Identification of a novel cuproptosis-related gene signature and integrative analyses in patients with lower-grade gliomas.

Background: Cuproptosis is a newly discovered unique non-apoptotic programmed cell death distinguished from known death mechanisms like ferroptosis, pyroptosis, and necroptosis. However, the prognostic value of cuproptosis and the correlation between cuproptosis and the tumor microenvironment (TME) in lower-grade gliomas (LGGs) remain unknown. Methods: In this study, we systematically investigated the genetic and transcriptional variation, prognostic value, and expression patterns of cuproptosis-related genes (CRGs). The CRG score was applied to quantify the cuproptosis subtypes. We then evaluated their values in the TME, prognostic prediction, and therapeutic responses in LGG. Lastly, we collected five paired LGG and matched normal adjacent tissue samples from Sun Yat-sen University Cancer Center (SYSUCC) to verify the expression of signature genes by quantitative real-time PCR (qRT-PCR) and Western blotting (WB). Results: Two distinct cuproptosis-related clusters were identified using consensus unsupervised clustering analysis. The correlation between multilayer CRG alterations with clinical characteristics, prognosis, and TME cell infiltration were observed. Then, a well-performed cuproptosis-related risk model (CRG score) was developed to predict LGG patients' prognosis, which was evaluated and validated in two external cohorts. We classified patients into high- and low-risk groups according to the CRG score and found that patients in the low-risk group showed significantly higher survival possibilities than those in the high-risk group (P<0.001). A high CRG score implies higher TME scores, more significant TME cell infiltration, and increased mutation burden. Meanwhile, the CRG score was significantly correlated with the cancer stem cell index, chemoradiotherapy sensitivity-related genes and immune checkpoint genes, and chemotherapeutic sensitivity, indicating the association with CRGs and treatment responses. Univariate and multivariate Cox regression analyses revealed that the CRG score was an independent prognostic predictor for LGG patients. Subsequently, a highly accurate predictive model was established for facilitating the clinical application of the CRG score, showing good predictive ability and calibration. Additionally, crucial CRGs were further validated by qRT-PCR and WB. Conclusion: Collectively, we demonstrated a comprehensive overview of CRG profiles in LGG and established a novel risk model for LGG patients' therapy status and prognosis. Our findings highlight the potential clinical implications of CRGs, suggesting that cuproptosis may be the potential therapeutic target for patients with LGG.

Deciphering a Novel Necroptosis-Related miRNA Signature for Predicting the Prognosis of Clear Cell Renal Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common histological and devastating subtype of renal cell carcinoma. Necroptosis is a form of programmed cell death that causes prominent inflammatory responses. miRNAs play a significant role in cancer progression through necroptosis. However, the prognostic value of necroptosis-related miRNAs remains ambiguous. In this study, 39 necroptosis-related miRNAs (NRMs) were extracted and 17 differentially expressed NRMs between normal and tumor samples were identified using data form The Cancer Genome Atlas (TCGA). After applying univariate Cox proportional hazard regression analysis and LASSO Cox regression model, six necroptosis-related miRNA signatures were identified in the training cohort and their expression levels were verified by qRT-PCR. Using the expression levels of these miRNAs, all patients were divided into the high- and low-risk groups. Patients in the high-risk group showed poor overall survival (P < 0.0001). Time-dependent ROC curves confirmed the good performance of our signature. The results were verified in the testing cohort and the entire TCGA cohort. Univariate and multivariate Cox regression models demonstrated that the risk score was an independent prognostic factor. Additionally, a predictive nomogram with good performance was constructed to enhance the implementation of the constructed signature in a clinical setting. We then employed miRBD, miRTarBase, and TargetScan to predict the target genes of six necroptosis-related miRNAs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that 392 potential target genes were enriched in cell proliferation-related biological processes. Six miRNAs and 59 differentially expressed target genes were used to construct an miRNA-mRNA interaction network, and 11 hub genes were selected for survival and tumor infiltration analysis. Drug sensitivity analysis revealed potential drugs that may contribute to cancer management. Hence, necroptosis-related genes play an important role in cancer biology. We developed, for the first time, a necroptosis-related miRNA signature to predict ccRCC prognosis.

Effects of interleukin 1ß on long noncoding RNA and mRNA expression profiles of human synovial fluid derived mesenchymal stem cells.

