Nanofiber-induced hierarchically-porous magnesium phosphate bone cements accelerate bone regeneration by inhibiting Notch signaling.

Magnesium phosphate bone cements (MPC) have been recognized as a viable alternative for bone defect repair due to their high mechanical strength and biodegradability. However, their poor porosity and permeability limit osteogenic cell ingrowth and vascularization, which is critical for bone regeneration. In the current study, we constructed a novel hierarchically-porous magnesium phosphate bone cement by incorporating extracellular matrix (ECM)-mimicking electrospun silk fibroin (SF) nanofibers. The SF-embedded MPC (SM) exhibited a heterogeneous and hierarchical structure, which effectively facilitated the rapid infiltration of oxygen and nutrients as well as cell ingrowth. Besides, the SF fibers improved the mechanical properties of MPC and neutralized the highly alkaline environment caused by excess magnesium oxide. Bone marrow stem cells (BMSCs) adhered excellently on SM, as illustrated by formation of more pseudopodia. CCK8 assay showed that SM promoted early proliferation of BMSCs. Our study also verified that SM increased the expression of OPN, RUNX2 and BMP2, suggesting enhanced osteogenic differentiation of BMSCs. We screened for osteogenesis-related pathways, including FAK signaing, Wnt signaling and Notch signaling, and found that SM aided in the process of bone regeneration by suppressing the Notch signaling pathway, proved by the downregulation of NICD1, Hes1 and Hey2. In addition, using a bone defect model of rat calvaria, the study revealed that SM exhibited enhanced osteogenesis, bone ingrowth and vascularization compared with MPC alone. No adverse effect was found after implantation of SM in vivo. Overall, our novel SM exhibited promising prospects for the treatment of critical-sized bone defects.

ZNF692 promotes osteosarcoma cell proliferation, migration, and invasion through TNK2-mediated activation of the MEK/ERK pathway.

BACKGROUND: Osteosarcoma is a diverse and aggressive bone tumor. Driver genes regulating osteosarcoma initiation and progression remains incompletely defined. Zinc finger protein 692 (ZNF692), a kind of Krüppel C2H2 zinc finger transcription factor, exhibited abnormal expression in different types of malignancies and showed a correlation with the clinical prognosis of patients as well as the aggressive characteristics of cancer cells. Nevertheless, its specific role in osteosarcoma is still not well understood. METHODS: We investigated the dysregulation and clinical significance of ZNF692 in osteosarcoma through bioinformatic method and experimental validation. A range of in vitro assays, including CCK-8, colony formation, EdU incorporation, wound healing, and transwell invasion tests, were conducted to assess the impact of ZNF692 on cell proliferation, migration, and invasion in osteosarcoma. A xenograft mouse model was established to evaluate the effect of ZNF692 on tumor growth in vivo. Western blot assay was used to measure the protein levels of MEK1/2, P-MEK1/2, ERK1/2, and P-ERK1/2 in cells that had been genetically modified to either reduce or increase the expression of ZNF692. The relationship between ZNF692 and tyrosine kinase non-receptor 2 (TNK2) were validated by qRT-PCR, chromatin immunoprecipitation and luciferase reporter assays. RESULTS: Expression of ZNF692 was increased in both human osteosarcoma tissues and cell lines. Furthermore, the expression of ZNF692 served as an independent predictive biomarker in osteosarcoma. The results of the survival analysis indicated that increased expression of ZNF692 was associated with worse outcome. Downregulation of ZNF692 inhibits the proliferation, migration, and invasion of osteosarcoma cells, whereas upregulation of ZNF692 has the opposite impact. Western blot assay indicates that reducing ZNF692 decreases phosphorylation of MEK1/2 and ERK1/2, whereas increasing ZNF692 expression enhances their phosphorylation. U0126, a potent inhibitor specifically targeting the MEK/ERK signaling pathway, partially counteracts the impact of ZNF692 overexpression on the proliferation, migration, and invasion of osteosarcoma cells. In addition, ZNF692 specifically interacts with the promoter region of TNK2 and stimulates the transcription of TNK2 in osteosarcoma cells. Forcing the expression of TNK2 weakens the inhibitory impact of ZNF692 knockdown on P-MEK1/2 and P-ERK1/2. Similarly, partly inhibiting TNK2 counteracts the enhancing impact of ZNF692 overexpression on the phosphorylation of MEK1/2 and ERK1/2. Functional tests demonstrate that the suppressive effects of ZNF692 knockdown on cell proliferation, migration, and invasion are greatly reduced when TNK2 is overexpressed. In contrast, the reduction of TNK2 hinders the ability of ZNF692 overexpression to enhance cell proliferation, migration, and invasion. CONCLUSION: ZNF692 promotes the proliferation, migration, and invasion of osteosarcoma cells via the TNK2-dependent stimulation of the MEK/ERK signaling pathway. The ZNF692-TNK2 axis might potentially function as a possible predictive biomarker and a promising target for novel therapeutics in osteosarcoma.

