Chondrocyte Apoptosis In Osteoarthritis Research Articles

MiR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflammatory response of osteoarthritis chondrocytes

To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA). Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2'-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay. After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G 2 phase decreased significantly, and the proportion of cells at G 1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes. miR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.

Retraction: MiR-27a promotes the autophagy and apoptosis of IL-1β treated-articular chondrocytes in osteoarthritis through PI3K/AKT/mTOR signaling.

Retraction: MiR-27a promotes the autophagy and apoptosis of IL-1β treated-articular chondrocytes in osteoarthritis through PI3K/AKT/mTOR signaling.

The p65-LOC727924-miR-26a/KPNA3-p65 regulatory loop mediates vasoactive intestinal peptide effects on osteoarthritis chondrocytes.

Loss and dysfunction of articular chondrocytes, which disrupt the homeostasis of extracellular matrix formation and breakdown, promote the onset of osteoarthritis (OA). Targeting inflammatory pathways is an important therapeutic strategy for OA. Vasoactive intestinal peptide (VIP) is an immunosuppressive neuropeptide with potent anti-inflammatory effects; however, its role and mechanism in OA remain unclear. In this study, microarray expression profiling from the Gene Expression Omnibus database and integrative bioinformatics analyses were performed to identify differentially expressed lncRNAs in OA samples. qRT-PCR validation of the top ten different expressed lncRNAs indicated that the expression level of intergenic non-protein coding RNA 2203 (LINC02203, also named LOC727924) was the highest in OA cartilage compared to normal cartilage. Hence, the LOC727924 function was further investigated. LOC727924 was upregulated in OA chondrocytes, with a dominant sub-localization in the cytoplasm. In OA chondrocytes, LOC727924 knockdown boosted cell viability, suppressed cell apoptosis, reactive oxygen species (ROS) accumulation, increased aggrecan and collagen II, decreased matrix metallopeptidase (MMP)-3/13 and ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4/5 levels, and reduced the levels of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6). LOC727924 could interact with the microRNA 26a (miR-26a)/ karyopherin subunit alpha 3 (KPNA3) axis by competitively targeting miR-26a for KPNA3 binding, therefore down-regulating miR-26a and upregulating KPNA3; in OA chondrocytes, miR-26a inhibition partially abolished LOC727924 knockdown effects on chondrocytes. miR-26a inhibited the nuclear translocation of p65 through targeting KPNA3 and p65 transcriptionally activated LOC727924, forming a p65-LOC727924-miR-26a/KPNA3-p65 regulatory loop to modulate OA chondrocyte phenotypes. In vitro, VIP improved OA chondrocyte proliferation and functions, down-regulated LOC727924, KPNA3, and p65 expression, and upregulated miR-26a expression; in vivo, VIP ameliorated destabilization of the medial meniscus (DMM)-induced damages on the mouse knee joint, down-regulated KPNA3, inhibited the nuclear translocation of p65. In conclusion, the p65-LOC727924-miR-26a/KPNA3-p65 regulatory loop modulates OA chondrocyte apoptosis, ROS accumulation, extracellular matrix (ECM) deposition, and inflammatory response in vitro and OA development in vivo, being one of the mechanisms mediating VIP ameliorating OA.

Ginkgolide K delays the progression of osteoarthritis by regulating YAP to promote the formation of cartilage extracellular matrix.

Osteoarthritis (OA) is a degenerative disease characterized by cartilage wear and degradation. Ginkgolide K (GK) is a natural compound extracted from Ginkgo biloba leaves and possesses anti-inflammatory and anti-apoptotic effects. We found that the biological characteristics of GK were highly consistent with those of OA medications. This study aimed to determine and verify the therapeutic effect of GK on OA and mechanism of its therapeutic effect. For the in vivo experiment, OA rats were regularly injected in the articular cavity with GK, and the curative effects were observed after 4 and 8 weeks. For the in vitro experiment, we treated OA chondrocytes with different concentrations of GK and then detected the related indices of OA. Through the in vivo and in vitro experiments, we found that GK could promote the production of major components of the cartilage extracellular matrix. Transcriptome sequencing revealed that GK may activate hypoxia-inducible factor 1 alpha via the hypoxia signaling pathway, which, in turn, activates yes-associated protein and inhibits apoptosis of OA chondrocytes. GK has a therapeutic effect on OA and, therefore, has the potential to be developed into a new drug for OA treatment.

