MgSiO3 Fiber Membrane Scaffold with Triggered Drug Delivery for Osteosarcoma Synergetic Therapy and Bone Regeneration.

In this research, a novel MgSiO3 fiber membrane (MSFM) loaded with indocyanine green (ICG) and doxorubicin (DOX) was prepared. Because of MgSiO3's unique lamellar structure composed of a silicon-oxygen tetrahedron, magnesium ion (Mg2+) moves easily and can be further replaced with other cations. Therefore, because of the positively charged functional group of ICG, MSFM has a rather high drug loading for ICG. In addition, there is electrostatic attraction between DOX (a cationic drug) and ICG (an anionic drug). Hence, after loading ICG, more DOX can be adsorbed into MSFM because of electrostatic interaction. The ICG endows the MSFM outstanding photothermal therapy (PTT) performance, and DOX as a chemotherapeutic drug can restrain tumor growth. On the one hand, H+ exchanged with the positively charged DOX based on the MgSiO3 special lamellar structure. On the other hand, the thermal effect could break the electrostatic interaction between ICG and DOX. Based on the above two points, both tumor acidic microenvironment and photothermal effect can trigger DOX release. What's more, in vitro and in vivo antiosteosarcoma therapy evaluations displayed a superior synergetic PTT-chemotherapy anticancer treatment and excellent biocompatibility of DOX&ICG-MSFM. Finally, the MSFM was proven to greatly promote cell proliferation, differentiation, and bone regeneration performance in vitro and in vivo. Therefore, MSFM provides a creative perspective in the design of multifunctional scaffolds and shows promising applications in controlled drug delivery, antitumor performance, and osteogenesis.

Identification of TNFRSF21 as an inhibitory factor of osteosarcoma based on a necroptosis-related prognostic gene signature and molecular experiments.

BACKGROUND: Osteosarcoma is one of the most common malignant bone tumors with bad prognosis. Necroptosis is a form of programmed cell death. Recent studies showed that targeting necroptosis was a new promising approach for tumor therapy. This study aimed to establish a necroptosis-related gene signature to evaluated prognosis and explore the relationship between necroptosis and osteosarcoma. METHODS: Data from The Cancer Genome Atlas was used for developing the signature and the derived necroptosis score (NS). Data from Gene Expression Omnibus served as validation. Principal component analysis (PCA), Cox regression, receiver operating characteristic (ROC) curves and Kaplan-Meier survival analysis were used to assess the performance of signature. The association between the NS and osteosarcoma was analyzed via gene set enrichment analysis, gene set variation analysis and Pearson test. Single-cell data was used for further exploration. Among the genes that constituted the signature, the role of TNFRSF21 in osteosarcoma was unclear. Molecular experiments were used to explore TNFRSF21 function. RESULTS: Our data revealed that lower NS indicated more active necroptosis in osteosarcoma. Patients with lower NS had a better prognosis. PCA and ROC curves demonstrated NS was effective to predict prognosis. NS was negatively associated with immune infiltration levels and tumor microenvironment scores and positively associated with tumor purity and stemness index. Single-cell data showed necroptosis heterogeneity in osteosarcoma. The cell communication pattern of malignant cells with high NS was positively correlated with tumor progression. The expression of TNFRSF21 was down-regulated in osteosarcoma cell lines. Overexpression of TNFRSF21 inhibited proliferation and motility of osteosarcoma cells. Mechanically, TNFRSF21 upregulated the phosphorylation levels of RIPK1, RIPK3 and MLKL to promote necroptosis in osteosarcoma. CONCLUSIONS: The necroptosis prognostic signature and NS established in this study could be used as an independent prognostic factor, TNFRSF21 may be a necroptosis target in osteosarcoma therapy.

Ortho-silicic Acid Prevents Glucocorticoid-Induced Femoral Head Necrosis by Promoting Akt Phosphorylation to Inhibit Endoplasmic Reticulum Stress-Mediated Apoptosis and Enhance Angiogenesis and Osteogenesis.