Surface functionalization of calcium magnesium phosphate cements with alginate sodium for enhanced bone regeneration via TRPM7/PI3K/Akt signaling pathway.

Although calcium­magnesium phosphate cements (CMPCs) have been widely applied to treating critical-size bone defects, their repair efficiency is unsatisfactory owing to their weak surface bioactivity and uncontrolled ion release. In this study, we lyophilized alginate sodium (AS) as a coating onto HAp/K-struvite (H@KSv) to develop AS/HAp/K-struvite (AH@KSv), which promotes bone regeneration. The compressive strength and hydrophilicity of AH@KSv significantly improved, leading to enhanced cell adhesion in vitro. Importantly, the SA coating enables continuous ions release of Mg2+ and Ca2+, finally leading to enhanced osteogenesis in vitro/vivo and different patterns of new bone ingrowth in vivo. Furthermore, these composites increased the expression levels of biomarkers of the TRPM7/PI3K/Akt signaling pathway via an equilibrium effect of Mg2+ to Ca2+. In conclusion, our study provides novel insights into the mechanisms of Mg-based biomaterials for bone regeneration.

Exploring the Osteogenic Potential of Zinc-Doped Magnesium Phosphate Cement (ZMPC): A Novel Material for Orthopedic Bone Defect Repair.

In orthopedics, the repair of bone defects remains challenging. In previous research reports, magnesium phosphate cements (MPCs) were widely used because of their excellent mechanical properties, which have been widely used in the field of orthopedic medicine. We built a new k-struvite (MPC) cement obtained from zinc oxide (ZnO) and assessed its osteogenic properties. Zinc-doped magnesium phosphate cement (ZMPC) is a novel material with good biocompatibility and degradability. This article summarizes the preparation method, physicochemical properties, and biological properties of ZMPC through research on this material. The results show that ZMPC has the same strength and toughness (25.3 ± 1.73 MPa to 20.18 ± 2.11 MPa), that meet the requirements of bone repair. Furthermore, the material can gradually degrade (12.27% ± 1.11% in 28 days) and promote osteogenic differentiation (relative protein expression level increased 2-3 times) of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. In addition, in vivo confirmation revealed increased bone regeneration in a rat calvarial defect model compared with MPC alone. Therefore, ZMPC has broad application prospects and is expected to be an important repair material in the field of orthopedic medicine.

A Bioactive Gelatin-Methacrylate Incorporating Magnesium Phosphate Cement for Bone Regeneration.

Maintaining proper mechanical strength and tissue volume is important for bone growth at the site of a bone defect. In this study, potassium magnesium phosphate hexahydrate (KMgPO4·6H2O, MPC) was applied to gelma-methacrylate hydrogel (GelMA) to prepare GelMA/MPC composites (GMPCs). Among these, 5 GMPC showed the best performance in vivo and in vitro. These combinations significantly enhanced the mechanical strength of GelMA and regulated the degradation and absorption rate of MPC. Considerably better mechanical properties were noted in 5 GMPC compared with other concentrations. Better bioactivity and osteogenic ability were also found in 5 GMPC. Magnesium ions (Mg2+) are bioactive and proven to promote bone tissue regeneration, in which the enhancement efficiency is closely related to Mg2+ concentrations. These findings indicated that GMPCs that can release Mg2+ are effective in the treatment of bone defects and hold promise for future in vivo applications.