A comprehensive analysis of biomarkers associated with synovitis and chondrocyte apoptosis in osteoarthritis.

Osteoarthritis (OA) is a chronic disease with high morbidity and disability rates whose molecular mechanism remains unclear. This study sought to identify OA markers associated with synovitis and cartilage apoptosis by bioinformatics analysis. A total of five gene-expression profiles were selected from the Gene Expression Omnibus database. We combined the GEO with the GeneCards database and performed Gene Ontology and Kyoto Encyclopedia of Genes and Genome analyses; then, the least absolute shrinkage and selection operator (LASSO) algorithm was used to identify the characteristic genes, and a predictive risk score was established. We used the uniform manifold approximation and projection (UMAP) method to identify subtypes of OA patients, while the CytoHubba algorithm and GOSemSim R package were used to screen out hub genes. Next, an immunological assessment was performed using single-sample gene set enrichment analysis and CIBERSORTx. A total of 56OA-related differential genes were selected, and 10 characteristic genes were identified by the LASSO algorithm. OA samples were classified into cluster 1 and cluster 2 subtypes byUMAP, and the clustering results showed that the characteristic genes were significantly different between these groups. MYOC, CYP4B1, P2RY14, ADIPOQ, PLIN1, MFAP5, and LYVE1 were highly expressed in cluster 2, and ANKHLRC15, CEMIP, GPR88, CSN1S1, TAC1, and SPP1 were highly expressed in cluster 1. Protein-protein interaction network analysis showed that MMP9, COL1A, and IGF1 were high nodes, and the differential genes affected the IL-17 pathway and tumor necrosis factor pathway. The GOSemSim R package showed that ADIPOQ, COL1A, and SPP1 are closely related to the function of 31 hub genes. In addition, it was determined that mmp9 and Fos interact with multiple transcription factors, and the ssGSEA and CIBERSORTx algorithms revealed significant differences in immune infiltration between the two OA subtypes. Finally, a qPCR experiment was performed to explore the important genes in rat cartilage and synovium tissues; the qPCR results showed that COL1A and IL-17A were both highly expressed in synovitis tissues and cartilage tissues of OA rats, which is consistent with the predicted results. In the future, common therapeutic targets might be found forsimultaneous remissions of both phenotypes of OA.

Retraction: MicroRNA-181 inhibits proliferation and promotes apoptosis of chondrocytes in osteoarthritis by targeting PTEN.

Retraction: MicroRNA-181 inhibits proliferation and promotes apoptosis of chondrocytes in osteoarthritis by targeting PTEN.

Achyranthes bidentata polysaccharides alleviate endoplasmic reticulum stress in osteoarthritis via lncRNA NEAT1/miR-377-3p pathway

Endoplasmic reticulum stress (ERS) has been identified to be an important factor leading to chondrocyte apoptosis in osteoarthritis (OA). Previous studies have confirmed that Achyranthes bidentata polysaccharides (ABPS) can inhibit chondrocyte apoptosis; however, the mechanism of action of ABPS on chondrocyte ERS remains unclear. Thus in this study, we aim to investigate whether ABPS could inhibit OA-associated chondrocyte apoptosis by regulating ERS, especially by observing the relationship between the lncRNA NEAT1 and miR-377-3p, to explore further the protective mechanism of ABPS in OA. In vitro and in vivo experiments showed that ABPS inhibited chondrocyte ERS by regulating the expression of lncRNA NEAT1 and miR-377-3p. Moreover, both lncRNA NEAT1 silencing and miR-377-3p inhibition could attenuate the therapeutic effect of ABPS on ERS. Dual-luciferase results indicated that miR-377-3p targets the lncRNA NEAT1 gene in mouse chondrocytes. Therefore, we concluded that ABPS could inhibit thapsigargin (TG)-induced chondrocyte ERS through the lncRNA NEAT1/miR-377-3p axis.

