ABSTRACT
Objective:To investigate the changes in hearing threshold of the acquired primary cholesteatoma of the middle ear with different degrees of eustachian tube dysfunction after balloon eustachian tuboplasty. Methods:This retrospective study included forty cases with middle ear cholesteatoma and eustachian tube dysfunction who underwent open mastoidectomy + tympanoplasty + balloon eustachian tuboplasty were enrolled. All patients were admitted from November 2020 to April 2022. The preoperative eustachian tube score of 0-2 were defined as the lower group, and the scores of 3-5 were defined as the higher group. Pure tone audiometry was measured preoperatively and 1, 3, 6 and 12 months postoperatively. The average value of bone conduction threshold and air conduction threshold of 250-4 000 Hz were calculated, and the air-bone gap was calculated simultaneously. SPSS 25.0 was used for statistical analysis. P<0.05 was considered statistically significant. Results:In the lower group, the air conduction threshold and air-bone gap at 3 months postoperatively were significantly decreased in comparison with those preoperativelyï¼P<0.05ï¼ï¼as was the air-bone gap at 6 months postoperativelyï¼P<0.05ï¼. In the higher group, the air conduction threshold and air-bone gap were significantly decreased at 3, 6 and 12 months postoperativelyï¼P<0.05ï¼. Conclusion:The air conduction threshold and air-bone gap of patients with the acquired primary cholesteatoma of the middle ear and eustachian tube dysfunction were significantly decreased after eustachian tube balloon dilatation. Hearing improvement lasted longer in patients with slight eustachian tube dysfunction.
Subject(s)
Audiometry, Pure-Tone , Cholesteatoma, Middle Ear , Eustachian Tube , Tympanoplasty , Humans , Eustachian Tube/physiopathology , Eustachian Tube/surgery , Retrospective Studies , Female , Cholesteatoma, Middle Ear/surgery , Male , Tympanoplasty/methods , Adult , Middle Aged , Auditory Threshold , Mastoidectomy/methods , Bone ConductionABSTRACT
[This retracts the article DOI: 10.21037/atm-21-6123.].
ABSTRACT
BACKGROUND: Osteosarcoma is a highly malignant bone tumor that exhibits rapid growth and early metastasis. Hypoxia plays a pivotal role in promoting the proliferation and metastasis of osteosarcoma through a series of molecular events, which are partially mediated and regulated by HIF-1α. However, the regulatory network associated with HIF-1α in osteosarcoma remains limited. Therefore, the objective of this study was to identify critical hypoxia-associated genes and investigate their effects and molecular mechanisms in osteosarcoma cells. METHODS: Through bioinformatics analysis, matrilin-4 (MATN4) was identified as a crucial gene associated with hypoxia. The expression of MATN4 and HIF-1α was assessed using immunohistochemistry, RT-qPCR, and western blotting. The proliferative capacity of osteosarcoma cells was assessed through the utilization of CCK-8, EDU staining, and colony formation assays. The effects of MATN4 on the mobility of OS cells were evaluated using wound-healing assays and transwell assays. The interaction between MATN4 and HIF-1α was detected through chromatin immunoprecipitation. RESULTS: MATN4 is overexpressed in osteosarcoma tissue and cells, particularly in osteosarcoma cells with high metastatic potential. Knockdown of MATN4 inhibits the proliferation, migration, and invasion abilities of osteosarcoma cells and reverses the promoting effects of hypoxia on these functions. Additionally, HIF-1α binds to MATN4 and upregulates its expression. Interestingly, knockdown of HIF-1α reduces the stimulatory effects of MATN4 overexpression on the proliferation, migration, and invasion of osteosarcoma cells under hypoxic conditions. CONCLUSIONS: Taken together, our results suggest that MATN4 is regulated by HIF-1α and confers a more aggressive phenotype on OS cells. This evidence suggests that MATN4 may act as a potential target for OS diagnosis and treatment.