Synovial fluid-derived mesenchymal stem cells (SFMSCs) play important regulatory roles in the physiological balance of the temporomandibular joint. Interleukin (IL)-1ß regulates the biological behavior of SFMSCs; however, the effects of IL-1ß on long noncoding RNA (lncRNA) and mRNA expression in SFMSCs in the temporomandibular joint are unclear. Here, we evaluated the lncRNA and mRNA expression profiles of IL-1ß-stimulated SFMSCs. Using microarrays, we identified 264 lncRNAs (203 upregulated, 61 downregulated) and 258 mRNAs (201 upregulated, 57 downregulated) that were differentially expressed after treatment with IL-1ß (fold changes ≥ 2, P < 0.05). Kyoto Encyclopedia of Genes and Genomes pathway analysis found that one of the most significantly enriched pathways was the NF-κB pathway. Five paired antisense lncRNAs and mRNAs, eight paired enhancer lncRNAs and mRNAs, and nine paired long intergenic noncoding RNAs and mRNAs were predicted to be co-expressed. A network constructed by the top 30 K-score genes was visualized and evaluated. We found a co-expression relationship between RP3-467K16.4 and IL8 and between LOC541472 and IL6, which are related to NF-κB pathway activation. Overall, our results provide important insights into changes in lncRNA and mRNA expression in IL-1ß-stimulated SFMSCs, which can facilitate the identification of potential therapeutic targets.

Histone deacetylase inhibitors attenuated interleukin-1ß-induced chondrogenesis inhibition in synovium-derived mesenchymal stem cells of the temporomandibular joint.

AIMS: In the repair of condylar cartilage injury, synovium-derived mesenchymal stem cells (SMSCs) migrate to an injured site and differentiate into cartilage. This study aimed to confirm that histone deacetylase (HDAC) inhibitors, which alleviate arthritis, can improve chondrogenesis inhibited by IL-1ß, and to explore its mechanism. METHODS: SMSCs were isolated from synovium specimens of patients undergoing temporomandibular joint (TMJ) surgery. Chondrogenic differentiation potential of SMSCs was evaluated in vitro in the control, IL-1ß stimulation, and IL-1ß stimulation with HDAC inhibitors groups. The effect of HDAC inhibitors on the synovium and condylar cartilage in a rat TMJ arthritis model was evaluated. RESULTS: Interleukin (IL)-1ß inhibited the chondrogenic differentiation potential of SMSCs, while the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and panobinostat (LBH589), attenuated inhibition of IL-1ß-induced SMSC chondrogenesis. Additionally, SAHA attenuated the destruction of condylar cartilage in rat TMJ arthritis model. IL-6 (p < 0.001) and matrix metalloproteinase 13 (MMP13) (p = 0.006) were significantly upregulated after IL-1ß stimulation, while SAHA and LBH589 attenuated IL-6 and MMP13 expression, which was upregulated by IL-1ß in vitro. Silencing of IL-6 significantly downregulated MMP13 expression and attenuated IL-1ß-induced chondrogenesis inhibition of SMSCs. CONCLUSION: HDAC inhibitors SAHA and LBH589 attenuated chondrogenesis inhibition of SMSC induced by IL-1ß in TMJ, and inhibition of IL-6/MMP13 pathway activation contributes to this biological progress. This study provides a theoretical basis for the application of HDAC inhibitors in the treatment of TMJ arthritis. Cite this article: Bone Joint Res 2022;11(1):40-48.

Rapamycin-Induced Autophagy Promotes the Chondrogenic Differentiation of Synovium-Derived Mesenchymal Stem Cells in the Temporomandibular Joint in Response to IL-1ß.

