Mesenchymal stem cell exosomes inhibit nucleus pulposus cell apoptosis via the miR-125b-5p/TRAF6/NF-κB pathway axis.

Intervertebral disc degeneration (IVDD) is the pathological basis of a range of degenerative spinal diseases and is the primary cause of lower back pain. Mesenchymal stem cell (MSC) transplantation inhibits IVDD progression. However, the specific mechanisms that underlie these effects remain unclear. In this study, candidate microRNAs (miRNAs) are screened using bioinformatics and high-throughput sequencing. TNF-α is used to induce nucleus pulposus cell (NPC) degeneration. MSC-derived exosomes (MSC-exosomes) are obtained using high-speed centrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis. Cell viability is determined by CCK-8 assay. Flow cytometry and TUNEL assays are used to detect cell apoptosis. The expression levels of miR-125b-5p are detected by RT-qPCR, and a dual-luciferase gene reporter assay confirms the downstream target genes of miR-125b-5p. Protein expression is determined by western blot analysis. Rat models are used to validate the function of miR-125b-5p in MSC-exosomes. The results show that miR-125b-5p is expressed at low levels in degenerated disc tissues compared with that in normal disc tissues; however, it is highly expressed in MSC-exosomes. Furthermore, MSC-exosomes are efficiently taken up by NPCs while miR-125b-5p is delivered into NPCs; thus, MSC-exosomes act as inhibitors of apoptosis in NPCs. Overexpression of miR-125b-5p downregulates TRAF6 expression and inhibits NF-κB activation. However, TRAF6 overexpression reverses these effects of miR-125b-5p. We demonstrate that MSC-exosomes attenuate IVDD in vivo by delivering miR-125b-5p. MSC-exosomes can deliver miR-125b-5p to target TRAF6, inhibit NF-κB activation, and attenuate the progression of IVDD.

ACSM5 inhibits ligamentum flavum hypertrophy by regulating lipid accumulation mediated by FABP4/PPAR signaling pathway.

BACKGROUND: Ligamentum flavum (LF) hypertrophy is the main cause of lumbar spinal canal stenosis (LSCS). Previous studies have shown that LF hypertrophy tissue exhibits abnormal lipid accumulation, but the regulatory mechanism remains unclear. The objective of this study was to explore the function and potential mechanism of ACSM5 in LF lipid accumulation. METHODS: To assess the ACSM5 expression levels, lipid accumulation and triglyceride (TG) level in LF hypertrophy and normal tissue, we utilized RT-qPCR, western blot, oil red O staining, and TG assay kit. The pearson correlation coefficient assay was used to analyze the correlation between ACSM5 levels and lipid accumulation or TG levels in LF hypertrophy tissue. The role of ACSM5 in free fatty acids (FFA)-induced lipid accumulation in LF cells was assessed in vitro, and the role of ACSM5 in LF hypertrophy in mice was verified in vivo. To investigate the underlying mechanisms of ACSM5 regulating lipid accumulation in LF, we conducted the mRNA sequencing, bioinformatics analysis, and rescue experiments. RESULTS: In this study, we found that ACSM5, which was significantly down-regulated in LF tissues, correlated with lipid accumulation. In vitro cell experiments demonstrated that overexpression of ACSM5 significantly inhibited FFA-induced lipid accumulation and fibrosis in LF cells. In vivo animal experiments further confirmed that overexpression of ACSM5 inhibited LF thickening, lipid accumulation, and fibrosis. Mechanistically, ACSM5 inhibited lipid accumulation of LF cells by inhibiting FABP4-mediated PPARγ signaling pathway, thereby improving hypertrophy and fibrosis of LF. CONCLUSIONS: our findings elucidated the important role of ACSM5 in the regulation of LF lipid accumulation and provide insight into potential therapeutic interventions for the treatment of LF hypertrophy. This study further suggested that therapeutic strategies targeting lipid deposition may be an effective potential approach to treat LF hypertrophy-induced LSCS.

ETS2 overexpression ameliorates cartilage injury in osteoarthritis by the ETS2/miR-155/STAT1/DNMT1 feedback loop pathway.

