CTGF regulates mineralization in human mature chondrocyte by controlling Pit-1 and modulating ANK via the BMP/Smad signalling.

OBJECTIVE: Connective tissue growth factor (CTGF) exhibits potent proliferative, differentiated, and mineralizing effects, and is believed to be contribute to cartilage mineralization in Osteoarthritis (OA). However, the underlying mechanism of chondrocyte mineralization induced by CTGF remains obscure. As a key regulator of mineral responses, type III phosphate transporter 1 (Pit-1) has been associated with the pathogenesis of articular mineralization. Therefore, the primary objective of this study was to investigate whether CTGF influences the development of mature chondrocyte mineralization and the underlying mechanisms governing such mineralization. METHODS: The effect of Connective tissue growth factor (CTGF) on human C-28/I2 chondrocytes were investigated. The chondrocytes were treated with CTGF or related inhibitors, and transfected with Overexpression and siRNA transfection of Type III Phosphate Transporter 1(Pit-1). Subsequently, the cells were subjected to Alizarin red S staining, PiPer Phosphate Assay Kit, Alkaline Phosphatase Diethanolamine Activity Kit, ELISA, RT-PCR or Western blot analysis. RESULTS: Stimulation with Connective tissue growth factor (CTGF) significantly upregulated the expression of the Type III Phosphate Transporter 1(Pit-1) and mineralization levels in chondrocytes through activation of α5ß1 integrin and BMP/Samd1/5/8 signaling pathways. Furthermore, treatment with overexpressed Pit-1 markedly increased the expression of Multipass Transmembrane Ankylosis (ANK) transporter in the cells. The inhibitory effect of CTGF receptor blockade using α5ß1 Integrin blocking antibody was demonstrated by significantly suppressed the expression of Pit-1 and ANK transporter, as well as chondrocyte mineralization. CONCLUSIONS: Our data indicate that Connective tissue growth factor (CTGF) plays a critical role inchondrocyte mineralization, which is dependent on the expression of the Type III Phosphate Transporter 1(Pit-1) and Multipass Transmembrane Ankylosis (ANK) transporter. Consequently, inhibition of CTGF activity may represent a novel therapeutic approach for the management of Osteoarthritis (OA).

Identification of Cell Subpopulations and Interactive Signaling Pathways From a Single-Cell RNA Sequencing Dataset in Osteosarcoma: A Comprehensive Bioinformatics Analysis.

Osteosarcoma is a type of highly aggressive bone tumor arising from primitive cells of mesenchymal origin in adults and is associated with a high rate of tumor relapse. However, there is an urgent need to clarify the molecular mechanisms underlying osteosarcoma development. The present study performed integrated bioinformatics analysis in a single-cell RNA sequencing dataset and explored the potential interactive signaling pathways associated with osteosarcoma development. Single-cell transcriptomic analysis of osteosarcoma tissues was performed by using the Seurat R package, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes was performed by using the clusterProfiler R package, and the cell-cell interaction analysis was performed by using the CellPhoneDB package. Our results showed that 11 clustered cell types were identified across 11 osteosarcoma tissues, with cell types including "osteoblastic", "myeloid", "osteoblastic_proli", "osteoclast", and "tumor-infiltrating lymphocytes (TILs)" as the main types. The DEGs between different cell types from primary, metastatic, and recurrent osteosarcomas were mainly enriched in the GO terms including "negative regulation of hydrolase activity", "regulation of peptidase activity", "regulation of binding", "negative regulation of proteolysis", and "negative regulation of peptidase activity" and in the KEGG pathways including "transcriptional misregulation in cancer", "cellular senescence", "apoptosis", "FoxO signaling pathway", "cell cycle", "NF-kappa B signaling pathway", "p53 signaling pathway", "pentose phosphate pathway", and "protein export". For the cell-cell communication network analysis, the different interaction profiles between cell types were detected among primary, metastatic, and recurrent osteosarcomas. Further exploration of the KEGG pathway revealed that these ligand/receptor interactions may be associated with the NF-κB signaling pathway and its interacted mediators. In conclusion, the present study for the first time explored the scRNA-seq dataset in osteosarcoma, and our results revealed the 11 clustered cell types and demonstrated the novel cell-cell interactions among different cell types in primary, metastatic, and recurrent osteosarcomas. The NF-κB signaling pathway may play a key role in regulating the TME of osteosarcoma. The present study may provide new insights into understanding the molecular mechanisms of osteosarcoma pathophysiology.

