Osteoblast hFOB Research Articles

Eicosapentaenoic Acid and Docosahexaenoic Acid as an Antimicrobial Agent in Orthopedics—An In Vitro Study About the Race for Surface

Background: The burden of prosthetic joint infection in combination with antibiotic-resistant bacterial strains is a rising dilemma for patients experiencing total joint replacements. Around 0.8–2% of patients experience prosthetic joint infections, while up to 21% of patients are considered fatal cases after 5 years. Staphylococcus aureus is one of the main reasons for prosthetic joint infections. Its capability of forming biofilms and developing mechanisms against antibiotics is one of the most dangerous clinical topics being currently discussed. Previous studies have shown the promising results of omega-3 fatty acids as an antimicrobial agent against Staphylococcus aureus. Though an antimicrobial effect has been examined, the influence of polyunsaturated fatty acids on Staphylococcus aureus in the presence of human osteoblasts has not been reported yet. In this study, we aimed to investigate the influence of omega-3 fatty acids on the biofilm formation of Staphylococcus aureus ATCC 29213 in the presence of hFOB 1.19 cells. The co-culture setup helped to examine the influence of omega-3 fatty acids on the race for surface to simulate prosthetic joint infections. Methods: In this study, we tested Staphylococcus aureus ATCC 29213 co-cultured with human fetal osteoblasts hFOB 1.19 in the presence of sub-MIC and MIC concentrations of docosahexaenoic acid (1.25 mg/L, 2.5 mg/L) and eicosapentaenoic acid (0.15 mg/L, 0.3 mg/L) after 1, 6 and 24 h of incubation. After establishing the co-culture, cell culture and biofilm, we performed colony-forming unit counting and cell counting to examine cell survivability. In addition, we carried out scanning electron microscopy to study the race for surface behaviour of the cells. Results: We found a protective influence of omega-3 fatty acids on osteoblasts when present in co-culture with Staphylococcus aureus after 6 h of incubation. Omega-3 fatty acids increase the cell survival of osteoblasts after 6 h in a co-culture with bacteria and are able to influence the race for surface. In this study, the strain of Staphylcoccus aureus ATCC 29213 showed signs of growth inhibition within the first 6 h. Conclusions: Omega-3 fatty acids can be a valuable antimicrobial agent in terms of decreasing the risk of on-site infection during surgery. Omega-3 fatty acids were shown to decrease the bacterial load within the first 6 h of incubation and increase the survivability of osteoblasts.

Deciphering the role of lipid metabolism and acetylation in osteosarcoma: A comprehensive molecular analysis.

Osteosarcoma, known for its rapid progression and high metastatic potential, poses significant challenges in adolescent oncology. This study delves into the roles of lipid metabolism and acetylation genes in the disease's pathogenesis. Utilizing gene set variation analysis, we examined 14 lipid metabolism-related pathways in osteosarcoma patients, identifying significant variances in three pathways between metastatic and primary cases. Additionally, differences in four acetylation genes between these groups were observed. A comprehensive analysis pinpointed 62 lipid metabolism-related genes, with 39 exhibiting significant correlations with acetylation genes, termed lipid metabolism acetylation (LMA) genes. Employing machine learning techniques like Lasso+RSF and GBM, we developed a predictive model for overall survival based on LMA genes. This model, with an average c-index of 0.771, focuses on three key genes: CYP2C8, PAFAH2, and ACOX3, whose prognostic value was confirmed through survival and receiver operating characteristic curve analyses. Quantitative RT-PCR results indicated higher expression levels of ACOX3 and PAFAH2 in OS cells (143B, HOS, MG63) than in osteoblasts (hFOB1.19), while CYP2C8 was lower in OS cells. Furthermore, drug sensitivity analysis through the pRRophetic algorithm suggested potential targeted therapies, revealing drugs with differential sensitivity based on LMA scores and varied treatment responses related to the expression of core genes. This study not only highlights the crucial role of lipid metabolism and acetylation in osteosarcoma but also offers a foundation for personalized treatment strategies, marking a notable advancement in combating this severe form of adolescent cancer.

