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

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

A novel biplanar positioning technique to guide iliosacral screw insertion: a retrospective study.

PURPOSE: To evaluate the safety and benefits of the biplanar position technique on operative time, radiation exposure, and screw placement accuracy. METHODS: In this study, we retrospectively evaluated the records of 64 patients with pelvic fractures (Tile B and C) between October 2020 and September 2021. According to the surgical methods selected by the patients, the patients were divided into a biplanar positioning technique group (biplanar group), a Ti-robot navigation group (Ti-robot group), and a traditional fluoroscopy-guided technique group (traditional group). Length of operation, blood loss, intra-operative radiation exposure fracture reduction, and the quality of screw positioning were compared among the three groups. RESULTS: One hundred three screws were implanted in 64 patients (biplanar group 22, Ti-robot group 21, traditional group 21). The average operation time was significantly less in the biplanar group (26.32 ± 6.32 min) than in the traditional group (79.24 ± 11.31 min), but significantly more than in the Ti-robot group (15.81 ± 3.9 min). The radiation exposure was similar in the biplanar group (740.53 ± 185.91 cGy/cm2) and Ti-robot group (678.44 ± 127.16 cGy/cm2), both of which were significantly more than in the traditional group (2034.58 ± 494.54 cGy/cm2). The intra-operative blooding loss was similar in the biplanar group (12.76 ± 3.77 mL) and the Ti-robot group (11.92 ± 4.67 mL), both of which were significantly less than in the traditional group (29.7 ± 8.01 mL). The Screw perforation was slightly lower in the biplanar group (94.1%) than in the Ti-robot group (97.2%) but was significantly higher than in the traditional group (75.7%). CONCLUSIONS: The biplanar positioning technique is as accurate and safe as computer-navigated systems for percutaneous iliosacral screw insertion, associated with shorter surgical time, lower intra-operative radiation exposure, and more accuracy compared to traditional fluoroscopy.

ADAM19 and TUBB1 Correlate with Tumor Infiltrating Immune Cells and Predicts Prognosis in Osteosarcoma.

BACKGROUND: Osteosarcoma is the most common type of primary malignant bone tumor. INTRODUCTION: This study aimed to explore potential key prognostic genes and their roles in osteosarcoma. METHODS: Three microarray datasets for osteosarcoma were downloaded from the GEO database. Differentially expressed genes (DEGs) were screened by the Limma package. Functional enrichment analysis was performed based on DAVID, GeneMANIA, and Metascape databases. Prognostic value of DEGs was elevated by survival analysis. CIBERSORT was used to assess the infiltrating abundance of 22 immune cells, followed by the Pearson correlation analysis between immune cells and prognosis-related genes. Gene set enrichment analysis and drug-gene interactions prediction were performed for prognosis-related genes. RESULTS: A total of 8 common up-regulated DEGs and 13 common down-regulated DEGs were screened in the GSE36001 and GSE56001 datasets. Enrichment analysis showed these DEGs were implicated in platelet activation, SMAD protein phosphorylation, lymphocyte/leukocyte/T cells activation, and cell migration. Survival analysis indicated that elevated expression of ADAM19 and TUBB1 were associated with a favorable prognosis. CIBERSORT algorithm revealed the higher infiltrating level of CD8 T cells, macrophages M0, and M2 in osteosarcoma. ADAM19 expression positively correlated with naïve B cells and negatively correlated with activated dendritic cells infiltrating abundance. TUBB1 expression positively correlated with gamma delta T cells while negatively correlated with helper follicular T cells infiltrating abundance. A total of 56 drugs were found to target TUBB1. CONCLUSION: ADAM19 and TUBB1 could be prognostic biomarkers in osteosarcoma. Both their expression correlates with tumor infiltrating immune cells. TUBB1 was a multi-drug target that might be a therapeutic target in osteosarcoma.

Bioinformatics integrated analysis to investigate candidate biomarkers and associated metabolites in osteosarcoma.