Long non-coding RNA DANCR modulates osteogenic differentiation by regulating the miR-1301-3p/PROX1 axis.

The balance of osteoblasts and marrow adipocytes from bone marrow mesenchymal stem cells (BM-MSCs) maintains bone health. Under aging or other pathological stimuli, BM-MSCs will preferentially differentiate into marrow adipocytes and reduce osteoblasts, leading to osteoporosis. Long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) participates in the osteogenic differentiation of human BM-MSCs, but the mechanism by which DANCR regulates the osteogenic differentiation of human BM-MSCs has not been fully explained. We observed that DANCR and prospero homeobox 1 (PROX1) were downregulated during osteogenic differentiation of human BM-MSCs, while miR-1301-3p had an opposite trend. DANCR overexpression decreased the levels of alkaline phosphatase, RUNX2, osteocalcin, Osterix in BM-MSCs after osteogenic induction, but DANCR silencing had the opposite result. Moreover, DANCR sponged miR-1301-3p to regulate PROX1 expression. miR-1301-3p overexpression reversed the suppressive role of DANCR elevation on the osteogenic differentiation of human BM-MSCs. Also, PROX1 elevation abolished the promoting role of miR-1301-3p overexpression on the osteogenic differentiation of human BM-MSCs. In conclusion, DANCR suppressed the osteogenic differentiation of human BM-MSCs through the miR-1301-3p/PROX1 axis, offering a novel mechanism by which DANCR is responsible for the osteogenic differentiation of human BM-MSCs.

TRPV1 acts as a Tumor Suppressor and is associated with Immune Cell Infiltration in Clear Cell Renal Cell Carcinoma: evidence from integrated analysis.

Differential expression of TRPV1 has been detected in many cancer types, including clear cell renal cell carcinoma (ccRCC). However, the clinical significance of TRPV1 expression profile in ccRCC has not been comprehensively elucidated. In this study, TRPV1 expression in ccRCC and other cancer types was analyzed based on data from the GEO and Oncomine databases. Immunohistochemical (IHC) staining was performed for further validation in human ccRCC tissue chips. Survival and correlation analyses of TRPV1 were conducted using Kaplan-Meier Plotter (KM-Plotter) and the Tumor IMmune Estimation Resource (TIMER) database. TRPV1 exhibited a low expression profile in 2 GEO datasets (GSE6344, GSE36895) and 4 Oncomine datasets (Gumz, Lenburg, Beroukhim 1 and Beroukhim 2), as also confirmed by IHC staining. Survival analysis indicated that high enrichment of TRPV1 significantly predicted a better overall survival (OS) and disease-free survival (DFS) of 1, 3, 5 and 10 years in ccRCC patients. TIMER analysis showed that TRPV1 copy number alterations (CNA) were closely related to immune cell infiltration. The detailed results indicated that TRPV1 expression was positively correlated with the infiltration level of CD4+ T cells, but negatively correlated with B cells, macrophages, and dendritic cells infiltration. In addition, TRPV1 might also be inversely related to abundance of the regulatory T cells (Treg) and the M2 subset of macrophages. Finally, we found that TRPV1 expression was tightly associated with several key molecules of the classical pathways in ccRCC, such as VHL, TP53, HIF1A, MTOR, MAPK1, MET, CTNNB1, etc. Our research work suggests that TRPV1 is a novel tumor suppressor and prognosis marker for ccRCC and is of great value for further exploration.

Identification of Potential Key Genes and Pathways in Enzalutamide-Resistant Prostate Cancer Cell Lines: A Bioinformatics Analysis with Data from the Gene Expression Omnibus (GEO) Database.

Enzalutamide (ENZ) has been approved for the treatment of advanced prostate cancer (PCa), but some patients develop ENZ resistance initially or after long-term administration. Although a few key genes have been discovered by previous efforts, the complete mechanisms of ENZ resistance remain unsolved. To further identify more potential key genes and pathways in the development of ENZ resistance, we employed the GSE104935 dataset, including 5 ENZ-resistant (ENZ-R) and 5 ENZ-sensitive (ENZ-S) PCa cell lines, from the Gene Expression Omnibus (GEO) database. Integrated bioinformatics analyses were conducted, such as analysis of differentially expressed genes (DEGs), Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, protein-protein interaction (PPI) analysis, gene set enrichment analysis (GSEA), and survival analysis. From these, we identified 201 DEGs (93 upregulated and 108 downregulated) and 12 hub genes (AR, ACKR3, GPER1, CCR7, NMU, NDRG1, FKBP5, NKX3-1, GAL, LPAR3, F2RL1, and PTGFR) that are potentially associated with ENZ resistance. One upregulated pathway (hedgehog pathway) and seven downregulated pathways (pathways related to androgen response, p53, estrogen response, TNF-α, TGF-ß, complement, and pancreas ß cells) were identified as potential key pathways involved in the occurrence of ENZ resistance. Our findings may contribute to further understanding the molecular mechanisms of ENZ resistance and provide some clues for the prevention and treatment of ENZ resistance.