Effect of luteolin on oxidative stress and inflammation in the human osteoblast cell line hFOB1.19 in an inflammatory microenvironment

BackgroundPeriapical lesions are characterized by periapical inflammation and damage to periapical tissues and eventually lead to bone resorption and even tooth loss. H2O2 is widely used in root canal therapy for patients with periapical inflammation. Luteolin possesses high anti-inflammatory, antioxidant, and anticancer potential. However, the underlying mechanism of the efficacy of H2O2 and luteolin on oxidative stress and inflammatory tissue has not been previously addressed. We aimed to investigate the anti-inflammatory and antioxidative effects of luteolin on H2O2-induced cellular oxidative inflammation.MethodsAfter human osteoblasts (hFOB1.19) were treated with lipopolysaccharide (LPS), luteolin, or H2O2, cell proliferation was analysed by using a cell counting kit-8 (CCK-8), cell apoptosis was measured by using flow cytometry, the production of reactive oxygen species (ROS) was evaluated by using an oxidation-sensitive probe DCFH-DA ROS assay kit, and the expression of genes and proteins was detected by using reverse transcription quantitative polymerase chain reaction (RT‒qPCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA).ResultsWe demonstrated that inflammation is closely related to oxidative stress and that the oxidative stress level in the inflammatory environment is increased. Luteolin inhibited the H2O2-induced increase in the expression of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor α (TNF-α) and significantly repressed the H2O2-induced increase in ROS, as well as markedly strengthened superoxide dismutase (SOD) activity in hFOB1.19 cells. Moreover, we detected that luteolin may inhibit H2O2-induced hFOB1.19 cell injury by suppressing the NF-κB pathway.ConclusionWe elucidated that luteolin protected human osteoblasts (hFOB1.19) from H2O2-induced cell injury and inhibited the production of proinflammatory cytokines by suppressing the NF-κB signalling pathway. Our findings provide a potential drug for treating H2O2-induced periodontitis and cell injury.

Aggregation of human osteoblasts unlocks self-reliant differentiation and constitutes a microenvironment for 3D-co-cultivation with other bone marrow cells

Skeletal bone function relies on both cells and cellular niches, which, when combined, provide guiding cues for the control of differentiation and remodeling processes. Here, we propose an in vitro 3D model based on human fetal osteoblasts, which eases the study of osteocyte commitment in vitro and thus provides a means to examine the influences of biomaterials, substances or cells on the regulation of these processes. Aggregates were formed from human fetal osteoblasts (hFOB1.19) and cultivated under proliferative, adipo- and osteoinductive conditions. When cultivated under osteoinductive conditions, the vitality of the aggregates was compromised, the expression levels of the mineralization-related gene DMP1 and the amount of calcification and matrix deposition were lower, and the growth of the spheroids stalled. However, within spheres under growth conditions without specific supplements, self-organization processes occur, which promote extracellular calcium deposition, and osteocyte-like cells develop. Long-term cultivated hFOB aggregates were free of necrotic areas. Moreover, hFOB aggregates cultivated under standard proliferative conditions supported the co-cultivation of human monocytes, microvascular endothelial cells and stromal cells. Overall, the model presented here comprises a self-organizing and easily accessible 3D osteoblast model for studying bone marrow formation and in vitro remodeling and thus provides a means to test druggable molecular pathways with the potential to promote life-long bone formation and remodeling.

HOXB5 promotes the progression and metastasis of osteosarcoma cells by activating the JAK2/STAT3 signalling pathway

ObjectiveTo investigate the involvement of the homeobox gene B5 (HOXB5) in the progression and metastasis of osteosarcoma. MethodsThe expression of HOXB5 in human osteosarcoma tissues and its correlation with clinical indicators were investigated using bioinformatics analysis and immunohistochemical labelling. Human osteosarcoma cells (HOS, MG63, U2OS, and Saos-2) and normal human osteoblasts (hFOB1.19) were cultivated. The expression of HOXB5 in these cells was detected using western blotting (WB) and RT‒PCR. Two cell lines exhibiting elevated HOXB5 expression were chosen and divided into three groups: the blank group (mock), control group (control) and transfection group (shHOXB5). The transfection group was infected with lentivirus expressing shRNAs targeting HOXB5. The transfection efficiency was detected by WB. Cell proliferation suppression was measured by CCK-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays; the percentage of apoptotic cells was determined by flow cytometry; and cell migration and invasion were detected via the Transwell chamber test. WB was utilized to determine the protein expression of genes linked to metastasis (MMP2, MMP9), apoptosis (Bax, Bcl-2), and the JAK2/STAT3 pathway (JAK2, p-JAK2, STAT3, p-STAT3). ResultsIn osteosarcoma tissues, HOXB5 expression was elevated and strongly correlated with distant metastasis. Silencing HOXB5 reduced the proliferation, migration and invasion of osteosarcoma cells; prevented the progression and metastasis of tumours in tumour-bearing nude mice; and reduced the activation of key proteins in the JAK2/STAT3 signalling pathway. ConclusionThrough the JAK2/STAT3 signalling pathway, HOXB5 plays a crucial role in the malignant progression of osteosarcoma and is a promising target for osteosarcoma treatment.