BACKGROUND: This study hoped to explore the potential biomarkers and associated metabolites during osteosarcoma (OS) progression based on bioinformatics integrated analysis. METHODS: Gene expression profiles of GSE28424, including 19 human OS cell lines (OS group) and 4 human normal long bone tissue samples (control group), were downloaded. The differentially expressed genes (DEGs) in OS vs. control were investigated. The enrichment investigation was performed based on DEGs, followed by protein-protein interaction network analysis. Then, the feature genes associated with OS were explored, followed by survival analysis to reveal prognostic genes. The qRT-PCR assay was performed to test the expression of these genes. Finally, the OS-associated metabolites and disease-metabolic network were further investigated. RESULTS: Totally, 357 DEGs were revealed between the OS vs. control groups. These DEGs, such as CXCL12, were mainly involved in functions like leukocyte migration. Then, totally, 38 feature genes were explored, of which 8 genes showed significant associations with the survival of patients. High expression of CXCL12, CEBPA, SPARCL1, CAT, TUBA1A, and ALDH1A1 was associated with longer survival time, while high expression of CFLAR and STC2 was associated with poor survival. Finally, a disease-metabolic network was constructed with 25 nodes including two disease-associated metabolites cyclophosphamide and bisphenol A (BPA). BPA showed interactions with multiple prognosis-related genes, such as CXCL12 and STC2. CONCLUSION: We identified 8 prognosis-related genes in OS. CXCL12 might participate in OS progression via leukocyte migration function. BPA might be an important metabolite interacting with multiple prognosis-related genes.

Immune-related prognostic genes signatures in the tumor microenvironment of sarcoma.

Sarcomas are a heterogeneous group of malignant mesenchymal neoplasms. This study aimed to investigate the immune-related prognostic gene signatures in the tumor microenvironment of sarcoma. The RNA-sequencing data and clinical phenotype data of 260 sarcoma samples and two normal samples were downloaded from The Cancer Genome Atla (TCGA) database. Tumor purity and immune cells infiltration were evaluated by Estimation of Stromal and Immune cells in Malignant Tumors using Expression data (ESTIMATE) deconvolution algorithm. Differentially expressed genes (DEGs) were screened in high vs. low immune score groups. Survival analysis was performed using Kaplan-Meier curve with log-rank test. Tumor infiltrating of immune cells was analyzed by Tumor Immune Estimation Resource (TIMER). High immune score and ESTIMATE score were associated with favorable prognosis. A total of 623 immune DEGs were screened. The majority of these genes (532 genes accounting for 85% of the DEGs) were up-regulated, and these genes were significantly enriched in various immune related biological processed and pathways, such as neutrophil activation, T cell activation, antigen processing and presentation. A total of 146 prognosis-related immune DEGs, and seven hub genes were identified, including B2M, HLA-DRB1, HLA-DRA, HLA-E, LCK, HLA-DPA1, and VAV1. Survival analysis showed that high expression of these genes was associated with a favorable prognosis. There were negative correlations between the expression of these hub genes and tumor purity, while positive correlations between expression of these hub genes and f infiltration levels of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages and dendritic cells. These results help to stratify patients with different immune subtypes and help to design immunotherapy strategies for these patients in sarcoma.

The effect of bone morphogenetic protein 2 composite materials combined with cannulated screws in treatment of acute displaced femoral neck fractures.

The risk of avascular necrosis (AVN) and nonunion after treatment of displaced femoral neck fractures is increased in patients aged <60 years. Therefore we established a new protocol for closed reduction and internal fixation (CRIF) using cannulated screws combined with bone morphogenetic protein 2 (BMP-2) composite materials to treat acute femoral neck fractures.This study enrolled 78 patients with acute femoral neck fractures between April 2014 and September 2016. We treated 46 patients with a mean age of 43.8 years in study group. These patients were treated by CRIF combined with BMP-2 composite materials. In control group, there were 32 patients with a mean age of 42.09 years. The patients were treated by CRIF without BMP-2. The duration between presentation and surgery, operative time, Harris score and complications were recorded.In study group, 43 patients were followed up with an average of 31.3 months. One patient suffered nonunion and three patients presented AVN. In control group, 28 patients were followed up with an average of 32.3 months, the rate of AVN and fracture nonunion were 25% (7/28) and 21.4% (6/28) respectively, significantly higher than those in study group (P < .05).Acute displaced femoral neck fractures can be treated with CRIF and BMP-2 composite materials in a minimally invasive manner. This technique was reproducible and had fewer complications.