Bioinformatics analysis of key genes and pathways in Hashimoto thyroiditis tissues.

Hashimoto thyroiditis (HT) is one of the most common autoimmune diseases, and the incidence of HT continues to increase. Long-term, uncontrollable HT results in thyroid dysfunction and even increases carcinogenesis risks. Since the origin and development of HT involve many complex immune processes, there is no effective therapy for HT on a pathogenesis level. Although bioinformatics analysis has been utilized to seek key genes and pathways of thyroid cancer, only a few bioinformatics studies that focus on HT pathogenesis and mechanisms have been reported. In the present study, the Gene Expression Omnibus dataset (GSE29315) containing 6 HT and 8 thyroid physiological hyperplasia samples was downloaded, and differentially expressed gene (DEG) analysis, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, protein-protein interaction analysis, and gene set enrichment analysis were performed. In total, 85 DEGs, containing 76 up-regulated and 9 down-regulated DEGS, were identified. The DEGs were mainly enriched in immune and inflammatory response, and the signaling pathways were involved in cytokine interaction and cytotoxicity. Moreover, ten hub genes were identified, and IFN-γ, IFN-α, IL6/JAK/STAT3, and inflammatory pathways may promote the origin and progression of HT. The present study indicated that exploring DEGs and pathways by bioinformatics analysis has important significance in understanding the molecular mechanisms of HT and providing potential targets for the prevention and treatment of HT.

Effect of porous tantalum on promoting the osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the MAPK/ERK signal pathway.

BACKGROUND: As an ideal new graft material, porous tantalum (pTa) has excellent mechanical properties and corrosion resistance and has received increased attention in the biomedical field because of its excellent cytocompatibility and ability to induce bone formation. However, the molecular mechanism of its potential to promote osteogenesis remains unclear, and very few reports have been published on this topic. METHODS: In this study, we first produced porous Ti6Al4V (pTi6Al4V) and pTa with the same pore size by three-dimensional printing combined with chemical vapour deposition. The number of adhesions between pTa and pTi6Al4V and bone marrow mesenchymal stem cells (BMSCs) after 1 day of culture was detected by the live/dead cell staining method. The proliferation activity of the two groups was determined after culture for 1, 3, 5 and 7 days by the cell counting kit-8 method. In addition, the osteogenic activity, mRNA expression levels of osteogenic genes alkaline phosphatase (ALP), osterix (OSX), collagen-I (Col-I), osteonectin (OSN) and osteocalcin (OCN) and protein expression levels of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signalling pathway marker p-ERK of the two groups cultured for 7, 14 and 21 days were determined by the ALP activity assay, real-time quantitative polymerase chain reaction (Q-PCR) and Western blotting, respectively. Subsequently, the two groups were treated with the MAPK/ERK-specific inhibitor U0126, and then, the mRNA expression levels of osteogenic genes and protein expression levels of p-ERK in the cultures were determined by Q-PCR and Western blotting, respectively. RESULTS: The live/dead cell staining and cell counting kit-8 assays showed that the adhesion and proliferation activities of BMSCs on pTa were significantly better than those on pTi6Al4V. In addition, the ALP activity assay and Q-PCR showed that pTa harboured osteogenic activity and that the osteogenic genes ALP, OSX, Col-I, OSN and OCN were highly expressed, and by Western blotting, the expression of p-ERK protein in the pTa group was also significantly higher than that in the pTi6Al4V group. Subsequently, using the MAPK/ERK-specific inhibitor U0126, Western blotting showed that the expression of p-ERK protein was significantly inhibited and that there was no difference between the two groups. Furthermore, Q-PCR showed that osteogenic gene expression and ALP expression levels were significantly increased in the pTa group, and there were no differences in the OSX, Col-I, OSN and OCN mRNA expression levels between the two groups. CONCLUSION: Overall, our research found that compared with the widely used titanium alloy materials, our pTa can promote the adhesion and proliferation of BMSCs, and the molecular mechanism of pTa may occur via activation of the MAPK/ERK signalling pathway to regulate the high expression of OSX, Col I, OSN and OCN osteogenic genes and promote the osteogenic differentiation of BMSCs in vitro. The translational potential of this article : Our self-developed pTa material produced by three-dimensional printing combined with the chemical vapour deposition method not only retains excellent biological activity and osteoinductive ability of the original tantalum metal but also saves considerably on material costs to achieve mass production of personalised orthopaedic implants with pTa as a stent and to accelerate the wide application of pTa implants in clinical practice, which have certain profound significance.