The Effect of Silk Fibroin/Poly(Lactic-Co-Glycolic Acid) Nanofiber Coating on Porous Calcium Phosphate-Based Composites for Osteoporosis Application

This study observed whether the silk fibroin/poly lactic-co-glycolic acid (SF/PLGA) nanofiber membrane three-dimensionally coated with porous calcium phosphate-based composite can promote the proliferation of osteoblasts, thereby improving osteoporosis. The SF/PLGA nanofiber membrane was used to three-dimensionally coated with the porous calcium phosphate-based composite to analyze proliferation of osteoblasts, hFOB1.19 expressions by Western blot, activity change of hFOB1.19 by CCK-8 assay. The morphology was observed with an electron microscope. Alizarin Red staining showed that the SF/PLGA nanofiber membrane three-dimensionally coated with porous calcium phosphatebased composite promoted osteogenic differentiation and upregulated the expression of osteogenic related genes. It inhibited adipogenic differentiation and the expression of adipogenic genes. SF/PLGA composite material promoted the viability of osteoblasts hFOB1.19, upregulatedGLUT3, downregulated the expression of various apoptosis-related proteins in hFOB1.19. SF/PLGA composite material can reduce the apoptosis efficiency of osteoblasts, promote the proliferation of osteoblasts, and inhibit apoptosis, thereby improving osteoporosis.

UBE2L3 Promotes Oxidative Stress-regulated Necroptosis to Accelerate Osteosarcoma Progression.

Osteosarcoma is a highly invasive bone marrow stromal tumor with limited treatment options. Oxidative stress plays a crucial role in the development and progression of tumors, but the underlying regulatory mechanisms are not fully understood. Recent studies have revealed the significant involvement of UBE2L3 in oxidative stress, but its specific role in osteosarcoma remains poorly investigated. This study aimed to explore the molecular mechanisms by which UBE2L3 promotes oxidative stress-regulated necroptosis to accelerate the progression of osteosarcoma using in vitro cell experiments. Human osteoblast hFOB1.19 cells and various human osteosarcoma cell lines (MG-63, U2OS, SJSA-1, HOS, and 143B) were cultured in vitro. Plasmids silencing UBE2L3 and negative control plasmids were transfected into U2OS and HOS cells. The cells were divided into the following groups: U2OS cell group, HOS cell group, si-NC-U2OS cell group, si-UBE2L3-U2OS cell group, si-NC-HOS cell group, and si-UBE2L3-HOS cell group. Cell viability and proliferation capacity were measured using the Tunnel method and clonogenic assay. Cell migration and invasion abilities were assessed by Transwell and scratch assays. Cell apoptosis was analyzed by flow cytometry, and ROS levels were detected using immunofluorescence. The oxidative stress levels in various cell groups and the expression changes of necroptosis-related proteins were assessed by PCR and WB. Through these experiments, we aim to evaluate the impact of oxidative stress on necroptosis and uncover the specific mechanisms by which targeted regulation of oxidative stress promotes tumor cell necroptosis as a potential therapeutic strategy for osteosarcoma. The mRNA expression levels of UBE2L3 in human osteosarcoma cell lines were significantly higher than those in human osteoblast hFOB1.19 cells (p <0.01). UBE2L3 expression was significantly decreased in U2OS and HOS cells transfected with si-UBE2L3, indicating the successful construction of stable cell lines with depleted UBE2L3. Tunnel assay results showed a significant increase in the number of red fluorescent-labeled cells in si-UBE2L3 groups compared to si-NC groups in both cell lines, suggesting a pronounced inhibition of cell viability. Transwell assay demonstrated a significant reduction in invasion and migration capabilities of si-UBE2L3 groups in osteosarcoma cells. The clonogenic assay revealed significant suppression of proliferation and clonogenic ability in both U2OS and HOS cells upon UBE2L3 knockdown. Flow cytometry confirmed that UBE2L3 knockdown significantly enhanced apoptosis in U2OS and HOS cells. Immunofluorescence results showed that UBE2L3 silencing promoted oxidative stress levels in osteosarcoma cells and facilitated tumor cell death. WB analysis indicated a significant increase in phosphorylation levels of necroptosis-related proteins, RIP1, RIP3, and MLKL, in both osteosarcoma cell lines after UBE2L3 knockdown. In addition, the expression of necrosis-associated proteins was inhibited by the addition of the antioxidant N-acetylcysteine (NAC). UBE2L3 is upregulated in osteosarcoma cells, and silencing of UBE2L3 promotes oxidative stress in these cells, leading to enhanced necroptosis and delayed progression of osteosarcoma.

