed in vitro using luciferase reporter constructs and electrophoretic mobility shift assays (EMSAs). The in vivo effect of nuclear receptor coactivator 3 (NCOA3) protein depletion on primary human OA articular cartilage chondrocytes was assessed using RNA interference.ResultsExpression of NCOA3 correlated with the genotype at rs6094710 (P = 0.006), and the gene demonstrated allelic expression imbalance (AEI) in individuals heterozygous for the SNP (mean AEI 1.21; P < 0.0001). In both instances, expression of the OA‐associated allele was reduced. In addition, there was reduced enhancer activity of the OA‐associated allele of rs116855380, a SNP in perfect LD with rs6094710 in luciferase assays (P < 0.001). EMSAs demonstrated a protein complex binding with reduced affinity to this allele. Depletion of NCOA3 led to significant changes (all P < 0.05) in the expression of genes involved in cartilage homeostasis.Conclusion NCOA3 is subject to a cis‐acting expression quantitative trait locus in articular cartilage, which correlates with the OA association signal and with the OA‐associated allele of the functional SNP rs116855380, a SNP that is located only 10.3 kb upstream of NCOA3. These findings elucidate the effect of the association of the 20q13 region on OA cartilage and provide compelling evidence of a potentially causal candidate SNP.