Cartilage defects in temporomandibular disorders (TMD) lead to chronic pain and seldom heal. Synovium-derived mesenchymal stem cells (SMSCs) exhibit superior chondrogenesis and have become promising seed cells for cartilage tissue engineering. However, local inflammatory conditions that affect the repair of articular cartilage by SMSCs present a challenge, and the specific mechanism through which the function remains unclear. Thus, it is important to explore the chondrogenesis of SMSCs under inflammatory conditions of TMD such that they can be used more effectively in clinical treatment. In this study, we obtained SMSCs from TMD patients with severe cartilage injuries. In response to stimulation with IL-1ß, which is well known as one of the most prevalent cytokines in TMD, MMP13 expression increased, while that of SOX9, aggrecan, and collagen II decreased during chondrogenic differentiation. At the same time, IL-1ß upregulated the expression of mTOR and decreased the ratio of LC3-II/LC3-I and the formation of autophagosomes. Further study revealed that rapamycin pretreatment promoted the migration of SMSCs and the expression of chondrogenesis-related markers in the presence of IL-1ß by inducing autophagy. 3-Benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), a new activator of mTOR, inhibited autophagy and increased the expression of p-GSK3ßser9 and ß-catenin, simulating the effect of IL-1ß stimulation. Furthermore, rapamycin reduced the expression of mTOR, whereas the promotion of LC3-II/LC3-I was blocked by the GSK3ß inhibitor TWS119. Taken together, these results indicate that rapamycin enhances the chondrogenesis of SMSCs by inducing autophagy, and GSK3ß may be an important regulator in the process of rapamycin-induced autophagy. Thus, inducing autophagy may be a useful approach in the chondrogenic differentiation of SMSCs in the inflammatory microenvironment and may represent a novel TMD treatment.

Knockdown of long non­coding RNA AK094629 attenuates the interleukin­1ß induced expression of interleukin­6 in synovium­derived mesenchymal stem cells from the temporomandibular joint.

Interleukin (IL)­1ß is a key promotor in the pathogenesis of temporomandibular joint osteoarthritis. Differentiation of stem cells to cartilage is a crucial repair mechanism of articular cartilage damage, and IL­1ß has been reported to impede the differentiation by upregulating the secretion of IL­6, an important inflammatory factor. Long non­coding RNAs (lncRNAs) regulate a number of physiological and pathological processes, but whether lncRNA AK094629 contributes to the IL­1ß mediated induction of inflammation remains unclear. Therefore, the aim of the present study was to investigate the effect of AK094629 on IL­1ß­induced IL­6 expression in synovial­derived mesenchymal stem cells (SMSCs) of the temporomandibular joints. The results of the present study demonstrated that the expression of AK094629 in the synovial tissue of patients with osteoarthritis was positively correlated with IL­1ß. In addition, IL­1ß upregulated the expression of AK094629 in the SMSCs in vitro, and AK094629 knockdown inhibited the IL­1ß mediated upregulation of IL­6. The present study also demonstrated that AK094629 knockdown downregulated the expression of the mitogen­activated protein kinase kinase kinase 4 (MAP3K4), which is upregulated by IL­1ß, whereas knockdown of MAP3K4 did not affect the expression of AK094629, but reversed the upregulation of IL­6 in SMSCs. In conclusion, AK094629 knockdown attenuated the expression of IL­1ß­regulated IL­6 in the SMSCs of the temporomandibular joint by inhibiting MAP3K4. Therefore, AK094629 may be a potential novel therapeutic target for the treatment of temporomandibular joint osteoarthritis.

Suberoylanilide Hydroxamic Acid Attenuates Interleukin-1ß-Induced Interleukin-6 Upregulation by Inhibiting the Microtubule Affinity-Regulating Kinase 4/Nuclear Factor-κB Pathway in Synovium-Derived Mesenchymal Stem Cells from the Temporomandibular Joint.

Synovium-derived mesenchymal stem cells (SMSCs) can migrate to the site of destroyed condylar cartilage and differentiate into chondrocytes to repair temporomandibular joint (TMJ) damage. Interleukin (IL)-1ß-induced IL-6 secretion has been shown to inhibit the chondrogenic potential of SMSCs. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) has recently been shown to be closely related to the inflammation induced by IL-1ß. However, the relationship between SAHA and IL-6 secretion induced by IL-1ß in SMSCs remains unclear. In this study, we evaluated the relationships between IL-1ß and IL-6 in synovial specimens from patients with TMD and in model rats with osteoarthritis (OA). We found that IL-1ß and IL-6 were positively correlated and that IL-6 expression in SMSCs increased with IL-1ß stimulation in vitro. Moreover, microtubule affinity-regulating kinase 4 (MARK4) was significantly upregulated in IL-1ß-stimulated SMSCs and in the synovium of rats with OA. MARK4 knockdown inhibited IL-6 secretion and nuclear factor (NF)-κB pathway activation in IL-1ß-stimulated SMSCs. SAHA attenuated IL-6 secretion in IL-1ß-induced SMSCs through NF-κB pathway inhibition, and MARK4 was also downregulated in SAHA-treated SMSCs. However, inhibition of the NF-κB pathway did not suppress MARK4 expression. Thus, these results showed that SAHA attenuated IL-6 secretion in IL-1ß-induced SMSCs through inhibition of the MARK4/NF-κB pathway.