SERPINH1 enhances the malignancy of osteosarcoma via PI3K-Akt signaling pathway.

BACKGROUND: Serpin Family H Member 1 (SERPINH1) may be involved in the regulation of occurrence and development of tumors. However, the role and mechanism of SERPINH1 in osteosarcoma remain poorly understood. The aim of this study is to investigate the expression and role of SRPINH1 in osteosarcoma and to elucidate its underlying mechanisms. METHODS: First, we examined the expression of SERPINH1 in osteosarcoma and analyzed publicly available datasets to investigate whether SERPINH1 expression was associated with the prognosis of osteosarcoma. Then we constructed SERPINH1 overexpression and knockdown systems in osteosarcoma cells, and examined the proliferation, migration and invasion ability of osteosarcoma cells after SERPINH1 expression changes using CCK-8 assay, wound healing assay and transwell invasion assay. In addition, we constructed a subcutaneous xenograft tumor model to study the function of SERPINH1 in vivo. We also examined the downstream pathways of SERPINH1 by functional analysis and performed subsequent validation. RESULTS: SERPINH1 was upregulated and associated with poor survival in patients with osteosarcoma. SERPINH1 promoted the proliferation, migration and invasion of osteosarcoma cells and promotes the growth of osteosarcoma in vivo by activating the PI3K-Akt signaling pathway. CONCLUSION: SERPINH1 partakes in the biological process of osteosarcoma as a tumor promotor and may be an emerging biomarker in osteosarcoma.

Construction of a prognostic risk score model based on the ARHGAP family to predict the survival of osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignancy of bone tumors. More and more ARHGAP family genes have been confirmed are to the occurrence, development, and invasion of tumors. However, its significance in osteosarcoma remains unclear. In this study, we aimed to identify the relationship between ARHGAP family genes and prognosis in patients with OS. METHODS: OS samples were retrieved from the TCGA and GEO databases. We then performed LASSO regression analysis and multivariate COX regression analysis to select ARHGAP family genes to construct a risk prognosis model. We then validated this prognostic model. We utilized ESTIMATE and CIBERSORT algorithms to calculate the stroma and immune scores of samples, as well as the proportions of tumor infiltrating immune cells (TICs). Finally, we conducted in vivo and in vitro experiments to investigate the effect of ARHGAP28 on osteosarcoma. RESULTS: We selected five genes to construct a risk prognosis model. Patients were divided into high- and low-risk groups and the survival time of the high-risk group was lower than that of the low-risk group. The high-risk group in the prognosis model constructed had relatively poor immune function. GSEA and ssGSEA showed that the low-risk group had abundant immune pathway infiltration. The overexpression of ARHGAP28 can inhibit the proliferation, migration, and invasion of osteosarcoma cells and tumor growth in mice, and IHC showed that overexpression of ARHGAP28 could inhibit the proliferation of tumor cells. CONCLUSION: We constructed a risk prognostic model based on five ARHGAP family genes, which can predict the overall survival of patients with osteosarcoma, to better assist us in clinical decision-making and individualized treatment.

RILP inhibits tumor progression in osteosarcoma via Grb10-mediated inhibition of the PI3K/AKT/mTOR pathway.

BACKGROUND: Rab-interacting lysosomal protein (RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of RILP in osteosarcoma cells and tissues to determine the effect of RILP on the biological behaviors of osteosarcoma cells and the underlying mechanism. METHODS: Tumor Immune Estimation Resource (TIMER) database, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used for bioinformatic analysis. Co-immunoprecipitation experiment was used to determine whether the two proteins were interacting. In functional tests, cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell invasion assay, Immunofluorescence (IF) assay and immunohistochemical (IHC) assay were performed. RESULTS: Overexpression of RILP significantly inhibited proliferation and impaired metastasis ability of osteosarcoma cells, while silencing of RILP showed the opposite trend. RNA-seq data analysis was applied in 143B cells and pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the PI3K/AKT pathway. We further verified that overexpression of RILP restrained the PI3K/AKT/mTOR signaling pathway and induced autophagy in osteosarcoma cells, while the opposite trend was observed when PI3K pathway activator 740Y-P was used. 3-Methyladenine (3-MA), a selective autophagy inhibitor, partially attenuated the inhibitory effect of RILP on the migration and invasion ability of osteosarcoma cells, suggesting the involvement of autophagy in epithelial-mesenchymal transition regulation in osteosarcoma cells. Growth factor receptor binding protein-10 (Grb10), an adaptor protein, was confirmed as a potential target of RILP to restrain the PI3K/AKT signaling pathway. We subcutaneously injected stably overexpressing 143B osteosarcoma cells into nude mice and observed that overexpression of RILP inhibited tumor growth by inhibiting the PI3K/AKT/mTOR pathway. CONCLUSION: Our study revealed that the expression of RILP was associated with favorable prognosis of osteosarcoma and RILP inhibits proliferation, migration, and invasion and promotes autophagy in osteosarcoma cells via Grb10-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. In the future, targeting RILP may be a potential strategy for osteosarcoma treatment.