Combined use of Serum miR-338-3p, Cartilage Oligomeric Matrix Protein and Chondroitin Sulfate-846 in the Early Diagnosis of Knee Osteoarthritis.

BACKGROUND: In this study, the serum levels of miR-338-3p, COMP, and CS-846 were detected in the patients with KOA and healthy controls, to explore the value of miR-338-3p, COMP, and CS-846 in the early diagnosis of KOA. METHODS: Real-time PCR was carried out to evaluate the level of miR-338-3p in KOA patients and healthy controls. Spearman's correlation coefficient was performed to examine the correlation between the expression of miR-338-3p, COMP, and CS-846. Receiver operating characteristic (ROC) curves were carried out to evaluate the diagnostic values of miR-338-3p, COMP, and CS-846 for KOA patients. RESULTS: In the current study, we first demonstrated that serum miR-338-3p, COMP, and CS-846 levels were increased in KOA patients compared to healthy controls. Moreover, the increase of miR-338-3p, COMP, and CS-846 levels positively correlated with VAS scores and joint space narrowing, suggesting miR-338-3p positively correlated with comprehensive disease severity. In addition, miR-338-3p, COMP, and CS-846 could be used as an independent biomarker for KOA patients. More importantly, combined use of miR-338-3p, COMP, and CS-846 demonstrates a higher diagnostic value with an AUC 0.926 for KOA patients. CONCLUSIONS: The combination of miR-338-3p, COMP, and CS-846 demonstrated higher diagnostic value for KOA patients, indicating their combination as novel and promising biomarkers for diagnosis and disease severity of KOA.

Mesenchymal stem cell-loaded porous tantalum integrated with biomimetic 3D collagen-based scaffold to repair large osteochondral defects in goats.

BACKGROUND: The body is unable to repair and regenerate large area bone defects. Moreover, the repair capacity of articular cartilage is very limited. There has long been a lack of effective treatments for osteochondral lesions. The engineered tissue with biphase synthetic for osteochondral repair has become one of the hot research fields over the past few years. In this study, an integrated biomanufacturing platform was constructed with bone marrow mesenchymal stem cells (BMSCs)/porous tantalum (pTa) associated with chondrocytes/collagen membranes (CM) to repair large osteochondral defects in load-bearing areas of goats. METHODS: Twenty-four goats with a large osteochondral defect in the femoral heads of the left hind legs were randomly divided into three groups: eight were treated with chondrocytes/CM-BMSCs/pTa, eight were treated with pure CM-pTa composite, and the other eight goats were untreated. The repair effect was assessed by X-ray, gross observation, and histomorphology for 16 weeks after the operation. In addition, the biocompatibility of chondrocytes/CM-BMSCs/pTa was observed by flow cytometry, CCK8, immunocytochemistry, and Q-PCR. The characteristics of the chondrocytes/CM-BMSCs/pTa were evaluated using both scanning electron microscopy and mechanical testing machine. RESULTS: The integrated repair material consists of pTa, injectable fibrin sealant, and CM promoted adhesion and growth of BMSCs and chondrocytes. pTa played an important role in promoting the differentiation of BMSCs into osteoblasts. Three-dimensional CM maintained the phenotype of chondrocytes successfully and expressed chondrogenic genes highly. The in vivo study showed that after 16 weeks from implantation, osteochondral defects in almost half of the femoral heads had been successfully repaired by BMSC-loaded pTa associated with biomimetic 3D collagen-based scaffold. CONCLUSIONS: The chondrocytes/CM-BMSCs/pTa demonstrated significant therapeutic efficacy in goat models of large osteochondral defect. This provides a novel therapeutic strategy for large osteochondral lesions in load-bearing areas caused by severe injury, necrosis, infection, degeneration, and tumor resection with a high profile of safety, effectiveness, and simplicity.