Anti-inflammatory effect of hesperidin enhances chondrogenesis of human mesenchymal stem cells for cartilage tissue repair.

BACKGROUND: Articular cartilage diseases are considered a major health problem, and tissue engineering using human mesenchymal stem cells (MSCs) have been shown as a promising solution for cartilage tissue repair. Hesperidin is a flavonoid extract from citrus fruits with anti-inflammatory properties. We aimed to investigate the effect of hesperidin on MSCs for cartilage tissue repair. MSCs were treated by hesperidin, and colony formation and proliferation assays were performed to evaluate self-renewal ability of MSCs. Alcian blue staining and Sox9 expression were measured to evaluate chondrogenesis of MSCs. Secretion of pro-inflammatory cytokines IFN-γ, IL-2, IL-4 and IL-10, and expression of nuclear factor kappa B (NF-κB) subunit p65 were also assessed. RESULTS: Hesperidin improved self-renewal ability and chondrogenesis of MSCs, inhibited secretion of pro-inflammatory cytokines IFN-γ, IL-2, IL-4 and IL-10, and suppressed the expression of p65. Overexpression of p65 was able to reverse the hesperidin inhibited secretions of pro-inflammatory cytokines, and abolish the enhancing effect of hesperidin on chondrogenesis of MSCs. CONCLUSION: Hesperidin could serve as a therapeutic agent to effectively enhance chondrogenesis of human MSCs by inhibiting inflammation to facilitate cartilage tissue repair.

Bindarit Attenuates Pain and Cancer-Related Inflammation by Influencing Myeloid Cells in a Model of Bone Cancer.

C-C motif chemokine ligand 2 (CCL2) is a small cytokine that functions in inflammation and cancer development. Bindarit, a CCL2 inhibitor, is a small anti-inflammatory molecule proven safe by phase II trials in type 2 diabetic nephropathy patients. As cancer-related inflammation is a cause of pain, we investigated whether Bindarit suppresses cancer-related inflammation and pain. We established a bone-cancer mouse model by inoculating cancer cells. After applying Bindarit, we preformed pain sensitivity tests and checked cancer development by X-ray. Using flow cytometry and qRT-PCR assays, we assessed the effect of Bindarit on peripheral blood monocyte mobilization and M2 macrophage polarization. We also investigated the targets of Bindarit using western blotting and luciferase assay. Bindarit-treated mice performed better in pain sensitivity tests compare to control mice. X-ray imaging showed that Bindarit-treated mice had fewer cancer cell colonies and smaller overall tumor burden. Bindarit reduced the number of monocytes in peripheral blood and down-regulated the expression of M2 macrophage polarization makers. Bindarit also inhibited IKKß phosphorylation. Bindarit efficiently relieves cancer-related pain and suppresses cancer development. Bindarit inhibits monocyte mobilization in peripheral blood as well as M2 macrophage polarization. IKKß is identified as a target of Bindarit.

Baicalin Inhibits Inflammatory Responses to Interleukin-1ß Stimulation in Human Chondrocytes.

A mix of flavonoids comprising baicalin (BA) and catechin showed effective impacts on controlling the progress of knee osteoarthritis (OA). However, little is known about the underlying mechanisms. The influences of BA on the transcriptional levels of a series of proinflammatory genes were measured using real-time reverse transcription and polymerase chain reaction (RT-PCR). The expressions of proteins involved in nuclear factor-κB (NF-κB) activation were detected by Western blot. The in vitro results were confirmed in a mouse OA model. We found that BA treatment led to remarkable reductions of OA-related proinflammatory gene expressions, including interleukin (IL)-6, tumor necrosis factor (TNF), chemokine (C-X-C motif) ligand 1 (CXCL1), and CXCL10. The transcriptional levels of inducible nitric oxide synthase (iNOS/Nos2), matrix metalloproteinases (MMP)3, and MMP13 were significantly inhibited by BA. We measured the productions of nitrite and prostaglandin E2 from human chondrocytes and BA was shown to reduce their productions. On the contrary, mRNA levels of aggrecan and collagen-II were enhanced by BA treatment. The inhibitory role of BA on OA may possibly be mediated by NF-κB signaling because of comparable decreases of phosphorylated (p)-p65 and p-IκBα and less p65 translocation in the nucleus after BA treatment. In OA mice model, BA significantly reduced synovitis scores and related gene expressions, including IL-6, TNF, CXCL1, CXCL10, MMP3, MMP13, and Nos2. In conclusion, BA suppresses the inflammatory responses of human chondrocytes to IL-1ß stimulation, and NF-κB signaling may be involved in the mechanisms of BA functions.