HDAC10 upregulation contributes to interleukin 1ß-mediated inflammatory activation of synovium-derived mesenchymal stem cells in temporomandibular joint.

Histone deacetylases (HDACs) are important in chronic inflammation, and inflammatory responses affect synovium-derived mesenchymal stem cell (SMSC) function in temporomandibular joint repair. However, the effect of HDACs on SMSC inflammatory activation remains unclear. In this study, temporomandibular joint fibroblast-like synoviocytes obtained from osteoarthritis patients met the minimal mesenchymal stem cell criteria. Interleukin 1ß (IL-1ß) upregulated IL-6 and IL-8 expression in SMSCs through nuclear factor-κB (NF-κB) pathway activation. IL-6 and IL-8 upregulation were blocked by broad-acting HDAC inhibitors SAHA and LBH589. MC1568 alleviated IL-1ß activation of SMSCs, whereas CI994 and FK228 produced a minimal or opposite effect in vitro. We also found HDAC10 was highly associated with localized IL-1ß expression in vivo and in vitro. HDAC10 knockdown alleviated IL-1ß-mediated SMSC activation and blocked NF-κB pathway activation. Conversely, HDAC10 overexpression promoted IL-6 and IL-8 expression and IL-1ß-mediated NF-κB pathway activation. In conclusion, HDAC10 upregulation contributed to IL-1ß-mediated inflammatory activation of SMSCs, indicating that HDAC10 may be a novel therapeutic target.

Synovial fluid­derived synovial fragments represent an improved source of synovial mesenchymal stem cells in the temporomandibular joint.

Surgery-obtained synovium specimens (SSSs) can provide a source of synovial mesenchymal stem cells (SMSCs) for experimental studies. However, these specimens contain diverse tissues, including the intima and subintima; therefore, these SMSCs are not entirely derived from the intima and their cell source is heterogeneous. The present study isolated synovial fragments (SFs) from synovial fluid dilutions extracted from patients with temporomandibular joint (TMJ) osteoarthrosis. Unlike SSSs, SFs, which are membranous and translucent, consist of only several cell layers, indicating the presence of only the intima. In the present study, SF cells (SFCs) and SSS cells (SSSCs) exhibited a homogeneous, fibroblast­like, spindle­shaped morphology after passaging in vitro. Furthermore, both cell types exhibited similar proliferative and differentiation potentials in vitro. However, SFCs exhibited more uniform surface markers compared with SSSCs when analysed by flow cytometry. Taken together, these results indicated that SFs contained a greater amount of unmixed intima than SSSs, and that SFCs exhibited more homogeneous characteristics than SSSCs, thereby offering an improved source of SMSCs in the TMJ.

IL-1ß impedes the chondrogenic differentiation of synovial fluid mesenchymal stem cells in the human temporomandibular joint.

Mesenchymal stem cell-based therapy has great therapeutic potential for temporomandibular joint (TMJ) cartilage repair. However, the behavior of mesenchymal stem cells in the inflammatory milieu following their delivery remains poorly understood. Synovial fluid-derived mesenchymal stem cells (SFMSCs) are a promising resource for TMJ cartilage repair, as they are easily obtained from patients with TMJ disorders (TMD). In this study, we obtained SFMSCs from patients with TMD and expanded them in vitro; we then stimulated the cells with interleukin (IL)­8, IL-1ß, IL-6, IL-10, tumor necrosis factor (TNF)-α and IL-12p. The cells expressed CD90, CD44, CD105 and CD73, and were negative for CD45, CD34, CD11b, CD19 and HLA-DR. They could be induced to differentiate into osteogenic, chondrogenic, adipogenic and neurogenic lineages in vitro. Only the levels of IL-6 and IL-8 were upregulated significantly following stimulation with IL-8, IL-1ß, IL-6, IL-10, TNF-α and IL-12p. Furthermore, IL-6 and IL-8 expression was driven mainly by IL-1ß-dependent nuclear factor-κB (NF-κB) pathway activation, and was independent of IL-8, IL-6, IL-10, TNF-α and IL-12p. IL-6 and IL-8 expression was inhibited completely by treatment with the NF-κB inhibitor, BAY11-7082. SRY-box 9 (SOX9) was downregulated and matrix metalloproteinase (MMP)13 was upregulated upon chondrogenic differentiation induced in the cells also exposed to IL-1ß. Sulfated glycosaminoglycan production was also reduced upon chondrogenic differentiation in the presence of IL-6, but not IL-8. Thus, IL-1ß in the inflammatory milieu is crucial in regulating SFMSCs. In doing so, IL-1ß impedes the chondrogenic differentiation of SFMSCs. The upregulation of IL-6 and NF-κB pathway activation also contribute to this biological behavior. The findings of our study indicate the potential adverse effects of IL-1ß on the chondrogenic differentiation of SFMSCs, and may thus provide new insight into the pathogenesis of TMD.