Osteoarthritis (OA) is the most common irreversible chronic joint dysfunction disease, which is pathologically characterized by disturbance of articular cartilage homeostasis leading to subsequent inflammatory response and cartilage extracellular matrix (ECM) degradation. Increasing evidence has demonstrated the dysregulation of transcription factors play crucial roles in the occurrence and development of osteoarthritis (OA), but the potential functions and mechanism of most transcription factors in OA has not been completely illuminated. In this study, we identified that transcription factor V-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) was significantly down-regulated in OA cartilage and IL-1ß-induced OA chondrocytes. Functional experiments in vitro demonstrated that the overexpressed ETS2 strikingly enhanced proliferation, outstandingly suppressed apoptosis, and dramatically reduced inflammation and ECM degradation in IL-1ß-induced OA chondrocytes, whereas the knockdown of ETS2 led to the opposite effects. Further in vivo studies have shown that up-regulated ETS2 dramatically ameliorates cartilage injury in DMM-induced OA mice. Mechanical studies have disclosed that DNMT1-mediated downregulation of ETS2 dramatically promotes STAT1 by inhibiting miR-155 transcription, and increased STAT1 initiates a feedback loop that may enhance DNMT1-mediated hypermethylation of ETS2 to inhibit ETS2 expression, thus forming a DNMT1/ETS2/miR-155/STAT1 feedback loop that inhibits MAPK signaling pathways and aggravates OA cartilage injury. In all, our results revealed that overexpression of ETS2 markedly ameliorated OA cartilage injury through the ETS2/miR-155/STAT1/DNMT1 feedback loop, providing a new perspective on the pathogenesis and therapeutic strategies for OA.

LncRNA XIST facilitates hypertrophy of ligamentum flavum by activating VEGFA-mediated autophagy through sponging miR-302b-3p.

BACKGROUND: Increasing evidences have shown that long non-coding RNAs (lncRNAs) display crucial regulatory roles in the occurrence and development of numerous diseases. However, the function and underlying mechanisms of lncRNAs in hypertrophy of ligamentum flavum (HLF) have not been report. METHODS: The integrated analysis of lncRNAs sequencing, bioinformatics analysis and real-time quantitative PCR were used to identify the key lncRNAs involved in HLF progression. Gain- and loss-function experiments were used to explore the functions of lncRNA X inactive specific transcript (XIST) in HLF. Mechanistically, bioinformatics binding site analysis, RNA pull-down, dual-luciferase reporter assay, and rescue experiments were utilized to investigate the mechanism by which XIST acts as a molecular sponge of miR-302b-3p to regulate VEGFA-mediated autophagy. RESULTS: We identified that XIST was outstandingly upregulated in HLF tissues and cells. Moreover, the up-regulation of XIST strongly correlated with the thinness and fibrosis degree of LF in LSCS patients. Functionally, knockdown of XIST drastically inhibited proliferation, anti-apoptosis, fibrosis and autophagy of HLF cells in vitro and suppressed hypertrophy and fibrosis of LF tissues in vivo. Intestinally, we uncovered that overexpression of XIST significantly promoted proliferation, anti-apoptosis and fibrosis ability of HLF cells by activating autophagy. Mechanistic studies illustrated that XIST directly medullated the VEGFA-mediated autophagy through sponging miR-302b-3p, thereby enhancing the development and progression of HLF. CONCLUSION: Our findings highlighted that the XIST/miR-302b-3p/VEGFA-mediated autophagy axis is involved in development and progression of HLF. At the same time, this study will complement the blank of lncRNA expression profiles in HLF, which laid the foundation for further exploration of the relationship between lncRNAs and HLF in the future.

TCF7/SNAI2/miR-4306 feedback loop promotes hypertrophy of ligamentum flavum.