Two different surgery approaches for treatment of thoracolumbar fracture.

BACKGROUND: To evaluate the safety and effectiveness of pedicle screw fixation for treatment of thoracolumbar fracture via Wiltse paraspinal approach. METHODS: From June 2009 to June 2012, we enrolled into our study 53 cases of thoracolumbar fractures (single segment compressive or burst fractures) without neurologic injury, among whom 28 were treated by mini-open operation via Wiltse approach (group A) and 25 by conventional posterior open surgery (group B). We recorded and compared the intraoperative and postoperative performance between the two groups. RESULTS: There was no serious complications occurring during our study, such as infection, blood vessel injury, spinal cord or nerve root injury. We did not find any statistically difference between the two groups in corrected rate of Cobb's angle or vertebral body height (P>0.05). However, the mini-open method had apparent advantages over the conventional open method in operative time, intraoperative blood loss, postoperative drainage, and postoperative improvement on visual analog scale (VAS) scores at 3 days, 1 month, and 6 months after operation (P<0.05). After 9-24-month follow-up, the number of patients with low back pain in group A was less than that in group B. CONCLUSION: In the premise of strict controlling surgery indications, pedicle screw fixation via Wiltse paraspinal approach has the advantages of simple operation and less trauma in treatment of single-segment thoracolumbar fracture without neurologic injury.

Which is the best alternative for displaced femoral neck fractures in the elderly?: A meta-analysis.

BACKGROUND: Treatment of displaced femoral neck fractures includes internal fixation and arthroplasty. However, whether arthroplasty or internal fixation is the primary treatment for displaced femoral neck fractures in elderly patients remains a subject for debate. The literature contains conflicting evidence regarding rates of mortality, revision surgery, major postoperative complications, and function in elderly patients with displaced femoral neck fractures treated either by internal fixation or arthroplasty (either hemiarthroplasty or THA). QUESTIONS/PURPOSE: We determined mortality, revision surgery rates, major surgical complications (which include infection, nonunion or early redisplacement, avascular necrosis, dislocation, loosening of the prosthesis, acetabular erosion, fracture below or around the implant, and other severe general complications such as deep vein thrombosis and pulmonary embolism), and function in patients treated with either internal fixation or arthroplasty for displaced femoral neck fractures in the elderly. METHODS: We searched PubMed, Embase, and the Cochrane Library for randomized controlled trials (RCTs) comparing internal fixation and arthroplasty. We identified 20 RCTs with 4508 patients meeting all the criteria for eligibility. We performed a meta-analysis of the major complications, reoperations, function, pain, and mortality. RESULTS: Compared with internal fixation, arthroplasty reduced the risk of the major complications (95% CI, 0.21-0.54 for 1 year; 95% CI, 0.16-0.31 for 5 years) and the incidence of reoperation 1 to 5 years after surgery (95% CI, 0.15-0.34 for 1 year; 95% CI, 0.08-0.24 for 5 years), and provided better pain relief (95% CI, 0.34-0.72). Function was superior (RR = 0.59; 95% CI, 0.44-0.79) for patients treated with arthroplasty than for patients treated by internal fixation. However, mortality 1 to 3 years after surgery was similar (95% CI, 0.96-1.23, p = 0.20 for 1 year; 95% CI, 0.91-1.17, p = 0.63 for 3 years). CONCLUSIONS: Arthroplasty can reduce the risk of major complications and the incidence of reoperation compared with internal fixation, and provide better pain relief and function, but it does not reduce mortality. LEVEL OF EVIDENCE: Level II, prognostic study. See the Guidelines for Authors for a complete description of levels of evidence.