Effect of Staphylococcal Nuclease and Tudor Domain Containing 1/SLC7A11 on the Occurrence and Development of Osteosarcoma by Inhibiting Ferroptosis

Objective To investigate the effect of staphylococcal nuclease and tudor domain containing 1(SND1) on the biological function of osteosarcoma cells and decipher the mechanism of SND1 in regulating ferroptosis in osteosarcoma cells via SLC7A11. Methods Human osteoblasts hFOB1.19 and osteosarcoma cell lines Saos-2,U2OS,HOS,and 143B were cultured,in which the expression level of SND1 was determined.Small interfering RNA was employed to knock down the expression of SND1(si-SND1) in the osteosarcoma cell line HOS and 143B.The CCK8 assay kit,colony formation assay,and Transwell assay were employed to examine the effect of SND1 expression on the biological function of osteosarcoma cells.Furthermore,we altered the expression of SND1 and SLC7A11 in osteosarcoma cells to investigate the effect of SND1 on osteosarcoma ferroptosis via SLC7A11. Results The mRNA and protein levels of SND1 in Saos-2,U2OS,HOS,and 143B cells were higher than those in hFOB1.19 cells(all P<0.01).Compared with the control group,transfection with si-SND1 down-regulated the expression level of SND1 in HOS and 143B cells(all P<0.01),decreased the viability of HOS and 143B cells,reduced the number of colony formation,and inhibited cell invasion and migration(all P<0.001).The ferroptosis inducer Erastin promoted the apoptosis of HOS and 143B cells,while the ferroptosis inhibitor Ferrostatin-1 improved the viability of HOS and 143B cells(all P<0.001).After SND-1 knockdown,Erastin reduced the viability of HOS and 143B cells,while Ferrostatin-1 restored the cell viability(all P<0.001).After treatment with Erastin in the si-SND1 group,the levels of iron and malondialdehyde were elevated,and the level of glutathione was lowered(all P<0.001).The results of in vivo experiments showed that SND1 knockdown inhibited the mass of the transplanted tumor in 143B tumor-bearing nude mice(P<0.001).Knocking down the expression of SND1 resulted in down-regulated SLC7A11 expression(all P<0.001) and increased ferroptosis in HOS and 143B cells(P<0.001,P=0.020). Conclusions SND1 presents up-regulated expression in osteosarcoma cells.It may inhibit ferroptosis by up-regulating the expression of SLC7A11,thereby improving the viability of osteosarcoma cells.

Mechanism of Circ_HECW2 regulating osteoblast apoptosis in osteoporosis by attenuating the maturation of miR-1224-5p

BackgroundOsteoporosis (OP) poses a significant clinical challenge with escalating morbidity. This study explores Circ_HECW2 expression in OP patients and its regulatory role in lipopolysaccharide (LPS)-induced osteoblast apoptosis.MethodsCirc_HECW2 expression in OP patient serum and healthy controls was quantified using RT-qPCR. Diagnostic value of Circ_HECW2 for OP was assessed via ROC curve. Pearson’s correlation model examined associations between indicators. Human osteoblasts HFOB1.19, treated with LPS, were analyzed for Circ_HECW2, pre-miR-1224, miR-1224-5p, and PDK2 mRNA levels. TUNEL assay determined cell apoptosis and Western blot assessed cleaved-caspase-3 protein levels. RNase R resistance assay and actinomycin D assay confirmed Circ_HECW2’s cyclic structure. RNA pull-down and dual-luciferase reporter assay verified binding relationships between Circ_HECW2 and miR-1224 and between miR-1224-5p and PDK2.ResultsCirc_HECW2 exhibited elevated expression in OP patients with diagnostic significance and a negative correlation with lumbar T-score. LPS co-culture increased Circ_HECW2 expression in HFOB1.19 cells, significantly elevating apoptosis index and cleaved-caspase-3. Circ_HECW2 downregulation inhibited HFOB1.19 apoptosis, reduced pre-miR-1224 expression, and elevated mature miR-1224-5p. Circ_HECW2 bound to pre-miR-1224, and inhibiting miR-1224-5p reversed the effect of Circ_HECW2 downregulation on osteoblast apoptosis. miR-1224-5p targeted PDK2 transcription.ConclusionCirc_HECW2, highly expressed in OP, holds diagnostic significance and reflects disease severity. Circ_HECW2 reduces mature miR-1224-5p by binding to pre-miR-1224, upregulating PDK2, and facilitating LPS-induced osteoblast apoptosis.Graphic

Exploring the role of lactylation-related genes in osteosarcoma: A deep dive into prognostic significance and therapeutic potential.