Long intergenic non-protein coding RNA 00707 regulates chondrocyte apoptosis and proliferation in osteoarthritis by serving as a sponge for microRNA-199-3p

ABSTRACT It is known that long intergenic non-protein coding RNA 00707 (LINC00707) promotes lipopolysaccharide (LPS)-injury and microRNA-199-3p (miR-199-3p) regulates chondrocyte proliferation and apoptosis. Both processes participate in osteoarthritis (OA). We predicted that LINC00707 and miR-199-3p may interact with each other. Therefore, LINC00707 and miR-199-3p may interact with each other to participate in OA. In this study, the expression of LINC00707 and miR-199-3p in both OA and normal articular cartilage tissues was analyzed using RT-qPCR. The subcellular location of LINC00707 and its direct interaction with miR-199-3p were explored by nuclear fractionation assay, RNA pull-down assay and Luciferase reporter assay, respectively. The role of LINC00707 and miR-199-3p in regulating the expression of each other was analyzed using an overexpression assay, followed by RT-qPCR. The role of LINC00707 and miR-199-3p in regulating OA chondrocyte proliferation and apoptosis was analyzed by BrdU assay and cell apoptosis assay, respectively. OA tissues exhibited increased expression of LINC00707 and decreased expression of miR-199-3p. LINC00707 directly interacted with miR-199-3p in cytoplasm. However, LINC00707 and miR-199-3p overexpression failed to affect each other’s expression. LPS treatment increased LINC00707 expression and decreased miR-199-3p expression in OA chondrocyte. LINC00707 overexpression increased the apoptosis of OA chondrocytes induced by LPS and suppressed the proliferation of OA chondrocytes. Moreover, LINC00707 suppressed the role of miR-199-3p in enhancing cell proliferation and suppressing cell apoptosis. In conclusion, LINC00707 can be detected in cytoplasm and it may sponge miR-199-3p to regulate chondrocyte proliferation and apoptosis in OA.

The lncRNA MIAT/miR-181a-5p axis regulates osteopontin (OPN)-mediated proliferation and apoptosis of human chondrocytes in osteoarthritis.

Osteoarthritis (OA) is a slow-progressing degenerative joint disease mainly characterized by progressive cartilage loss and subchondral bone remodeling. Osteopontin (OPN) is a matrix extracellular glyco-phosphoprotein capable of regulating the expression levels of multiple factors linked with OA pathogenesis. This study explores the upstream regulatory molecular mechanism of OPN on proliferation and apoptosis of human chondrocytes in OA. Chondrocytes were isolated from OA cartilage and identified by toluidine blue staining and immunofluorescent staining of type II collagen. An MTT assay was used for cell viability, and a BrdU assay was applied for DNA synthesis. Cell apoptosis was detected by a flow cytometry assay. A lncRNA MIAT/miR-181a-5p/OPN axis regulating OA chondrocyte proliferation and apoptosis were identified. miR-181a-5p directly targeted OPN and inhibited OPN expression in OA chondrocytes. miR-181a-5p overexpression inhibited OA chondrocyte viability, suppressed DNA synthesis, and promoted apoptosis. OPN overexpression exerted opposite effects on OA chondrocytes and significantly attenuated the roles of miR-181a-5p overexpression in OA chondrocytes. A total of six long non-coding RNAs (lncRNAs) were predicted to target miR-181a-5p, and MIAT was the most up-regulated in OA cartilage tissues among the six lncRNAs. Through direct targeting, MIAT inhibited miR-181a-5p expression. MIAT silencing inhibited cell viability, suppressed DNA synthesis, and promoted cell apoptosis. Moreover, miR-181a-5p inhibition partially reversed the effects of MIAT silencing on OA chondrocytes. The lncRNA MIAT/miR-181a-5p/OPN axis could modulate OA chondrocyte proliferation and apoptosis. The comprehensive function of this axis on OA requires further in vivo and clinical investigations.