BACKGROUND: Hypertrophy of ligamentum flavum (HLF) is the mainly cause of lumbar spinal stenosis (LSS), but the precise mechanism of HLF formation has not been fully elucidated. Emerging evidence indicates that transcription factor 7 (TCF7) is the key downstream functional molecule of Wnt/ß-catenin signaling, which participated in regulating multiple biological processes. However, the role and underlying mechanism of TCF7 in HLF is still unclear. METHODS: We used mRNAs sequencing analysis of human LF and subsequent confirmation with RT-qPCR, western blot and immunohistochemistry to identified the TCF7 in HLF tissues and cells. Then effect of TCF7 on HLF progression was investigated both in vitro and in vivo. Mechanically, chromatin immunoprecipitation, dual-luciferase reporter assays, and rescue experiments were used to validate the regulation of TCF7/SNAI2/miR-4306 feedback loop. RESULTS: Our results identified for first time that the TCF7 expression was obviously elevated in HLF tissues and cells compared with control, and also found that TCF7 expression had significant positive correlation with LF thickness and fibrosis score. Notably, TCF7 inhibition suppressed the hyper-proliferation and fibrosis phenotype of HLF cells in vitro and ameliorated progression of HLF in mice in vivo, whereas TCF7 overexpression promoted hyper-proliferation and fibrosis phenotype of HLF cells in vitro. Our data further revealed that TCF7 interacted with SNAI2 promoter to transactivated the SNAI2 expression, thereby promoting hyper-proliferation and fibrosis phenotype of HLF cells in vitro. Furthermore, miR-4036 negatively regulated by SNAI2 could negatively feedback regulate TCF7 expression by directly binding to TCF7 mRNA 3'-UTR, thus inhibiting the hyper-proliferation and fibrosis phenotype of HLF cells in vitro. CONCLUSIONS: Our study demonstrated that TCF7 inhibition could suppress HLF formation by modulating TCF7/SNAI2/miR-4306 feedback loop, which might be considered as a novel potential therapeutic target for HLF.

Immune cell infiltration and the genes associated with ligamentum flavum hypertrophy: Identification and validation.

Ligamentum flavum hypertrophy (LFH) is a common cause of spinal stenosis. The aim of the current study was to identify the differentially expressed genes (DEGs) in LFH and the molecular mechanisms underlying the development of and immune responses to LFH. The gene expression omnibus (GEO) database was used to obtain the GSE113212 dataset, and the DEGs were derived from microarray data. To identify critical genes and signaling pathways, gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network analyses were performed, followed by immune cell infiltration and Friends analyses using the retrieved datasets. The results were validated using quantitative real-time PCR. The 1530 DEGs identified comprised 971 upregulated and 559 downregulated genes. KEGG analysis revealed that DEGs were mostly enriched in the PI3K-Akt signaling pathway, while PPI network analysis identified tumor necrosis factor, interleukin (IL)-6, IL-10, epidermal growth factor receptor, and leptin as important nodes, which was validated by qPCR and IHC in human LFH tissues in vitro. A significant positive correlation was found between key LFH immune-related DEGs and several immune cell types, including T and B cells. The findings of the present study might lead to novel therapeutic targets and clinical approaches, as they provide insights into the molecular mechanisms of LFH.

m6A Regulator-Mediated RNA Methylation Modification Patterns Regulate the Immune Microenvironment in Osteoarthritis.

Epigenetic regulation, particularly RNA n6 methyl adenosine (m6A) modification, plays an important role in the immune response. However, the regulatory role of m6A in the immune microenvironment in osteoarthritis (OA) remains unclear. Accordingly, we systematically studied RNA modification patterns mediated by 23 m6A regulators in 38 samples and discussed the characteristics of the immune microenvironment modified by m6A. Next, we constructed a novel OA m6A nomogram, an m6A-transcription factor-miRNA network, and a drug network. Healthy and OA samples showed distinct m6A regulatory factor expression patterns. YTHDF3 expression was upregulated in OA samples and positively correlated with type II helper cells and TGFb family member receptors. Furthermore, three different RNA modification patterns were mediated by 23 m6A regulatory factors; in Mode 3, the expression levels of YTHDF3, type II T helper cells, and TGFb family member receptors were upregulated. Pathways related to endoplasmic reticulum oxidative stress and mitochondrial autophagy showed a strong correlation with the regulatory factors associated with Mode 3 and 23 m6A regulatory factors. Through RT-qPCR we validated that SREBF2 and EGR1 as transcription factors of YTHDF3 and IGF2BP3 are closely associated with the development of OA, hsa-miR-340 as a miRNA for YTHDF3 and IGF2BP3 was involved in the development of OA, we also detected the protein expression levels of IGF2BP3, YTHDF3, EGR1 and SREBF2 by western blotting, and the results were consistent with PCR. Overall, the constructed nomogram can facilitate the prediction of OA risk.