Osteosarcoma (OS), notorious for its complex pathogenesis and formidable prognosis, represents a significant medical quandary. This research embarked on a quest to unravel the implications of lactylation-related genes (LRGs) in OS, offering a novel lens through which to interpret its intricacies. A meticulous evaluation of 329 LRGs within the TARGET dataset spotlighted 27 paramount genes, intricately intertwined with survival. These genes highlighted metabolic processes-particularly amino acid metabolism-as key players, as evidenced in both GO and KEGG analyses. Utilizing consensus clustering and principal component analysis, the 93 OS samples were segmented into two distinct groups, differing notably in overall and event-free survival. Cluster 2 demonstrated a heightened immune response, contrasting the other cluster. Machine learning techniques, like generalized boosted model, CoxBoost, and RSF, spotlighted MYC and GOT2 as critical genes. Using multivariate Cox regression, a risk model was developed, categorizing patients into high and low-risk groups, each displaying varied survival patterns. Additionally, a contrast was observed between MYC and GOT2's associations with HLA molecules, emphasizing their distinct roles in antigen presentation. Potential therapeutic avenues were identified for each risk group, with special attention to mutations in MYC, particularly amplifications, hinting at its role in tumor progression. Finally, delving deeper into the role of MYC, Western blot analyses exhibited amplified myc protein levels in OS cells U-2 and MG-63 when juxtaposed against human osteoblastic cells Hfob1.19. A focused knockdown of myc in OS cells subsequently confirmed its influence on cell proliferation and migration, with reduced myc expression resulting in inhibited cell activities. Furthermore, immunofluorescence assays corroborated myc's heightened expression in OS cells relative to normal osteoblastic cells. In summary, this study accentuates the vital role of LRGs and specifically MYC in OS, ushering in a horizon of tailored therapeutic strategies.

Exosomes promote hFOB1.19 proliferation and differentiation via LINC00520

BackgroundOsteoporosis remains a significant clinical challenge worldwide. Recent studies have shown that exosomes stimulate bone regeneration. Thus, it is worthwhile to explore whether exosomes could be a useful therapeutic strategy for osteoporosis. The purpose of this study was to investigate the effects of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs) on osteoblast proliferation and differentiation.MethodsExosomes were isolated from hucMSCs. Bioinformatics analysis was performed to identify the differentially expressed lncRNAs in myeloma-derived mesenchymal stem cells. Plasmids encoding LINC00520 or short hairpin RNA of LINC00520 were transfected into hucMSCs and then exosomes were isolated. After human osteoblasts hFOB1.19 were exposed to the obtained exosomes, cell survival, cell cycle, apoptosis and calcium deposits of hFOB1.19 cell were detected by MTT, 7-aminoactinomycin D, Annexin V-FITC/propidium iodide and Alizarin red staining, respectively.ResultsIn hFOB1.19 cells, 10 × 109/mL hucMSC-derived exosomes inhibited cell proliferation, arrested cell cycle, and promoted apoptosis, while hucMSCs or 1 × 109/mL exosomes promoted cell proliferation, accelerated cell cycle, and promoted calcium deposits and the expression of OCN, RUNX2, collagen I and ALP. In hFOB1.19 cells, exosomes from hucMSCs with LINC00520 knockdown reduced the survival and calcium deposits, arrested the cell cycle, and enhanced the apoptosis, while exosomes from hucMSCs overexpressing LINC00520 enhance the proliferation and calcium deposits and accelerated the cell cycle.ConclusionsLINC00520 functions as a modulator of calcium deposits, and exosomes derived from hucMSCs overexpressing LINC00520 might be a novel therapeutic approach for osteoporosis.

Peonidin-3-O-glucoside and Resveratrol Increase the Viability of Cultured Human hFOB Osteoblasts and Alter the Expression of Genes Associated with Apoptosis, Osteoblast Differentiation and Osteoclastogenesis.

High-throughput RNA-sequencing can determine the impact of nutrients and their combinations on gene transcription levels in osteocytes, and clarify the biological pathways associated with their impact on bone tissues. Previously, we reported that resveratrol (RES) and peonidin-3-O-glucoside (POG) increased osteoblastogenesis, as well as reduced osteoclastogenesis in transgenic teleost fish models. Here, we perform whole-genome transcriptomic profiling of osteoblasts treated with POG or RES to provide a comprehensive understanding of alterations in gene expression and the molecular mechanisms involved. Cultured human fetal osteoblastic hFOB 1.19 cells were treated with the test compounds, and then RNA was used to prepare RNA-seq libraries, that were sequenced using a NovaSeq 6000. Treatment with POG or RES increased osteoblast proliferation and reduced apoptosis. Transcriptomic profiling showed that of the 29,762 genes investigated, 3177 were differentially expressed (1481 upregulated, 1696 downregulated, FDR ≤ 0.05) in POG-treated osteoblasts. In the RES-treated osteoblasts, 2288 genes were differentially expressed (DGEs, 1068 upregulated, 1220 downregulated, FDR ≤ 0.05). Ingenuity® Pathway Analysis (IPA) of DGEs from RES or POG-treated osteoblasts revealed significant downregulation of the apoptosis, osteoarthritis and HIF1α canonical pathways, and a significant reduction in Rankl mRNA expression. The data suggest that RES and POG have both anabolic and anticlastogenic effects.