Circular RNA Derived from Vacuolar ATPase Assembly Factor VMA21 Suppresses Lipopolysaccharide-Induced Apoptosis of Chondrocytes in Osteoarthritis (OA) by Decreasing Mature miR-103 Production.

Circular RNA derived from vacuolar ATPase assembly factor (VMA21) has been proven to be an inflammation suppressor in many diseases, while its role in osteoarthritis (OA) is unknown. We predicted that VMA21 participates in OA via interacting with miR-103, an OA promoter. Therefore, we analyzed the crosstalk between VMA21 and miR-103 in OA. In this study, the levels of VMA21, pre-miR-103, and mature miR-103 in synovial fluid samples from OA patients (n = 56) and controls (n = 56) were analyzed using RT-qPCR. Nuclear and cytoplasm samples were prepared from chondrocytes, and VMA21 expression was detected by RT-PCR. RNA-RNA pulldown assay was applied to analyze the direct interaction between VMA21 and pre-miR-103. The involvement of VMA21 and miR-103 in lipopolysaccharide (LPS)-induced chondrocyte apoptosis and viability was analyzed using cell apoptosis assay and 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, respectively. We found that compared to the control group, VMA21 expression was decreased in OA, and miR-103 maturation was increased in OA. VMA21 could be detected in both nuclear and cytoplasm, and VMA21 directly interacted with pre-miR-103. VMA21 overexpression reduced miR-103 maturation. VMA21 suppressed the role of miR-103 in enhancing chondrocyte apoptosis and reducing cell viability after LPS treatment. In conclusion, VMA21 might suppress LPS-induced chondrocyte apoptosis in OA by decreasing the production of mature miR-103.

CircTMOD3 promotes lipopolysaccharide-induced chondrocyte apoptosis in osteoarthritis by sponging miR-27a

The progression of osteoarthritis (OA) requires the involvement of lipopolysaccharide (LPS)-induced inflammation, in which circTMOD3 plays an important role. We predicted that circTMOD3 could interact with miR-27a to inhibit LPS-induced chondrocyte apoptosis and explored the interaction between circTMOD3 and miR-27a in OA. Total RNAs were isolated from cartilage tissue samples from both OA patients (n = 62) and controls (n = 62) and subjected to RT-qPCRs to determine circTMOD3 and miR-27a (mature and premature) expression. Subcellular location of circTMOD3 and its interaction with premature miR-27a were analyzed using subcellular fractionation assay and RNA-RNA pulldown assay, respectively. CircTMOD3 was overexpressed in chondrocytes to study its role in miR-27a maturation. The roles of circTMOD3 and miR-27a in LPS-induced chondrocyte apoptosis were analyzed using cell apoptosis assay. CircTMOD3 and premature miR-27a levels were increased while mature miR-27a level was decreased in OA. CircTMOD3 was located in both nuclear and cytoplasm fractions of chondrocytes. CircTMOD3 directly interacted with premature miR-27a and promoted LPS-induced chondrocyte apoptosis, while miR-27a inhibited LPS-induced chondrocyte apoptosis. Moreover, circTMOD3 overexpression suppressed miR-27a maturation and reduced the inhibitory effects of miR-27a on LPS-induced chondrocyte apoptosis. CircTMOD3 suppresses miR-27a maturation in OA to promote chondrocyte apoptosis induced by LPS.