Identification of Core Genes and Screening of Potential Targets in Intervertebral Disc Degeneration Using Integrated Bioinformatics Analysis.

Background: Intervertebral disc degeneration (IDD), characterized by diverse pathological changes, causes low back pain (LBP). However, prophylactic and delaying treatments for IDD are limited. The aim of our study was to investigate the gene network and biomarkers of IDD and suggest potential therapeutic targets. Methods: Differentially expressed genes (DEGs) associated with IDD were identified by analyzing the mRNA, miRNA, and lncRNA expression profiles of IDD cases from the Gene Expression Omnibus (GEO). The protein-protein interaction (PPI) network, Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis as well as miRNA-lncRNA-mRNA networks were conducted. Moreover, we obtained 71 hub genes and performed a comprehensive analysis including GO, KEGG, gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), Disease Ontology (DO), methylation analysis, receiver operating characteristic (ROC) curve analysis, immune infiltration analysis, and potential drug identification. We finally used qRT-PCR to verify 13 significant DEGs in normal and degenerative nucleus pulposus cells (NPCs). Results: We identified 305 DEGs closely related to IDD. The GO and KEGG analyses indicated that changes in IDD are significantly associated with enrichment of the inflammatory and immune response. GSEA analysis suggested that cell activation involved in the inflammatory immune response amide biosynthetic process was the key for the development of IDD. The GSVA suggested that DNA repair, oxidative phosphorylation, peroxisome, IL-6-JAK-STAT3 signaling, and apoptosis were crucial in the development of IDD. Among the 71 hub genes, the methylation levels of 11 genes were increased in IDD. A total of twenty genes showed a high functional similarity and diagnostic value in IDD. The result of the immune cell infiltration analysis indicated that seven genes were closely related to active natural killer cells. The most relevant targeted hub genes for potential drug or molecular compounds were MET and PIK3CD. Also, qRT-PCR results showed that ARHGAP27, C15orf39, DEPDC1, DHRSX, MGAM, SLC11A1, SMC4, and LINC00887 were significantly downregulated in degenerative NPCs; H19, LINC00685, mir-185-5p, and mir-4306 were upregulated in degenerative NPCs; and the expression level of mir-663a did not change significantly in normal and degenerative NPCs. Conclusion: Our findings may provide new insights into the functional characteristics and mechanism of IDD and aid the development of IDD therapeutics.

Bioinformatics-Led Discovery of Osteoarthritis Biomarkers and Inflammatory Infiltrates.

The molecular mechanisms of osteoarthritis, the most common chronic disease, remain unexplained. This study aimed to use bioinformatic methods to identify the key biomarkers and immune infiltration in osteoarthritis. Gene expression profiles (GSE55235, GSE55457, GSE77298, and GSE82107) were selected from the Gene Expression Omnibus database. A protein-protein interaction network was created, and functional enrichment analysis and genomic enrichment analysis were performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) databases. Immune cell infiltration between osteoarthritic tissues and control tissues was analyzed using the CIBERSORT method. Identify immune patterns using the ConsensusClusterPlus package in R software using a consistent clustering approach. Molecular biological investigations were performed to discover the important genes in cartilage cells. A total of 105 differentially expressed genes were identified. Differentially expressed genes were enriched in immunological response, chemokine-mediated signaling pathway, and inflammatory response revealed by the analysis of GO and KEGG databases. Two distinct immune patterns (ClusterA and ClusterB) were identified using the ConsensusClusterPlus. Cluster A patients had significantly lower resting dendritic cells, M2 macrophages, resting mast cells, activated natural killer cells and regulatory T cells than Cluster B patients. The expression levels of TCA1, TLR7, MMP9, CXCL10, CXCL13, HLA-DRA, and ADIPOQSPP1 were significantly higher in the IL-1ß-induced group than in the osteoarthritis group in an in vitro qPCR experiment. Explaining the differences in immune infiltration between osteoarthritic tissues and normal tissues will contribute to the understanding of the development of osteoarthritis.