Construction and validation of a novel cuproptosis-mitochondrion prognostic model related with tumor immunity in osteosarcoma.

The purpose of this study was to develop a new prognostic model for osteosarcoma based on cuproptosis-mitochondrion genes. The data of osteosarcoma were obtained from TARGET database. By using Cox regression and LASSO regression analysis, a novel risk score was constructed based on cuproptosis-mitochondrion genes. Kaplan-Meier, ROC curve and independent prognostic analyses were performed to validate the risk score in GSE21257 dataset. Then, a predictive nomogram was constructed and further validated by calibration plot, C-index and ROC curve. Based on the risk score, all patients were divided into high-risk and low-risk group. GO and KEGG enrichment, immune correlation and drug sensitivity analyses were performed between groups. Real-time quantitative PCR verified the expression of cuproptosis-mitochondrion prognostic model genes in osteosarcoma. And we explored the function of FDX1 in osteosarcoma by western blotting, CCK8, colony formation assay, wound healing assay and transwell assays. A total of six cuproptosis-mitochondrion genes (FDX1, COX11, MFN2, TOMM20, NDUFB9 and ATP6V1E1) were identified. A novel risk score and associated prognostic nomogram were constructed with high clinical application value. Strong differences in function enrichment and tumor immune microenvironment were shown between groups. Besides, the correlation of cuproptosis-mitochondrion genes and drug sensitivity were revealed to search for potential therapeutic target. The expression of FDX1, COX11, MFN2, TOMM20 and NDUFB9 at mRNA level was elevated in osteosarcoma cells compared with normal osteoblast hFOB1.19. The mRNA expression level of ATP6V1E1 was decreased in osteosarcoma. Compared with hFOB1.19, western blotting revealed that the expression of FDX1 was significantly elevated in osteosarcoma cells. Functional experiments indicated that FDX1 mainly promoted the migration of osteosarcoma rather than proliferation. We developed a novel prognostic model of osteosarcoma based on cuproptosis-mitochondrion genes, which provided great guidance in survival prediction and individualized treatment decision making for patients with osteosarcoma.

Prognostic and immunological significance of an M1 macrophage-related gene signature in osteosarcoma.

As the most abundant infiltrating immune cells in the tumor microenvironment (TME), tumor-associated macrophages (TAMs) are pivotal in tumor development and treatment. The present investigation endeavors to explore the potential of M1 macrophage-related genes (MRGs) as biomarkers for assessing risk in individuals with osteosarcoma. RNA-sequence data and clinical data were derived from TCGA and GEO databases. The CIBERSORT method was utilized to discern subtypes of tumor-infiltrating immune cells. Identification of MRGs was achieved through Pearson correlation analysis. A prognostic risk model for MRGs was developed using Cox and LASSO regression analyses. A tripartite gene signature comprising CD37, GABRD, and ARHGAP25 was an independent prognostic indicator and was employed to develop a risk score model. The internal and external validation cohort confirmed the results. The area under the ROC curve (AUC) was determined for survival periods of 1 year, three years, and five years, yielding values of 0.746, 0.839, and 0.850, respectively. The C-index of the risk score was found to be superior to clinicopathological factors. GO/KEGG enrichment showed that the differences between high- and low-risk groups were predominantly associated with immune response pathways. Immune-related analysis related to proportions of immune cells, immune function, and expression levels of immune checkpoint genes all showed differences between the high- and low-risk groups. The qRT-PCR and Western blotting results indicate that CD37 expression was markedly higher in MG63 and U2OS cell lines when compared to normal osteoblast hFOB1.19. In U2OS cell line, GABRD expression levels were significantly upregulated. ARHGAP25 expression levels were elevated in both 143B and U2OS cell lines. In summary, utilizing a macrophage genes signature demonstrates efficacy in predicting both the prognosis and therapy response of OS. Additionally, immune analysis confirms a correlation between the risk score and the tumor microenvironment. Our findings, therefore, provide a cogent account for the disparate prognoses observed among patients and furnish a justification for further inquiry into biomarkers and anti-tumor treatment strategies.

FGF23 promotes proliferation, migration and invasion by regulating miR-340-5p in osteosarcoma