Zyxin Inhibits the Proliferation, Migration, and Invasion of Osteosarcoma via Rap1-Mediated Inhibition of the MEK/ERK Signaling Pathway.

Zyxin (ZYX) is an actin-interacting protein with unknown biological functions in patients with osteosarcoma. This research sought to understand how ZYX affects the biological behavior of osteosarcoma cells and to identify the associated mechanism. Firstly, ZYX expression was decreased in osteosarcoma, and its higher expression indicated better outcomes in patients with osteosarcoma. ZYX overexpression significantly inhibited the proliferation, migration, and invasion of osteosarcoma cells, whereas ZYX silencing resulted in the opposite trend. Subsequently, we found that the Rap1 signaling pathway was significantly correlated with ZYX expression as reported in The Cancer Genome Atlas's database using bioinformatic analysis. Moreover, we found that ZYX overexpression regulated the Rap1/MEK/ERK axis, and osteosarcoma cell growth, migration, and invasion were consequently restrained. Additionally, by administering tumor cells subcutaneously to nude mice, a mouse model of transplanted tumors was created. Compared to the control group, the ZYX overexpression group's tumors were lighter and smaller, and the ZYX/Rap1 axis was activated in the ZYX overexpression group. Taken together, our results suggest that ZYX inhibits osteosarcoma cell proliferation, migration, and invasion by regulating the Rap1/MEK/ERK signaling pathway. ZYX might be crucial in the clinical management of osteosarcoma and is a promising novel therapeutic target in patients with this disease.

TRIM26 inhibited osteosarcoma progression through destabilizing RACK1 and thus inactivation of MEK/ERK signaling.

Osteosarcoma is a highly aggressive malignant tumor that is common in the pediatric population and has a high rate of disability and mortality. Recent studies have suggested that the tripartite motif-containing family genes (TRIMs) play critical roles in oncogenesis in several cancers. TRIM26, one of the TRIMs family genes, was more frequently reported to exert a tumor-suppressive role, while its detailed functional roles in the osteosarcoma progression were still unknown and require further investigation. Herein, we found that TRIM26 was markedly downregulated in osteosarcoma tissues and cells. Survival analysis revealed that higher expression of TRIM26 was associated with better prognosis and its expression was an independent protective factor in osteosarcoma. Functional analysis demonstrated that overexpression of TRIM26 inhibited osteosarcoma cell proliferation and invasion via inhibiting the EMT process and MEK/ERK signaling. In contrast, the silence of TRIM26 caused the opposite effect. RACK1, a member of the Trp-Asp repeat protein family, was identified as a novel target of TRIM26. TRIM26 could interact with RACK1 and accelerate the degradation of RACK1, thus inactivation of MEK/ERK signaling. Overexpression of RACK1 could attenuate the inhibitory effect of TRIM26 overexpression on p-MEK1/2 and p-ERK1/2, and silence of RACK1 could partly impair the effect of TRIM26 knockdown-induced upregulation of p-MEK1/2 and p-ERK1/2. Further, a series of gain- and loss-of-function experiments showed that decreased malignant behaviors including cell proliferation and invasion in TRIM26-upregulated cells were reversed when RACK1 was overexpressed, whereas RACK1 knockdown diminished the increased malignant phenotypes in TRIM26-silenced osteosarcoma cells. In conclusion, our study indicated that TRIM26 inhibited osteosarcoma progression via promoting proteasomal degradation of RACK1, thereby resulting in inactivation of MEK/ERK signaling, and impeding the EMT process.