Subject(s)
Bone Neoplasms , Cell Proliferation , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit , Osteosarcoma , Humans , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Bone Neoplasms/metabolism , Cell Line, Tumor , Cell Movement , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Neoplasm Metastasis , Osteosarcoma/genetics , Osteosarcoma/pathology , Osteosarcoma/metabolismABSTRACT
Tumour-associated macrophages (TAMs), encompassing M1 and M2 subtypes, exert significant effects on osteosarcoma (OS) progression and immunosuppression. However, the impacts of TAM-derived biomarkers on the progression of OS remains limited. The GSE162454 profile was subjected to single-cell RNA (scRNA) sequencing analysis to identify crucial mediators between TAMs and OS cells. The clinical features, effects and mechanisms of these mediators on OS cells and tumour microenvironment were evaluated via biological function experiments and molecular biology experiments. Phosphodiesterase 4C (PDE4C) was identified as a pivotal mediator in the communication between M2 macrophages and OS cells. Elevated levels of PDE4C were detected in OS tissues, concomitant with M2 macrophage level, unfavourable prognosis and metastasis. The expression of PDE4C was observed to increase during the conversion process of THP-1 cells to M2 macrophages, which transferred the PDE4C mRNA to OS cells through exosome approach. PDE4C increased OS cell proliferation and mobility via upregulating the expression of collagens. Furthermore, a positive correlation was observed between elevated levels of PDE4C and increased TIDE score, decreased response rate following immune checkpoint therapy, reduced TMB and diminished PDL1 expression. Collectively, PDE4C derived from M2 macrophages has the potential to enhance the proliferation and mobility of OS cells by augmenting collagen expression. PDE4C may serve as a valuable biomarker for prognosticating patient outcomes and response rates following immunotherapy.
Subject(s)
Bone Neoplasms , Cyclic Nucleotide Phosphodiesterases, Type 4 , Immunotherapy , Macrophages , Osteosarcoma , Tumor Microenvironment , Humans , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/genetics , Bone Neoplasms/immunology , Bone Neoplasms/pathology , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4/genetics , Gene Expression Regulation, Neoplastic , Immunotherapy/methods , Macrophages/metabolism , Macrophages/immunology , Neoplasm Metastasis , Osteosarcoma/pathology , Osteosarcoma/immunology , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/therapy , Prognosis , Tumor Microenvironment/immunology , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/metabolismABSTRACT
Osteoarthritis (OA) is a degenerative disease closely associated with Anoikis. The objective of this work was to discover novel transcriptome-based anoikis-related biomarkers and pathways for OA progression.The microarray datasets GSE114007 and GSE89408 were downloaded using the Gene Expression Omnibus (GEO) database. A collection of genes linked to anoikis has been collected from the GeneCards database. The intersection genes of the differential anoikis-related genes (DEARGs) were identified using a Venn diagram. Infiltration analyses were used to identify and study the differentially expressed genes (DEGs). Anoikis clustering was used to identify the DEGs. By using gene clustering, two OA subgroups were formed using the DEGs. GSE152805 was used to analyse OA cartilage on a single cell level. 10 DEARGs were identified by lasso analysis, and two Anoikis subtypes were constructed. MEgreen module was found in disease WGCNA analysis, and MEturquoise module was most significant in gene clusters WGCNA. The XGB, SVM, RF, and GLM models identified five hub genes (CDH2, SHCBP1, SCG2, C10orf10, P FKFB3), and the diagnostic model built using these five genes performed well in the training and validation cohorts. analysing single-cell RNA sequencing data from GSE152805, including 25,852 cells of 6 OA cartilage.
Subject(s)
Anoikis , Osteoarthritis , Humans , Anoikis/genetics , Machine Learning , Cadherins , Osteoarthritis/diagnosis , Osteoarthritis/genetics , Sequence Analysis, RNA , Shc Signaling Adaptor ProteinsABSTRACT
Hematopoietic stem cells (HSCs) sustain hematopoiesis during homeostasis and regeneration. However, their limited availability poses a challenge for protein analysis. Here, we present a protocol for performing high-sensitivity western blot on HSCs using two techniques that enhance HSC isolation from mice and boost sensitivity for low cell numbers. We describe steps for isolating murine bone marrow cells, antibody staining, and cell sorting and post-sort analysis. We then detail a western blot procedure suitable for low numbers of HSCs. For complete details on the use and execution of this protocol, please refer to Li et al (2022).1,2.