Circular RNA CCDC66 Regulates Osteoarthritis Progression by Targeting miR-3622b-5p

Objective: CircCCDC66 is involved in cancer progression, but its role in osteoarthritis (OA) remains unknown. This study was carried out to explore the biological role of circCCDC66 in OA and its underlying mechanism. Methods: The expression levels of miR-3622b-5p and circCCDC66 in OA cartilage tissues were detected by qRT-PCR. Cell Counting Kit-8 (CCK8) and flow cytometry were used to detect the chondrocyte viability and apoptosis. The expression of chondrocyte inflammatory factors (IL-6 and TNF-α) was measured by ELISA. The target genes of circCCDC66 and miR-3622b-5p were analyzed by bioinformatics analysis and luciferase reporter gene assay. The relationship between circCCDC66 and miR-3622b-5p was analyzed by bioinformatics analysis and luciferase reporter gene assay. Results: It was found that circCCDC66 expression in OA cartilage tissues was upregulated. CircCCDC66 overexpression inhibited proliferation and promoted apoptosis of chondrocytes and increased IL-6 and TNF-α levels in chondrocytes. miR-3622b-5p was predicted to be a downstream target gene of circCCDC66, and circCCDC66 overexpression inhibited miR-3622b-5p expression in chondrocytes. Moreover, miR-3622b-5p expression was downregulated in OA cartilage tissues. miR-3622b-5p overexpression increased chondrocyte proliferation, inhibited chondrocyte apoptosis, and enhanced the expression of IL-6 and TNF-α in chondrocytes. In addition, circCCDC66 overexpression enhanced SIRT3 expression in chondrocytes, while miR-3622b-5p overexpression inhibited SIRT3 expression in chondrocytes. Conclusion: CircCCDC66 promoted OA chondrocyte apoptosis by regulating the miR-3622b-5p/SIRT3 axis. CircCCDC66 may be a new therapeutic target of OA.

Long non-coding RNA musculin antisense RNA 1 promotes proliferation and suppresses apoptosis in osteoarthritic chondrocytes via the microRNA-369-3p/Janus kinase-2/ signal transducers and activators of transcription 3 axis

ABSTRACT Increasing evidence indicates that long non-coding RNAs (lncRNAs) play critical roles in osteoarthritis (OA). The present study aimed to investigate the underlying molecular mechanism of lncRNA musculin antisense RNA 1 (MSC-AS1) in OA. RT-qPCR was used to detect MSC-AS1 levels in cartilage tissues from patients with OA. The effects of MSC-AS1 knockdown on the viability and apoptosis in OA were evaluated via CCK-8 and TUNEL assays. The StarBase database was used to predict the binding sites between microRNA (miR)-369-3p and MSC-AS1 or JAK2, which were confirmed via the dual-luciferase reporter assay. The results demonstrated that MSC-AS1 expression was downregulated in OA. Functional analysis indicated that the addition of MSC-AS1 promoted viability and inhibited inflammation and the apoptosis of chondrocytes. In addition, MSC-AS1 regulated the survival of OA chondrocytes by sponging miR-369-3p. JAK2 was confirmed as a direct target of miR-369-3p, and MSC-AS1 regulated JAK2/STAT3 signaling via miR-369-3p in OA chondrocytes. Taken together, our results suggest that MSC-AS1 may regulate the miR-369-3p/JAK2/STAT3 signaling pathway to accelerate the viability, and inhibit inflammation and cell apoptosis in OA chondrocytes.

Hyaluronic Acid Modified Curcumin-Loaded Chitosan Nanoparticles Inhibit Chondrocyte Apoptosis to Attenuate Osteoarthritis via Upregulation of Activator Protein 1 and RUNX Family Transcription Factor 2.