Isoquercitrin restrains the proliferation and promotes apoptosis of human osteosarcoma cells by inhibiting the Wnt/ß-catenin pathway.

Currently, chemotherapeutic drugs are widely used for the treatment of osteosarcoma. However, many of these drugs exhibit shortcomings such as poor efficacy, high toxicity, and tolerance. Isoquercitrin (ISO) is a traditional Chinese medicine that has been proved to exert good therapeutic effects on various tumors; however, its role in osteosarcoma has not been reported. Here, we observed that ISO exerted a marked inhibitory effect on the occurrence and development of osteosarcoma in a time- and dose-dependent manner. First, we determined that ISO significantly inhibited proliferation, induced EMT-related migration and invasion and induced apoptosis of osteosarcoma cells in vitro. Concurrently, we also observed that both ß-catenin and its downstream genes (c-Myc, CyclinD1, and Survivin) were significantly down-regulated. To verify if the anti-tumor effect of ISO was related to the Wnt/ß-catenin signaling pathway, we altered the protein expression level of ß-catenin using recombinant lentivirus, then we observed that the effects of ISO on the proliferation, metastasis, and apoptosis of osteosarcoma cells were significantly reversed. Additionally, we used a nude mouse xenograft model and observed that ISO significantly inhibited the growth of osteosarcoma and improved the survival rate of the animal models. In conclusion, this study demonstrates that ISO can exert anti-tumor effects in part by inhibiting the Wnt/ß-catenin signaling pathway, thus providing a new potential therapeutic strategy for the treatment of osteosarcoma.

Carboxymethyl chitosan-alginate enhances bone repair effects of magnesium phosphate bone cement by activating the FAK-Wnt pathway.

There is a continuing need for artificial bone substitutes for bone repair and reconstruction, Magnesium phosphate bone cement (MPC) has exceptional degradable properties and exhibits promising biocompatibility. However, its mechanical strength needs improved and its low osteo-inductive potential limits its therapeutic application in bone regeneration. We functionally modified MPC by using a polymeric carboxymethyl chitosan-sodium alginate (CMCS/SA) gel network. This had the advantages of: improved compressive strength, ease of handling, and an optimized interface for bioactive bone in-growth. The new composites with 2% CMCS/SA showed the most favorable physicochemical properties, including mechanical strength, wash-out resistance, setting time, injectable time and heat release. Biologically, the composite promoted the attachment and proliferation of osteoblast cells. It was also found to induce osteogenic differentiation in vitro, as verified by expression of osteogenic markers. In terms of molecular mechanisms, data showed that new bone cement activated the Wnt pathway through inhibition of the phosphorylation of ß-catenin, which is dependent on focal adhesion kinase. Through micro-computed tomography and histological analysis, we found that the MPC-CMCS/SA scaffolds, compared with MPC alone, showed increased bone regeneration in a rat calvarial defect model. Overall, our study suggested that the novel composite had potential to help repair critical bone defects in clinical practice.

Functionalized Magnesium Phosphate Cement Induces In Situ Vascularized Bone Regeneration via Surface Lyophilization of Chondroitin Sulfate.

Bone defect repair poses significant challenges in orthopedics, thereby increasing the demand for bone substitutes. Magnesium phosphate cements (MPCs) are widely used for bone defect repair because of their excellent mechanical properties and biodegradability. However, high crystallinity and uncontrolled magnesium ion (Mg2+) release limit the surface bioactivity of MPCs in bone regeneration. Here, we fabricate chondroitin sulfate (CS) as a surface coating via the lyophilization method, namely CMPC. We find that the CS coating is uniformly distributed and improves the mechanical properties of MPC through anionic electrostatic adsorption, while mediating degradation-related controlled ion release of Mg2+. Using a combination of in vitro and in vivo analyses, we show that the CS coating maintained cytocompatibility while increasing the cell adhesion area of MC3T3-E1s. Furthermore, we display accelerated osteogenesis and angiogenesis of CMPC, which are related to appropriate ion concentration of Mg2+. Our findings reveal that the preparation of a lyophilized CS coating is an effective method to promote surface bioactivity and mediate Mg2+ concentration dependent osteogenesis and angiogenesis, which have great potential in bone regeneration.