Glucocorticoid-induced osteonecrosis of the femoral head (SONFH) is the most prevalent form of secondary osteonecrosis affecting the femoral head. Glucocorticoids can cause damage to both vascular endothelial cells and osteoblasts. Previous studies have demonstrated that silicon can improve the resistance of vascular endothelial cells to oxidative stress and positively impact bone health. However, the impact of silicon on SONFH has yet to be investigated. We examined the influence of ortho-silicic acid (OSA, Si(OH)4) on the apoptosis and proliferation of vascular endothelial cells after glucocorticoid induction. Additionally, we evaluated the expression of apoptosis-related genes such as cleaved-caspase-3, Bcl-2 and Bax. The impact of glucocorticoids and OSA on the function of vascular endothelial cells was evaluated through wound healing, transwell and angiogenesis assays. Osteogenic function was subsequently evaluated through alizarin red staining, alkaline phosphatase staining and expression levels of osteogenic genes like RUNX2 and ALP. Moreover, we investigated the potential role of OSA in vivo using the SONFH animal model. At concentrations below 100 µM, OSA exhibits no toxicity on vascular endothelial cells and effectively reverses glucocorticoid-induced apoptosis in these cells. OSA increases the resilience of vascular endothelial cells against oxidative stress and enhances osteoblast differentiation. Our study revealed that glucocorticoids activate endoplasmic reticulum stress, a process that mediates the apoptosis of vascular endothelial cells. OSA ameliorated the endoplasmic reticulum stress associated with glucocorticoids through the increased expression of p-Akt levels. In vivo, OSA treatment effectively improved SONFH by enhancing vascular endothelial cell function and promoting osteogenic differentiation. OSA counteracted the adverse effects of glucocorticoids both in vitro and in vivo, demonstrating a beneficial therapeutic effect on SONFH.

M2 macrophage-derived exosomal miR-486-5p influences the differentiation potential of bone marrow mesenchymal stem cells and osteoporosis.

BACKGROUND: An imbalance between osteogenesis and adipogenesis in bone marrow mesenchymal stem cells (BMMSCs) can cause osteoporosis. Macrophage-derived exosomes (MD-Exos) and microRNAs (miRNAs) enriched in exosomes participate in the differentiation of BMMSCs. METHODS: Bioinformatics methods were used to analyze differentially expressed miRNAs. We cocultured M2 macrophages and BMMSCs to examine the biological function of exosomal microRNA-486-5p (miR-486-5p) on BMMSCs differentiation. Gain-of-function experiments related to osteogenesis were designed to investigate the effects of exosomes carrying miR-486-5p on an ovariectomized (OVX) mice model and the direct impact of miR-486-5p on BMMSCs. A dual luciferase experiment was performed to demonstrate the target gene of miR-486-5p. RESULTS: Bioinformatics analysis identified high expression of miRNA-486 in M2 macrophage-derived exosomes (M2D-Exos). The in vitro results demonstrated that M2 macrophage-derived exosomal miR-486-5p enhanced osteogenic capacity but inhibited the adipogenesis of BMMSCs. The direct effect of miR-486-5p on BMMSCs showed the same effects. Animal experiments revealed that exosomal miR-486-5p rescued bone loss of OVX mice. SMAD2 was characterized as a target gene of miR-486-5p. Pathway analysis showed that M2 macrophage-derived exosomal miR-486-5p stimulated osteogenic differentiation via the TGF-ß/SMAD2 signalling pathway. CONCLUSIONS: Taken together, M2 macrophage-derived exosomal miR-486-5p influences the differentiation potential of BMMSCs through the miR-486-5p/SMAD2/TGF-ß signalling pathway and osteoporosis.

Arginine methylation of PPP1CA by CARM1 regulates glucose metabolism and affects osteogenic differentiation and osteoclastic differentiation.

BACKGROUND: The imbalance between osteoblasts and osteoclasts may lead to osteoporosis. Osteoblasts and osteoclasts have different energy requirements, with aerobic glycolysis being the prominent metabolic feature of osteoblasts, while osteoclast differentiation and fusion are driven by oxidative phosphorylation. METHODS: By polymerase chain reaction as well as Western blotting, we assayed coactivator-associated arginine methyltransferase 1 (CARM1) expression in bone tissue, the mouse precranial osteoblast cell line MC3T3-E1 and the mouse monocyte macrophage leukaemia cell line RAW264.7, and expression of related genes during osteogenic differentiation and osteoclast differentiation. Using gene overexpression (lentivirus) and loss-of-function approach (CRISPR/Cas9-mediated knockout) in vitro, we examined whether CARM1 regulates osteogenic differentiation and osteoblast differentiation by metabolic regulation. Transcriptomic assays and metabolomic assays were used to find the mechanism of action of CARM1. Furthermore, in vitro methylation assays were applied to clarify the arginine methylation site of PPP1CA by CARM1. RESULTS: We discovered that CARM1 reprogrammed glucose metabolism in osteoblasts and osteoclasts from oxidative phosphorylation to aerobic glycolysis, thereby promoting osteogenic differentiation and inhibiting osteoclastic differentiation. In vivo experiments revealed that CARM1 significantly decreased bone loss in osteoporosis model mice. Mechanistically, CARM1 methylated R23 of PPP1CA, affected the dephosphorylation of AKT-T450 and AMPK-T172, and increased the activities of phosphofructokinase-1 and pructose-2,6-biphosphatase3, causing an up-regulation of glycolytic flux. At the same time, as a transcriptional coactivator, CARM1 regulated the expression of pyruvate dehydrogenase kinase 3, which resulted in the inhibition of pyruvate dehydrogenase activity and inhibition of the tricarboxylic acid cycle, leading to a subsequent decrease in the flux of oxidative phosphorylation. CONCLUSIONS: These findings reveal for the first time the mechanism by which CARM1 affects both osteogenesis and osteoclast differentiation through metabolic regulation, which may represent a new feasible treatment strategy for osteoporosis.