Subject(s)
Hematopoiesis , Hematopoietic Stem Cells , Animals , Mice , Hematopoietic Stem Cells/metabolism , Cell Separation , Bone Marrow Cells , Blotting, WesternABSTRACT
PURPOSE: Osteosarcoma is one of the leading causes of cancer mortality in children and teenagers. Dysregulation of lipid metabolism has been reported to involve tumor progression. Our previous evidence has revealed that circular RNA hsa_circ_0000073 enhanced the proliferation and metastasis of osteosarcoma cells. However, the effect of hsa_circ_0000073 on the lipid metabolism of osteosarcoma remains unclear. In this paper, we focused on the effect of hsa_circ_0000073 in lipid metabolism and investigated a network among hsa_circ_0000073/ miR-1184 /FADS2 in osteosarcoma, which provides a new idea to treat osteosarcoma. METHODS: The osteosarcoma and its adjacent tissue samples were collected for further validation. qRT-PCR or western blot was employed to detect the expression of hsa_circ_0000073, miR-1184, and FADS2 in OS cells and tissues. Microarray analysis, mass spectrometry, metabolomics analysis, and bioinformatics analysis were used to explore the potential function and target gene of hsa_circ_0000073. Oil red o, Nile red staining, and Triglyceride content assay were adopted to confirm the effect of hsa_circ_0000073 on the lipid metabolism of OS. Dual-luciferase reporter assays and RNA immunoprecipitation were applied to construct and validate the ceRNA network of hsa_circ_0000073. The xenograft mouse model was taken to verify the effect of hsa_circ_0000073 on lipid metabolism in vivo. RESULTS: The results confirmed that hsa_circ_0000073 was raised in the tumor tissues more than its adjacent tissue. Moreover, the higher expression of hsa_circ_0000073 was associated with worse survival rates, advanced clinical stage, large tumor size, and metastasis. After hsa_circ_0000073 silence, the gene chip and metabolomics result implied that hsa_circ_0000073 expression is positively correlated with a 91 genes signature and 78 metabolites in MG-63 and Saos-2 cells. The bioinformatics analysis indicated that hsa_circ_0000073 might involve in the biological processes of lipid metabolism. Further loss and gain of function experiments affirmed that hsa_circ_0000073 could impact cell lipid synthesis. Mechanically, hsa_circ_0000073 favored the expression of FADS2 genes by sponging miR-1184. Consistent with these observations, silencing of hsa_circ_0000073 inhibited lipid synthesis in vivo xenograft mouse model. CONCLUSIONS: Our study revealed that hsa_circ_0000073 contributed to the lipid synthesis of osteosarcoma by decreasing the expression of miR-1184, thereby increasing FADS2, which provides new insights into treating osteosarcoma.
Subject(s)
Bone Neoplasms , Fatty Acid Desaturases , MicroRNAs , Osteosarcoma , RNA, Circular , Animals , Humans , Mice , Bone Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Fatty Acid Desaturases/genetics , Lipids , MicroRNAs/genetics , Osteosarcoma/pathology , RNA, Circular/geneticsABSTRACT
BACKGROUND: Post-traumatic related limb osteomyelitis (PTRLO) is a complex bone infection. Currently, there are no available microbial data on a national scale that can guide appropriate antibiotic selection, and explore the dynamic changes in dominant pathogens over time. This study aimed to conduct a comprehensive epidemiological analysis of PTRLO in China. METHODS: The study was approved by the Institutional Research Board (IRB), and 3526 PTRLO patients were identified from 212 394 traumatic limb fracture patients at 21 hospitals between 1 January 2008 and 31 December 2017. A retrospective analysis was conducted to investigate the epidemiology of PTRLO, including changes in infection rate (IR), pathogens, infection risk factors and antibiotic resistance and sensitivity. RESULTS: The IR of PTRLO increased gradually from 0.93 to 2.16% (Z=14.392, P <0.001). Monomicrobial infection (82.6%) was significantly higher than polymicrobial infection (17.4%) ( P <0.001). The IR of Gram-positive (GP) and Gram-negative (GN) pathogens showed a significant increase from the lowest 0.41% to the highest 1.15% (GP) or 1.62% (GN), respectively. However, the longitudinal trend of GP vs. GN's composition did not show any significance (Z=±1.1918, P >0.05). The most prevalent GP strains were Methicillin-sensitive Staphylococcus aureus (MSSA) (17.03%), Methicillin-resistant Staphylococcus aureus (MRSA) (10.46%), E. faecalis (5.19%) and S. epidermidis (4.87%). In contrast, the dominant strains GN strains were Pseudomonas Aeruginosa (10.92%), E. cloacae (10.34%), E. coli (9.47%), Acinetobacter Baumannii (7.92%) and Klebsiella Pneumoniae (3.33%). In general, the high-risk factors for polymicrobial infection include opened-fracture (odds ratio, 2.223), hypoproteinemia (odds ratio, 2.328), and multiple fractures (odds ratio, 1.465). It is important to note that the antibiotics resistance and sensitivity analysis of the pathogens may be influenced by complications or comorbidities. CONCLUSIONS: This study provides the latest data of PTRLO in China and offers trustworthy guidelines for clinical practice. (China Clinical Trials.gov number, ChiCTR1800017597).