CircDOCK1 Regulates miR-186/DNMT3A to Promote Osteosarcoma Progression.

BACKGROUND: Circular RNAs (circRNAs), as a class of endogenous RNAs, are implicated in osteosarcoma (OS) progression. However, the functional properties of circDOCK1 in OS have been largely unexplored. The present study demonstrated the regulatory mechanism of circDOCK1 in OS. METHODS: QRT-PCR and Western blots were used to determine the abundances of circDOCK1, miR-186, and DNMT3A. Cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), colony formation, Transwell, and wound healing assays were used to examine cellular multiplication, motility, and invasion. Luciferase reporter analysis, RNA immunoprecipitation (RIP), and pull-down assays were used to verify target relationships. Xenograft models were used to analyze in vivo function. RESULTS: OS tissues and cells showed high levels of circDOCK1. By knocking down circDOCK1, cellular multiplication, motility, and invasion were suppressed. Furthermore, silencing circDOCK1 suppressed the growth of tumor xenografts. According to mechanistic studies, miR-186 targets DNA methyltransferases 3A (DNMT3A) directly and acts as a circDOCK1 target. Furthermore, circDOCK1 upregulated DNMT3A expression through sponging miR-186 to regulate the progression of OS. CONCLUSIONS: CircDOCK1 promotes OS progression by interacting with miR-186/DNMT3ADNMT3A, representing a novel therapeutic approach.

Identification of a novel gene signature with regard to ferroptosis, prognosis prediction, and immune microenvironment in osteosarcoma.

Ferroptosis is a novel form of non-apoptotic cell death that mainly results from the iron-dependent lethal accumulation of lipid peroxidation products. Here, we defined differentially expressed genes between control and RSL3-treated osteosarcoma cells as ferroptosis-associated genes (FAGs). These FAGs were then subjected to weighted gene correlation network analysis (WGCNA), and we found that the turquoise module, containing 71 FAGs, was markedly related to the patient's vital status. After that, FAGs in the turquoise module were utilized to construct a prognostic multigene (COL5A2, HOXB4, and UNC5B) signature for risk stratification in osteosarcoma. Validation in internal and external cohorts indicated the accuracy and clinical applicability of this signature in predicting the prognosis of patients with osteosarcoma. Univariate and multivariate Cox regression analyses suggested that the signature-derived risk score is an independent indicator of patient prognosis. Immunological analysis indicated that significant variations in stromal and ESTIMATE scores, as well as tumor purity, were found when the high- and low-risk groups were compared. Regarding immune cell infiltration, the proportion of activated CD4 memory T cells was significantly lower in the high-risk group than that in the low-risk group. The ssGSEA results suggested that CD8+ T, Tfh, and Th1 cell scores were consistently lower in the high-risk group than those in the low-risk group. In terms of immune-related activities, the high-risk group had considerably lower scores for promoting inflammation, T-cell co-inhibition, and T-cell co-stimulation than the low-risk group, indicating the differential immunological state of the high- and low-risk groups. Of the three FAGs included in the signature, the expression of COL5A2, HOXB4, and UNC5B was higher in the high-risk groups, and the expression of COL5A2 and UNC5B was negatively associated with patient prognosis. Additionally, the mRNA levels of COL5A2 and HOXB4 were lower and those of UNC5B were higher in RSL3-treated cells than in control cells. In all, we systematically analyzed the transcriptional changes of osteosarcoma cells induced by RSL3 and constructed a novel three-gene signature with regard to ferroptosis, prognosis prediction, and immune microenvironment. We also identified COL5A2, HOXB4, and UNC5B as potential therapeutic targets and important regulators of ferroptosis in osteosarcoma.

TIAM2 promotes proliferation and invasion of osteosarcoma cells by activating the JAK2/STAT3 signaling pathway.