Development and validation of a N6 methylation regulator-related gene signature for prognostic and immune response prediction in non-small cell lung cancer.

N6 methylation (m6A) has been reported to play an important role in tumor progression. Non-small cell lung cancer (NSCLC) is the predominant pathological type of lung cancer with a high mortality rate. The purpose of this study was to develop and validate a N6 methylation regulator-related gene signature for assessing prognosis and response to immunotherapy in NSCLC. Data from The Cancer Genome Atlas was used as the training cohort. Data from Gene Expression Omnibus and Xena served as the two validation cohorts. We performed Cox regression, last absolute shrinkage and selection operator, receiver operating characteristic curves and Kaplan-Meier survival analysis to generate and validate a prognostic signature based on m6A regulator-related genes. We explored the association between the signature and tumor microenvironment including genomic mutation, immune cell infiltration and tumor mutation burden. We also analyzed the association between the signature and immunotherapy. Finally, among the genes that constituted the signature, GGA2 was the only favorable factor for NSCLC prognosis. Molecular experiments were used to explore GGA2 function in NSCLC. We generated a prognostic signature based on seven m6A regulator-related genes (GGA2, CD70, BMP2, GPX8, YWHAZ, NOG and TEAD4). And the data from three cohorts showed that the signature could effectively assess prognosis in NSCLC. Patients with high risk scores had the higher mutational load and lower immune infiltration levels and were more likely to not respond to immunotherapy. The experiments revealed overexpression of GGA2 inhibited proliferation and motility of NSCLC cells. Mechanically, GGA2 downregulated METTL3 expression and thus reduced m6A abundance in NSCLC. This study developed and validated a prognostic signature based on m6A regulator-related genes, providing useful insights for the management of NSCLC. And GGA2 may be a target of m6A regulation.

WTAP-Mediated m6A RNA Methylation Regulates the Differentiation of Bone Marrow Mesenchymal Stem Cells via the miR-29b-3p/HDAC4 Axis.

N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the progression of osteoporosis (OP), providing novel insights into the pathogenesis of OP. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been studied in OP. Here we explored the biological role and underlying mechanism of WTAP in OP and the differentiation of bone marrow mesenchymal stem cells (BMMSCs). We demonstrated that WTAP was expressed at low levels in bone specimens from patients with OP and OVX mice. Functionally, WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs in vitro and in vivo. In addition, microRNA-29b-3p (miR-29b-3p) was identified as a downstream target of WTAP. M6A modifications regulated by WTAP led to increased miR-29b-3p expression. WTAP interacted with the microprocessor protein DGCR8 and accelerated the maturation of pri-miR-29b-3p in an m6A-dependent manner. Target prediction and dual-luciferase reporter assays identified the direct binding sites of miR-29b-3p with histone deacetylase 4 (HDAC4). WTAP-mediated m6A modification promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs through the miR-29b-3p/HDAC4 axis. Furthermore, WTAP-mediated m6A methylation negatively regulates osteoclast differentiation. Collectively, our study first identified a critical role of WTAP-mediated m6A methylation in BMMSC differentiation and highlighted WTAP as a potential therapeutic target for OP treatment.

WTAP-mediated m6A modification modulates bone marrow mesenchymal stem cells differentiation potential and osteoporosis.