BackgroundIncreasing evidences have been indicated that FGF23 is associated with the biological behavior of malignant tumors, but its role in osteosarcoma and the specific mechanism need to be elucidated. The purpose of this study is to investigate the effects of FGF23 on the proliferation, migration and invasion of osteosarcoma cells, and the possible molecular mechanisms.MethodsWestern blot was used to detect differences in FGF23 expression in osteosarcoma cells MG-63 and U2-OS and osteoblasts hFOB1.19. FGF23-overexpressing adenoviruses and FGF-silencing plasmids were transfected into osteosarcoma cells, and transfection efficiency was verified using Western blot. MTT and colony formation assays were performed to detect osteosarcoma cell proliferation. Cell cycle was measured by flow cytometry. Scratch assay, holographic imaging cell analyzer Holomonitor ® M4 and transwell were applied to detect cell migration and invasion. Dual-luciferase reporter assay was performed to validate the interaction between FGF23 and miR-340-5p. Changes in miR-340-5p mRNA levels were measured by QRT-PCR.ResultsFGF23 is highly expressed in osteosarcoma cells compared to hFOB1.19. Overexpression of FGF23 significantly promoted the proliferation, migration and invasion of MG-63 and U2-OS cells. MiR-340-5p is a target of FGF23. Transfection of miR-340-5p mimics reversed the promoting effects of FGF23 on proliferation, migration and invasion of MG-63 and U2-OS cells.ConclusionFGF23 promotes osteosarcoma cell proliferation, migration and invasion by targeting miR-340-5p gene expression.

Development and validation of a cuproptosis-related lncRNA model correlated to the cancer-associated fibroblasts enable the prediction prognosis of patients with osteosarcoma

BackgroundOsteosarcoma is the most common primary pediatric and adolescent bone malignancy. An imbalance in copper homeostasis caused by copper ion accumulation could increase intracellular toxicity and regulate cancer cell growth. This study aimed to identify long non-coding RNAs (lncRNAs) associated with cuproptosis to predict prognosis and target drug use to improve patient survival. MethodsRNA sequencing and relevant clinical information of ninety-three osteosarcoma patients were obtained from the TARGET database. We then identified thirteen prognostic cuproptosis-related lncRNAs(CRLncs) using coexpression and univariate Cox analyses. The prognostic risk model with three CRLncs was constructed using the least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analysis. Patients were divided into low-risk and high-risk subgroups using the median risk score. The tumor microenvironment (TME) and immune status of identified subgroups were analyzed using ESTIMATE, CIBERSORT, MCP-counter, xCELL, EPIC, and ssGSEA analyses. Functional analyses were conducted to elucidate the underlying mechanisms, including GO, KEGG, GSVA, and GSEA analyses. Also, the relationships between the model, tumor immunity, and drug sensitivity were explored. Lastly, the expression level of ZNF37BP, AL353759.1, and AC005034.5 was validated in vitro. ResultsWe constructed a model containing three CRLncs (ZNF37BP, AL353759.1, and AC005034.5) and validated its excellent prognostic and predictive power. The AUC curves for 1-year, 3-year, and 5-year survival probabilities were 0.76, 0.84, and 0.89, respectively. Patients in the high-risk group had a shorter overall survival (OS) time than those in the low-risk. The stroma score and cancer-associated fibroblasts (CAFs) were significantly higher in the low-risk group. Immune cells such as T cells CD4 naive, T cells gamma delta, NK cells resting, dendritic cells resting, and mast cells activated were significantly upregulated in the high-risk group. Based on functional analyses, the PI3K-Akt pathway was identified as a critical metabolic pathway in osteosarcoma. Additionally, we obtained three potentially effective drugs for OS: erlotinib, MP470, and WH-4-023 targeting the PI3K-Akt pathway. The expression level of ZNF37BP was significantly elevated in OS cell lines than in normal osteoblast hFOB1.19 cells, and that of ATP7A, LIPT1, AL353759.1, and AC005034.5 were decreased considerably in OS cell lines. ConclusionCuproptosis-related lncRNAs are correlated with the CAFs of osteosarcoma, and this could serve as a foundation for OS survival prediction and treatment.

MiRNA-181a-5p inhibits proliferation and migration of osteosarcoma cell line HOS by targeting HOXB4

To study the effects and mechanisms of miR-181a-5p on the proliferation, cycle and migration of HOS osteosarcoma cells. Real-time quantitative PCR was used to detect the expression of miR-181a-5p and HOXB4 in osteoblast hFOB1.19 cell line and osteosarcoma cell lines (HOS, U2OS, MG63). miR-181a-5p mimics and miR-181a-5p inhibitors were respectively transfected into HOS cells by Lipofectamine 2000, and miR NC group was set as control group. CCK-8 method was used to detect the change in cell proliferation. Flow cytometry was used to detect the changes in cell cycles. Wound healing experiments and Transwell migration experiments were used to detect the changes in cell migration ability. The target gene of miR-181a-5p was predicted by Targetscan website and validated by Dual-luciferase reporter gene system and Western blot. Compared with osteoblast hFOB1.19, miR-181a-5p was low expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05), while HOXB4 was high expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05). Compared with the miR NC group, over expression of miR-181a-5p inhibited the proliferation and migration of osteosarcoma HOS cells, and the number of cells in S phase decreased(P<0.05). However, knockdown miR-181a-5p promoted the proliferation and migration of osteosarcoma HOS cells, the cells in S phase increased(P<0.05). Bioinformatics prediction and Dual-luciferase reporter gene system validate HOXB4 as a downstream target gene of miR-181a-5p(P<0.05). Western blot showed that miR-181a-5p over expression or knockdown significantly down-regulated or up-regulated HOXB4 expressions in the HOS cells respectively(P<0.05). miR-181a-5p is down expressed in osteosarcoma cells, and over-expression miR-181a-5p inhibits the proliferation, cell cycle and migration ability of osteosarcoma cells by targeting HOXB4.