Hyaluronic acid (HA) and curcumin (CUR) have been previously utilized for osteoarthritis (OA) treatment. CUR-loaded chitosan nanoparticles (CUR@CS NPs) and HA CUR@CS NPs were synthesized in our research to ascertain the synergistic impacts of HA and CUR-loaded NPs on OA treatment. CUR@CS NPs and HA CUR@CS NPs were synthesized with evaluation of their particle size, potential, PDI, encapsulation efficiency, drug loading and surface coating as well as HA binding rate. The in vitro CUR release curve and stability of HA-CUR@CS NPs were measured. Chondrocytes were isolated from the cartilages of OA patients, followed by cell uptake assay. The chondrocyte viability and apoptosis were determined. Subsequently, the knee OA model was established, followed by H&E, Safranin O/Fast green staining and micro-CT. HA CUR@CS NPs improved CUR stability and bioavailability. CUR@CS NPs and HA-CUR@CS NPs were successfully characterized and could further be internalized by chondrocytes. CUR@CS NPs promoted tBHP-induced chondrocyte viability and inhibited chondrocyte apoptosis. HA-CUR@CS NPs upregulated the AP-1 and RUNX2 transcription levels to activate Hedgehog pathway, which subsequently blocked the Notch pathway. Mechanically, HA-CUR@CS NPs sustained release and long-lasting effect and long-term retention in the joint cavity and downregulated the expression of several pro-inflammatory cytokines in vivo. HA-CUR@CS NPs exhibited superior effects in the preceding experiments than CUR@CS NPs. Altogether, HA-CUR@CS NPs may restrict inflammation and chondrocyte apoptosis in OA through upregulation of AP-1 and RUNX2.

Mesenchymal stem cell-derived exosome mediated long non-coding RNA KLF3-AS1 represses autophagy and apoptosis of chondrocytes in osteoarthritis

ABSTRACT Osteoarthritis is a degenerative joint disease and a leading cause of adult disability. Our previous study has reported that mesenchymal stem cell-derived exosomes (MSC-Exo) mediated long non-coding RNA KLF3-AS1 improves osteoarthritis. This study aims to investigate the molecular mechanism of KLF3-AS1 in osteoarthritis. Chondrocytes were treated with IL-1β to induce chondrocyte injury, followed by MSC-Exo treatment. We found that MSC-Exo enhanced KLF3-AS1 expression in IL-1β-treated chondrocytes. IL-1β treatment reduced cell viability and enhanced apoptosis in chondrocytes. MSC-Exo-mediated KLF3-AS1 promoted cell viability and repressed apoptosis of IL-1β-treated chondrocytes. Rapamycin (autophagy activator) promoted cell viability and suppressed apoptosis of chondrocytes by activating autophagy. Moreover, KLF3-AS1 interacted with YBX1 in chondrocytes. MSC-Exo-mediated KLF3-AS1 activated PI3K/Akt/mTOR signaling pathway, which was abrogated by YBX1 silencing. MSC-Exo-mediated KLF3-AS1 repressed autophagy and apoptosis of chondrocytes by activating PI3K/Akt/mTOR signaling pathway. In conclusion, our data demonstrate that MSC-Exo-mediated KLF3-AS1 inhibits autophagy and apoptosis of IL-1β-treated chondrocyte through PI3K/Akt/mTOR signaling pathway. KLF3-AS1 activates PI3K/Akt/mTOR signaling pathway by targeting YBX1 to improve the progression of osteoarthritis. Thus, this work suggests that MSC-Exo-mediated KLF3-AS1 may be a potential therapeutic target for osteoarthritis.

Long non-coding RNA LINC00265 promotes proliferation, apoptosis, and inflammation of chondrocytes in osteoarthritis by sponging miR-101-3p

Long non-coding RNAs (lncRNAs) play a part in a wide variety of diseases, including osteoarthritis (OA). This study was designed to investigate the biological role of lncRNA LINC00265 in OA and its underlying mechanisms. We examined the levels of LINC00265 and miR-101-3p using RT-qPCR, inflammatory factors using ELISA, and caspase-3, c-caspase-3, Bcl-2, Bax, and MMP-13 levels using Western blot in normal and OA chondrocytes, analysed the relationship between LINC00265 and miR-101-3p using bioinformatics analysis and luciferase reporter assays, performed loss- and gain-of-function analyses. The results showed that (1) LINC00265 expression was increased in OA chondrocytes, (2) si-LINC00265 inhibited OA chondrocyte apoptosis and inflammation, and (3) LINC00265 overexpression promoted normal and OA chondrocyte apoptosis and inflammation. Furthermore, we predicted and confirmed that miR-101-3p was a target of LINC00265. LINC00265 negatively regulated miR-101-3p in OA chondrocytes and LINC00265 promoted OA and normal chondrocyte apoptosis via miR-101-3p. Overall, lncRNA LINC00265 regulates chondrocyte apoptosis by acting as a sponge of miR-101-3p in OA.