An imbalance in the differentiation potential of bone marrow mesenchymal stem cells (BMSCs) is an important pathogenic mechanism underlying osteoporosis (OP). N6-methyladenosine (m6A) is the most common post-transcriptional modification in eukaryotic cells. The role of the Wilms' tumor 1-associated protein (WTAP), a member of the m6A functional protein family, in regulating BMSCs differentiation remains unknown. We used patient-derived and mouse model-derived samples, qRT-PCR, western blot assays, ALP activity assay, ALP, and Alizarin Red staining to determine the changes in mRNA and protein levels of genes and proteins associated with BMSCs differentiation. Histological analysis and micro-CT were used to evaluate developmental changes in the bone. The results determined that WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. We used co-immunoprecipitation (co-IP), RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), RNA pulldown, and dual-luciferase assay to explore the direct mechanism. Mechanistically, the expression of WTAP increased during osteogenic differentiation and significantly promoted pri-miR-181a and pri-miR-181c methylation, which was recognized by YTHDC1, and increased the maturation to miR-181a and miR-181c. MiR-181a and miR-181c inhibited the mRNA expression of SFRP1, promoting the osteogenic differentiation of BMSCs. Our results demonstrated that the WTAP/YTHDC1/miR-181a and miR-181c/SFRP1 axis regulated the differentiation fate of BMSCs, suggesting that it might be a potential therapeutic target for osteoporosis.

Characterization of the functional effects of ferredoxin 1 as a cuproptosis biomarker in cancer.

Background: Cuproptosis is a recently discovered form of programmed cell death. Ferredoxin 1 (FDX1) is a key gene that mediates this process. However, the role of FDX1 in human tumors is not clear. Methods: We comprehensively analyzed the differential expression and genetic alterations of FDX1 using multiomics data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database. Subsequently, we explored the association between FDX1 and tumor parameters such as genomic instability, RNA methylation modifications, immune infiltration and pathway activity. In addition, we performed functional enrichment analysis and assessed the sensitivity potential of FDX1-related drugs. Finally, we experimentally verified the functional effects of FDX1. Results: The analysis revealed differential expression of FDX1 in a variety of tumors. By analyzing the association of FDX1 expression with genomic instability, immune cell infiltration, signaling pathway etc. We explored the role of FDX1 in regulating cell activity. Also, we evaluated the function of FDX1 in biologic process and drug sensitivity. Our experimental results demonstrated that FDX1 exerts its antitumor effects through cuproptosis in liver hepatocellular carcinoma and non-small cell lung cancer cell lines. Conclusion: Our study reveals the functional effects of FDX1 in tumors and deepens the understanding of the effects of FDX1. We validated the inhibitory effect of FDX1 in copper induced cell-death, confirming the role of FDX1 as a cuproptosis biomarker.

Long noncoding RNA KCNQ1OT1 inhibits osteoclast differentiation by regulating the miR-128-3p/NFAT5 axis.

Noncoding RNAs play an important role in regulating osteoclast differentiation. We investigated whether and how potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1), a long noncoding RNA, regulates osteoclast differentiation. We found that the expression of KCNQ1OT1 was downregulated in osteoporotic bone tissue. Then transfection of KCNQ1OT1 overexpression vectors or small interfering RNAs showed that the proliferation, migration, and osteoclast differentiation of RAW 264.7 cells were inhibited by KCNQ1OT1 upregulation, while they were promoted by KCNQ1OT1 knockdown. Interestingly, we found and confirmed that miR-128-3p was a target of KCNQ1OT1 using online databases, dual luciferase reporter assays and quantitative real-time polymerase chain reaction, and that it inhibited the expression of miR-128-3p. Moreover, we confirmed that miR-128-3p directly targeted nuclear factor of activated T cell 5 (NFAT5), a protein that combines with osteoprotegerin and thus regulates osteoclastogenesis with the presence of the receptor activator of nuclear factor κB ligand. Furthermore, we demonstrated that both the knockdown of KCNQ1OT1 and the overexpression of miR-128-3p attenuate the expression of NFAT5, while upregulating the osteoclastogenesis markers c-Fos, NFATc1, and Ctsk. The results from overexpression of KCNQ1OT1 and the inhibition of miR-128-3p were contrary to the above. Finally, we found that the inhibition of osteoclast differentiation by KCNQ1OT1 overexpression could be rescued using a miR-128-3p mimic, while the enhancement of migration and osteoclast differentiation by si-NFAT5 could be reversed with a miR-128-3p inhibitor. These results suggested that KCNQ1OT1 regulates the osteoclast differentiation via the miR-128-3p/NFAT5 axis.