Integrative analysis of genome-wide DNA methylation and single-nucleotide polymorphism identified ACSM5 as a suppressor of lumbar ligamentum flavum hypertrophy.

BACKGROUND: Hypertrophy of ligamentum flavum (HLF) is a common lumbar degeneration disease (LDD) with typical symptoms of low back pain and limb numbness owing to an abnormal pressure on spinal nerves. Previous studies revealed HLF might be caused by fibrosis, inflammatory, and other bio-pathways. However, a global analysis of HLF is needed severely. METHODS: A genome-wide DNA methylation and single-nucleotide polymorphism analysis were performed from five LDD patients with HLF and five LDD patients without HLF. Comprehensive integrated analysis was performed using bioinformatics analysis and the validated experiments including Sanger sequencing, methylation-specific PCR, qPCR and ROC analysis. Furthermore, the function of novel genes in ligamentum flavum cells (LFCs) was detected to explore the molecular mechanism in HLF through knock down experiment, overexpression experiment, CCK8 assay, apoptosis assay, and so on. RESULTS: We identified 69 SNP genes and 735 661 differentially methylated sites that were enriched in extracellular matrix, inflammatory, and cell proliferation. A comprehensive analysis demonstrated key genes in regulating the development of HLF including ACSM5. Furthermore, the hypermethylation of ACSM5 that was mediated by DNMT1 led to downregulation of ACSM5 expression, promoted the proliferation and fibrosis, and inhibited the apoptosis of LFCs. CONCLUSION: This study revealed that DNMT1/ACSM5 signaling could enhance HLF properties in vitro as a potential therapeutic strategy for HLF.

Exosomes derived from PC-3 cells suppress osteoclast differentiation by downregulating miR-148a and blocking the PI3K/AKT/mTOR pathway.

Prostate cancer is a leading malignancy in men that can also disrupt the bone tissue balance. Among all urological cancers, prostate cancer is associated with the highest rate of bone metastases, which can greatly reduce a patient's quality of life. In recent years, cell-derived exosomes, which can contain a wide range of biologically active molecules, have been reported as a novel method of communication among individual cells. However, the specific role that exosomes serve in this disease has not been fully elucidated. The prostate cancer cell line PC-3 were applied in the present study, where its exosomes were isolated to explore their potential effects on osteoclast differentiation. Exosomes are extracellular vesicles secreted by cells. The size of exosomes is 30-150 nm. They have double membrane structure and saucer-like morphology. They contain rich contents (including nucleic acid, protein and lipid) and participate in molecular transmission between cells. The combined results of tartrate-resistant acid phosphatase staining (to identify osteoclasts obtained from human peripheral blood mononuclear cells), reverse transcription-quantitative PCR and western blotting showed that PC-3-derived exosomes attenuated osteoclast differentiation by downregulating marker genes associated with osteoclastic maturation, including V-maf musculoaponeurotic fibrosarcoma oncogene homolog B, matrix metalloproteinase 9 and integrin ß3. microRNA (miR)-148a expression was also found to be downregulated in osteoclasts by PC-3-derived exosomes. In addition, the mTOR and AKT signaling pathways were blocked after exposure to these PC-3 cell-derived exosomes. Therefore, results from the present study suggest that miR-148a mimics may be a new therapeutic approach for the prevention of prostate cancer bone metastases.

Cadmium chloride enhances cisplatin sensitivity in osteosarcoma cells by reducing FOXM1 expression.

Osteosarcoma is a highly malignant disease and is associated with a poor patient prognosis and a high mortality rate. Disease prognosis significantly correlates with chemotherapeutic responses. Cadmium is a heavy metal with specific effects on bone, but its benefits for osteosarcoma treatment have not been characterized. In the present study, cadmium chloride was used to treat MG63 osteosarcoma cells, and their gene expression profiles were assessed by GeneChip technology. We found that forkhead box protein M1 (FOXM1) was downregulated by cadmium chloride, and lentiviral­mediated silencing of FOXM1 confirmed a role for this factor in the cisplatin resistance of MG63 cells. In nude mice, cadmium chloride enhanced the sensitivity of osteosarcoma to cisplatin, an effect mediated by FOXM1. Collectively, these data indicate that cadmium chloride can alter the sensitivity of osteosarcoma cells to cisplatin through FOXM1, highlighting it as a potential therapeutic target and prognostic factor for osteosarcoma.