STEAP2 promotes osteosarcoma progression by inducing epithelial–mesenchymal transition via the PI3K/AKT/mTOR signaling pathway and is regulated by EFEMP2

ABSTRACT This study was designed to explore the prognostic significance and functionality of STEAP2 (six-transmembrane epithelial antigen of prostate 2) in osteosarcomas and determine whether EFEMP2 (Epidermal growth factor-containing fibulin-like extracellular matrix protein 2) targets STEAP2 to facilitate osteosarcoma cell infiltration and migration. STEAP2 expression in peritumoral tissues, osteosarcoma, benign fibrous dysplasia, osteosarcoma cells, normal osteoblastic hFOB cells, and various invasive subclones was evaluated using IHC, ICC, and qRT-PCR. We also evaluated the association between STEAP2 expression and disease outcome using Kaplan–Meier analyses and then investigated STEAP2 regulation and its functional effects using both in vitro and in vivo assays. The results revealed that the upregulation of STEAP2 in osteosarcoma tissues positively correlated with both the malignant osteosarcoma phenotype and poor patient outcomes. In addition, STEAP2 expression induced epithelial–mesenchymal transition (EMT) via the PI3K/AKT/mTOR axis and facilitated osteosarcoma cell infiltration and migration. Changes in EFEMP2 expression resulted in correlating changes in STEAP2 expression, with EFEMP2-overexpressing osteosarcoma cells exhibiting a less invasive phenotype and reduced EMT following STEAP2 inhibition. It is also worth noting that although EFEMP2 overexpression activated the PI3K/AKT/mTOR pathway promoting EMT, it did not affect osteosarcoma cells in which STEAP2 or Akt was knocked down. Thus, we can conclude that STEAP2 acts as an oncogene in osteosarcoma progression, while EFEMP2 enables PI3K/AKT/mTOR axis initiation and EMT by partly targeting STEAP2, thereby facilitating osteosarcoma cell infiltration and migration.

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

EFEMP1 binds to STEAP1 to promote osteosarcoma proliferation and invasion via the Wnt/β-catenin and TGF-β/Smad2/3 signal pathways

PurposeTo investigate the prognostic value and function of six-transmembrane epithelial antigen of prostate 1 (STEAP1) in osteosarcoma and determine whether EFEMP1 mediates its effects. MethodsIHC (immunohistochemistry)/ICC (immunocytochemistry) in conjunction with RT-qPCR (quantitative real-time polymerase chain reaction) were employed to assess the expression of STEAP1 in paratumoral tissues, osteosarcoma, benign fibrous dysplasia, osteosarcoma cells, normal osteoblastic hFOB cells, as well as various invasive subclones. The association of STEAP1 with outcome was examined with Kaplan–Meier graph among the osteosarcoma population. The effects of the down-regulation and up-regulation of STEAP1 on the biological behavior of osteosarcoma cells were studied through in-vitro and in-vivo functional tests. ResultsUp-regulation of STEAP1 in the osteosarcoma tissues, whose correlations with the malignant osteosarcoma phenotype and the poor patient outcome were positive. In addition, STEAP1 induced the epithelial–mesenchymal transition (EMT) via the Wnt/β-catenin and TGF-β/Smad2/3 pathways and facilitated the osteosarcoma cell infiltration and migration. An increase or decrease in EFEMP1 expression directly promoted or inhibited the expression of STEAP1. In osteosarcoma cells overexpressing EFEMP1, STEAP1 knockdown significantly inhibited cell invasion, EMT process, and increased activity of Wnt/β-catenin and TGF-β/Smad2/3 signaling pathways. Although exogenous EFEMP1 could stimulate the Wnt/β-catenin and TGF-β/Smad2/3 pathways to promote the EMT, it had not effect on osteosarcoma cells with STEAP1 knockdown. Collectively, similar to EFEMP1, STEAP1 acted like an oncogene in the osteosarcoma progression. ConclusionEFEMP1 enabled the Wnt/β-catenin and TGF-β/Smad2/3 axises initiation and EMT elicitation by targeting STEAP1, thereby facilitating the osteosarcoma cell infiltration and migration. These results are expected to contribute to the search for new targeted drugs able to effectively inhibit invasion and metastasis and improve prognosis in osteosarcoma.