T-cell lymphoma invasion and metastasis 2 (TIAM2) plays a critical role in the malignancy development of many human cancers. However, the specific regulatory mechanism of TIAM2 in osteosarcoma has not yet been explored. In this study, we investigated how TIAM2 affects the proliferation and invasion of osteosarcoma cells and the underlying molecular mechanism. We performed data mining of publicly available datasets to examine whether the expression of TIAM2 is associated with osteosarcoma. We knocked down the expression of and overexpressed TIAM2 in osteosarcoma cells. The proliferative capacity of cells in each group was determined by the Cell Counting Kit-8 assay. Wound healing and Transwell invasion assays were performed to evaluate the migration and invasion abilities of the TIAM2 knockdown and overexpressed osteosarcoma cells. Determination of the function of TIAM2 in vivo was performed in nude mice. Data mining confirmed that TIAM2 expression is associated with poor prognosis in osteosarcoma. TIAM2 expression levels were significantly higher in osteosarcoma cells, and TIAM2 expression knockdown reduced proliferation and invasion abilities. Animal experiments demonstrated that TIAM2 promotes tumor growth. Additional experiments suggested that TIAM2 was significantly related to the activation of the JAK2/STAT3 pathway, and subsequent mechanistic experiments further confirmed this. Our findings suggest that TIAM2 promotes the proliferation and invasion capacities of osteosarcoma cells by activating the JAK2/STAT3 signaling pathway. TIAM2 may serve as a potential prognostic target for patients with OS.

Long non-coding RNA NR2F2-AS1 regulates human osteosarcoma growth and metastasis through miR-425-5p-mediated HMGB2.

BACKGROUND: Multiple studies have revealed that long non-coding RNA (lncRNA) NR2F2-AS1 plays a role in affecting cancer cell proliferation and metastasis. Here, both in vitro and in vivo experiments were performed for investigating the function and mechanism of NR2F2-AS1 in human osteosarcoma (OS). METHODS: The NR2F2-AS1 level in human OS tissues and adjacent non-tumor tissues was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The NR2F2-AS1 overexpression model was constructed in OS cells, then cell proliferation, invasion, and apoptosis were monitored. The OS xenograft model was established in nude mice using NR2F2-AS1-overexpressed OS cells. The downstream target genes of NR2F2-AS1 were predicted. qRT-PCR and Western blot were implemented to validate the profiles of miR-425-5p and HMGB2. The targeting link between NR2F2-AS1 and miR-425-5p, miR-425-5p and HMGB2 was further probed by dual-luciferase reporter experiment. RESULTS: In comparison to adjacent non-tumor tissues, OS tissues showed upregulated NR2F2-AS1 expression. Higher NR2F2-AS1 level was predominantly correlated with worse clinical stages. In vivo and in vitro tests corroborated that NR2F2-AS1 overexpression spurred OS cell proliferation, growth, invasion, and choked apoptosis. Mechanistically, NR2F2-AS1 hampered miR-425-5p expression as its competitive endogenous RNA (ceRNA). Thus, NR2F2-AS1 facilitated the HMGB2 expression. However, miR-425-5p inhibited HMGB2 expression by targeting the latter. CONCLUSION: NR2F2-AS1 expedited the evolution of OS by elevating HMGB2 levels through sponging miR-425-5p. The NR2F2-AS1/miR-425-5p/HMGB2 regulatory axis is a promising target in treating human OS.

Unbiased transcriptome mapping and modeling identify candidate genes and compounds of osteoarthritis.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage loss, subchondral bone remodeling, and synovial inflammation. Given that the current therapies for advanced OA patients are limited, the understanding of mechanisms and novel therapies are urgently needed. In this study, we employed the weighted gene co-expression network (WGCNA) method and the connectivity map (CMap) database to identify the candidate target genes and potential compounds. Four groups of co-expressing genes were identified as the OA-related modules. The biological annotations of these modules indicated some critical hallmarks of OA and aging, such as mitochondrial dysfunctions and abnormal energy metabolism, and the signaling pathways, such as MAPK, TNF, and PI3K/Akt signaling pathways. Some genes, such as RELA and GADD45B, were predicted to extensively involve these critical pathways, indicating their potential functions in OA mechanisms. Moreover, we constructed the co-expressing networks of modules and identified the hub genes based on network topology. GADD45B, MAFF, and MYC were identified and validated as the hub genes. Finally, anisomycin and MG-262 were predicted to target these OA-related modules, which may be the potential drugs for OA therapy. In conclusion, this study identified the significant modules, signaling pathways, and hub genes relevant to OA and highlighted the potential clinical value of anisomycin and MG-262 as novel therapies in OA management.