Calcyclin (S100A6) Attenuates Inflammatory Response and Mediates Apoptosis of Chondrocytes in Osteoarthritis via the PI3K/AKT Pathway

ObjectiveTo clarify the regulatory effect of Calcyclin (S100A6) on chondrocytes apoptosis and its relationship with progression of osteoarthritis in an effort to explore potential therapeutic targets for osteoarthritis.MethodImmunofluorescence assay was produced to identify the rat chondrocyte sample and western blots assay was detected the expression changes of S100A6 between control group and osteoarthritis model which induced by interleukin‐1β. Adenovirus were transfected into the chondrocytes in vitro, in order to regulate the S100A6 expression. The influence of S100A6 on inflammatory reaction of osteoarthritis was detected by RT‐PCR. Also, Caspase‐3 activity assay and TUNEL assay were performed to evaluate the apoptosis changes. In addition, RT‐PCR and western blots were performed to verify that S100A6 mediated the PI3K/AKT signaling pathway. Through the usage of pathway regulator, we detected S100A6 produced the effect by mediating the PI3K/AKT pathway.ResultsWe determined the expression of S100A6 decreased in osteoarthritis model, the relative expression level in osteoarthritis model was about 0.5 fold compared with control group. Through adenovirus transfection we revealed that the inflammatory factors of osteoarthritis (interleukin‐6 and matrix metalloproteinase‐13) showed a negative correlation with the S100A6 expression. The relative expression level of interleukin‐6 and matrix metalloproteinase‐13 were 1.534 and 1.259 when S100A6 was up‐regulated and the values were up to 2.445 and 2.074, respectively, when S100A6 was down‐regulated. Also, the data verified the apoptosis could be reduced when the S100A6 was up‐regulated and be activated when the S100A6 was down‐regulated, the Caspase‐3 activity was 16.512 U/μg and 24.45 U/μg respectively. Similar results were shown in TUNEL assay, the apoptosis index was 4.46% and 31.44%, respectively. Additionally, the results of polymerase chain reaction and western blots both demonstrated that the expression level of PI3K and AKT were increased when S100A6 was up‐regulated, conversely the expression level of those two signal modules were reduced if the S100A6 was down‐regulated. More importantly, the apoptosis triggered by S100A6 can be offset by the PI3K/AKT pathway inhibitor and activator (LY294002 and IGF‐1), the values of Caspase‐3 activity and apoptosis index became close to the untreated osteoarthritis group. The experimental results in this study were statistically significant.ConclusionWe investigated that Calcyclin (S100A6) relieved the inflammation and mediated the chondrocyte apoptosis through PI3K/AKT pathway and we confirmed that S100A6 might be an attractive therapeutic target.

Cryptotanshinone interferes with chondrocyte apoptosis inosteoarthritis by inhibiting the expression of miR‑574‑5p.

Chondrocyte apoptosis is an important factor in the development and progression of osteoarthritis (OA). Cryptotanshinone (CTS) can inhibit chondrocyte apoptosis, but the specific mechanism remains unknown. The aim of the present study was to explore how CTS may affect chondrocyte apoptosis. Reverse transcription-quantitative PCR and western blotting were used to validate microRNA (miR)-574-5p, YY1-associated factor 2 (YAF2), Bcl-2 and Bax expression levels. H&E, Safranin O and TUNEL staining assays were used to evaluate the apoptosis of arthritic chondrocytes in vivo. A Cell Counting Kit-8 assay and flow cytometry were performed to detect cell proliferation and apoptosis of chondrocytes in vitro. The methylation level of the miR-574-5p promoter was measured via methylation specific PCR. The degree of chondrocyte apoptosis and the expression levels of YAF2 and Bcl-2 were decreased in the mice with OA, and were increased in the OA + CTS mice, while the expression levels of miR-574-5p and Bax showed opposite changes. Furthermore, the degree of chondrocyte apoptosis and the expression levels of the aforementioned key factors in chondrocytes were consistent with those observed in vivo. The methylation degree of the miR-574-5p promoter was increased by the addition of CTS, and was reduced after the addition of a methylation inhibitor, 5-aza-CdR, indicating that CTS could regulate the methylation of miR-574-5p promoter. The present study suggested that CTS could downregulate the expression of miR-574-5p by regulating its methylation, and thus, could improve YAF2 expression and affect chondrocyte apoptosis.