Mesenchymal Stem Cells Obtained from Synovial Fluid Mesenchymal Stem Cell-Derived Induced Pluripotent Stem Cells on a Matrigel Coating Exhibited Enhanced Proliferation and Differentiation Potential.

Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) serve as a promising source for cell-based therapies in regenerative medicine. However, optimal methods for transforming iPSCs into MSCs and the characteristics of iPSC-MSCs obtained from different methods remain poorly understood. In this study, we developed a one-step method for obtaining iPSC-MSCs (CD146+STRO-1+ MSCs) from human synovial fluid MSC-derived induced iPSCs (SFMSC-iPSCs). CD146-STRO-1-SFMSCs were reprogrammed into iPSCs by transduction with lentivirus-mediated Sox2, Oct-3/4, klf4, and c-Myc. SFMSC-iPSCs were maintained with mTeSR1 medium in Matrigel-coated culture plates. Single dissociated cells were obtained by digesting the SFMSC-iPSCs with trypsin. The dissociated cells were then plated into Matrigel-coated culture plate with alpha minimum essential medium supplemented with 10% fetal bovine serum, 1× Glutamax, and the ROCK inhibitor Y-27632. Cells were then passaged in standard cell culture plates with alpha minimum essential medium supplemented with 10% fetal bovine serum and 1× Glutamax. After passaging in vitro, the cells showed a homogenous spindle-shape similar to their ancestor cells (SFMSCs), but with more robust proliferative activity. Flow cytometric analysis revealed typical MSC surface markers, including expression of CD73, CD90, CD105, and CD44 and lack of CD45, CD34, CD11b, CD19, and HLA-DR. However, these cells were positive for CD146 and stro-1, which the ancestor cells were not. Moreover, the cells could also be induced to differentiate in osteogenic, chondrogenic, and adipogenic lineages in vitro. The differentiation potential was improved compared with the ancestor cells in vitro. The cells were not found to exhibit oncogenicity in vivo. Therefore, the method presented herein facilitated the generation of STRO-1+CD146+ MSCs from SFMSC-iPSCs exhibiting enhanced proliferation and differentiation potential.

Synovium fragment-derived cells exhibit characteristics similar to those of dissociated multipotent cells in synovial fluid of the temporomandibular joint.

Multipotent mesenchymal stem cells (MSCs) found in the synovial fluid (SFMSCs) of the tempromandibular joint (TMJ) remain poorly understood. During TMJ arthrocentesis, we discovered that synovial fluid collected from some patients with TMJ disorders contained not only SFMSCs but also synovium fragments (SFs). In this study, we attempted to characterize both the SFMSCs and SF-derived cells (SFCs) in order to further understand the role of MSCs in the synovial fluid of the TMJ. The SFs were membranous and translucent and consisted of several cell layers, indicating that their origin was only from the intima. SFCs were obtained by digestion of the SFs and subsequently expanded in vitro. SFMSCs were enriched by centrifugation of the synovial fluid and expanded in vitro. SFCs and SFMSCs displayed a similar fibroblast-like, spindle-shaped morphology, and we observed that some SFMSCs grew out of small tissue masses in culture. Flow cytometric analysis showed that both groups of cells expressed similar surface markers, including CD90, CD44, CD105, and CD73. However, both were negative for Stro-1, CD146, CD45, CD34, CD11b, CD19, and HLA-DR. Immunofluorescent staining showed that both SFs and SFMSCs expressed vascular cell adhesion molecule 1. Both SFCs and SFMSCs could be induced to differentiate down osteogenic, chondrogenic, adipogenic, and neurogenic lineages in vitro. Together, our results indicate that the intima is the most likely tissue origin of SFMSCs in the TMJ. Moreover, the SFs are composed of only intima and thus offer an improved source of synovium-derived MSCs compared to synovium specimens obtained by surgery, which contain both intima and subintima.