XIST sponges miR-320d to promote chordoma progression by regulating ARF6.

Background: Long non-coding RNAs (lncRNAs) have been demonstrated to play important roles in various tumors, including chordoma. The purpose of this study was to investigate the role and mechanism of lncRNA X-inactive specific transcript (XIST) in chordoma. Methods: RNA levels and protein levels were measured by real-time quantitative polymerase chain reaction (RT­qPCR) and western blot assay, respectively. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay. Tanswell assay was used to examine cell migration and invasion. Cellular glycolysis was examined via the measurement of extracellular acidification rate (ECAR) and lactate production. The interaction between microRNA-320d (miR-320d) and XIST or ADP-ribosylation factor 6 (ARF6) was predicted by bioinformatics analysis and verified by a dual-luciferase reporter and RNA-pull down assays. The xenograft tumor model was used to explore the biological function of XIST in vivo. Results: XIST was overexpressed in chordoma tissues. XIST knockdown suppressed chordoma cell proliferation, migration, invasion, and glycolysis. XIST acted as a sponge of miR-320d. Moreover, miR-320d overexpression inhibited the proliferation, migration, invasion, and glycolysis of chordoma cells. ARF6 was a direct target of miR-320d, and XIST upregulated ARF6 expression via sponging miR-320d. Furthermore, overexpression of ARF6 reversed the inhibitory effects of XIST knockdown on chordoma cell proliferation, migration, invasion, and glycolysis. Importantly, XIST silencing blocked xenograft tumor growth in vivo. Conclusion: XIST knockdown inhibited chordoma progression via regulating the miR-320d/ARF6 axis, providing a novel insight into chordoma pathogenesis.

Identification of a Solute Carrier Family-Based Signature for Predicting Overall Survival in Osteosarcoma.

Given the important role of SLC family in essential physiological processes including nutrient uptake, ion transport, and waste removal, and that their dysregulation was found in distinct forms of cancer, here we identified a novel gene signature of SLC family for patient risk stratification in osteosarcoma. Gene expression data and relevant clinical materials of osteosarcoma samples were retrieved from The Cancer Genome Atlas (TCGA) database. Prognosis-related SLC genes were identified by performing univariate Cox regression analysis and were utilized to construct a four-SLC gene signature in osteosarcoma. It allowed patients to be classified into high- and low-risk groups, and Kaplan-Meier survival analysis in the training, testing, entire, and external GSE21257 cohorts suggested that the overall survival of patients in high-risk group was consistently worse than that in low-risk group, suggesting the promising accuracy and generalizability of the SLC-based signature in predicting the prognosis of patients with osteosarcoma. Moreover, univariate and multivariate Cox regression analyses indicated that the derived risk score was the only independent prognostic factor for osteosarcoma patients in TCGA and GSE21257 cohorts. Besides, a prognostic nomogram comprising the derived risk score and clinical features including gender and age was developed for clinical decision-making. Functional enrichment analyses of the differentially expressed genes between high- and low-risk group revealed that immune-related biological processes and pathways were significantly enriched. Estimation of tumor immune microenvironment using ESTIMATE algorithm revealed that patients with lower risk score had higher stromal, immune, and ESTIMATE score, and lower tumor purity. ssGSEA analyses indicated that the scores of various immune subpopulations including CD8+ T cells, DCs, and TIL were lower in high-risk group than these in low-risk group in both cohorts. As for the related immune functions, the scores of APC co-inhibition, CCR, check-point, T cell co-stimulation, and Type II IFN response were lower in high-risk group than these in low-risk group in both cohorts. In all, we identified a novel prognostic signature based on four SLC family genes that accurately predicted overall survival in osteosarcoma patients. Furthermore, the signature is linked to differences in immunological status and immune cell infiltrations in the tumor microenvironment.