KIF21B Expression in Osteosarcoma and Its Regulatory Effect on Osteosarcoma Cell Proliferation and Apoptosis Through the PI3K/AKT Pathway.

Osteosarcoma (OS) is the most common malignancy that occurs mainly during childhood and adolescence; however, no clear molecular or biological mechanism has been identified. In this study, we aimed to explore new biomarkers for the early diagnosis, targeted treatment, and prognostic determination of osteosarcoma. We first used bioinformatics analysis to show that KIF21B can be used as a biomarker for the diagnosis and prognosis of osteosarcoma. We then examined the expression of KIF21B in human osteosarcoma tissues and cell lines using immunohistochemistry, western blotting, and qRT-PCR. It was found that KIF21B expression was significantly upregulated in osteosarcoma tissues and cell lines. After knocking down the expression of KIF21B in the osteosarcoma cell lines 143B and U2-OS, we used cell fluorescence counting, CCK-8 assays, flow cytometry, and TUNEL staining to examine the effects of KIF21B on osteosarcoma cell proliferation and apoptosis. The results demonstrated that knocking down KIF21B in 143B and U2-OS cells could increase cell apoptosis, inhibit cell proliferation, and reduce tumor formation in nude mice. Subsequently, we used gene chips and bioinformatics to analyze the differential gene expression caused by knocking down KIF21B. The results showed that KIF21B may regulate OS cell proliferation and apoptosis by targeting the PI3K/AKT pathway. We then examined the expression of PI3K/AKT- and apoptosis-related proteins using western blotting. KIF21B knockdown inhibited the PI3K pathway, downregulated Bcl-2, and upregulated Bax. Moreover, the use of PI3K/AKT pathway agonists reversed the regulatory effect of KIF21B on the apoptosis and proliferation of 143B and U2-OS cells. In conclusion, our results indicated that KIF21B plays a key role in osteosarcoma. Low KIF21B expression might indirectly increase the apoptosis and inhibit the proliferation of osteosarcoma cells through the PI3K/AKT pathway.

The effects of tourniquet use in total knee arthroplasty: a randomized, controlled trial.

PURPOSE: Tourniquets are still widely used in total knee arthroplasty (TKA), although they may be associated with several adverse effects. An observer-blinded, randomized, controlled trial was performed to evaluate the effects of tourniquet use in TKA. METHODS: Fifty participants who underwent staged bilateral TKA were recruited for this study. The first-side TKA was randomly allocated to either long-duration tourniquet use or short-duration tourniquet use followed by a 3-month washout period and crossover to the other tourniquet strategy for the opposite-side TKA. Blood loss was monitored perioperatively. The operating time, allogeneic blood transfusion rate, thigh pain, knee pain, limb swelling, clinical outcome as measured by the Likert-type Western Ontario and McMaster University (WOMAC) score, straight leg raising and knee active range of motion (ROM) were also recorded. RESULTS: The long-duration tourniquet group exhibited reduced total blood loss [-99.1 ml, 95 % confidence interval (CI) -168.1 to -30.1, P = 0.0411] and intraoperative blood loss (-225.2 ml, 95 % CI -369.5 to -80.9, P = 0.0071) compared with the short-duration tourniquet group. However, there were greater postoperative blood loss (69.6 ml, 95 % CI 21.1 to 118.2, P = 0.0282) and hidden blood loss (52.8 ml, 95 % CI 10.5 to 95.1, P = 0.0332) in the long-duration tourniquet group. The short-duration tourniquet group showed better outcomes for thigh and knee pain, limb swelling, WOMAC score at 6-week follow-up, straight leg raising and knee ROM. Similar allogeneic blood transfusion rates were observed for both groups. CONCLUSION: Total and intraoperative blood losses were reduced with the long-duration tourniquet use, whereas the short-duration tourniquet use would reduce postoperative and hidden blood losses without increasing the allogeneic blood transfusion rate. In addition, short-duration tourniquet use would result in faster recovery and less pain during the early rehabilitation period following TKA. LEVEL OF EVIDENCE: I.