Potential of miR-25-3p in protection of chondrocytes: emphasis on osteoarthritis.

Osteoarthritis (OA) is the most prevailing musculoskeletal dysfunction triggered by lesions in synovial membranes and articular cartilage. MicroRNAs (miRNAs) have emerged as crucial regulators participated in many biological processes, such as osteoarthritis. This study was undertaken to address the role of miR-25-3p in the apoptosis of rat chondrocytes under an OA-like condition and its underlying mechanism. OA cellular model was established in rat chondrocytes by TNF-a induction. Then, qRTPCR and Western blotting were utilized for evaluation of the expressions of miR-25-3p and insulin-like growth factor-binding protein 7 (IGFBP7), CCK-8 assay for inspection of chondrocyte viability, flow cytometry for assessment of cell apoptosis rate, Western blotting for the detection of cleaved caspase-3 level and dual-luciferase reporter gene assay for verification of the targeting relationship between miR-25-3p and IGFBP7. The miR-25-3p expression was decreased and IGFBP7 was elevated in TNF-a-induced rat chondrocytes. The miR-25-3p inhibited chondrocyte apoptosis and IGFBP7 promoted apoptosis as evidenced by enhanced cell viability and suppressed cell apoptosis in OA chondrocytes after miR-25-3p overexpression or IGFBP7 knockdown. The miR-25-3p facilitated chondrocyte viability and repressed cell apoptosis in OA by negatively regulating IGFBP7. MiR-25-3p negatively regulates IGFBP7 to promote chondrocyte proliferation and restrain chondrocyte apoptosis. Our findings suggest that the regulation of IGFBP7 by miR-25-3p may emerge as a novel therapeutic regimen for OA.

LINC02288 promotes chondrocyte apoptosis and inflammation through miR-374a-3p targeting RTN3.

Dysregulation of long non-coding RNAs (lncRNAs) is related to the occurrence of osteoarthritis (OA). In the present study, we explored the role of LINC02288 and its regulatory mechanism in OA development. GSE113825 was obtained from Gene Expression Omnibus (GEO) database and analyzed to identify the differentially expressed lncRNAs in OA. Gene enrichment analyses and Kyoto Encyclopedia of Genes and Genomes biological process analysis were performed through Metascape (http://metascape.org/gp). The interactions among LINC02288, miR-374a-3p and RTN3 were determined using RNA immunoprecipitation (RIP) assays and dual luciferase reporter assays. Chondrocyte apoptosis was examined using flow cytometry. Western blot assays were conducted to assess the pro-apoptotic and anti-apoptotic markers. We identified a total of 4,491 differentially expressed lncRNAs. We focused on LINC02288 as the top-ranked up-regulated lncRNA in OA as indicated by a significant p-value. LINC02288 was significantly up-regulated, which was further verified by a real-time polymerase chain reaction. Down-regulation of LINC02288 significantly reduced the apoptosis of OA chondrocytes induced by interleukin-1β and the production of pro-inflammatory cytokines. These effects were further verified in an OA rat model. An RIP assay and dual luciferase assay further confirmed that LINC02288 served as a sponge of miR-374a-3p. Moreover, the overexpression of RTN3 could partially reverse the effects of LINC02288 knockdown, mediating inhibitory effects on chondrocyte apoptosis and the inflammatory response. Down-regulation of LINC02288 alleviated OA development in an in vivo OA animal model. Our findings indicate that LINC02288 contributes to OA progression by targeting the miR-374a-3p/RTN3 axis, which might provide a promising molecular therapy strategy for OA.