Subject(s)
Coinfection , Fractures, Open , Methicillin-Resistant Staphylococcus aureus , Osteomyelitis , Humans , Retrospective Studies , Escherichia coli , Coinfection/drug therapy , Microbial Sensitivity Tests , Anti-Bacterial Agents/therapeutic use , China/epidemiology , Osteomyelitis/epidemiology , Osteomyelitis/etiology , Osteomyelitis/drug therapyABSTRACT
Inflammation and ferroptosis crosstalk complexly with immune microenvironment of hepatocellular carcinoma (HCC), thus affecting the efficacy of immunotherapy. Herein, our aim was to identify the inflammation-associated ferroptosis (IAF) biomarkers for contributing HCC. A total of 224 intersecting DEGs identified from different inflammation- and ferroptosis-subtypes were set as IAF genes. Seven of them including ADH4, APOA5, CFHR3, CXCL8, FTCD, G6PD and PON1 were used for construction of a risk model which classified HCC patients into two groups (high and low risk). HCC patients in the high-risk group exhibited shorter survival rate and higher immune score, and were predicted to have higher respond rate in immune checkpoint inhibition (ICI) therapy. Levels of the seven genes were significantly changed in HCC tissues in comparison to adjacent tissues. After inserting the gene expression into the risk model, we found that the risk model exhibited the higher diagnostic value for distinguish HCC tissues compared each single gene. Furthermore, HCC tissues from our research group with high-risk score exhibited more cases of microsatellite instability (MSI), heavier tumour mutational burden (TMB), higher expression level of PDL1 and cells with CD8. Knockdown of APOA5 reduced HCC cell proliferation combining with elevating inflammation and ferroptosis levels. In conclusion, we considered APOA5 maybe a novel target for suppressing HCC via simultaneously elevating inflammation and ferroptosis levels, and signature constructed by seven IAF genes including ADH4, APOA5, CFHR3, CXCL8, FTCD, G6PD and PON1 can act as a biomarker for optimising the diagnosis, prognosis evaluation and immunotherapy options in HCC patients.
Subject(s)
Carcinoma, Hepatocellular , Ferroptosis , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/therapy , Ferroptosis/genetics , Liver Neoplasms/diagnosis , Liver Neoplasms/genetics , Liver Neoplasms/therapy , Immunotherapy , Inflammation/genetics , Tumor Microenvironment/genetics , AryldialkylphosphataseABSTRACT
Background: Anoikis is a specialized form of programmed apoptosis that occurs in two model epithelial cell lines and plays an important role in tumors. However, the prognostic value of anoikis-related lncRNA (ARLncs) in osteosarcoma (OS) has not been reported. Methods: Based on GTEx and TARGET RNA sequencing data, we carried out a thorough bioinformatics analysis. The 27 anoikis-related genes were obtained from the Gene Set Enrichment Analysis (GSEA). Univariate Cox regression and least absolute shrinkage and selection operator (LASSO) analysis were successively used to screen for prognostic-related ARLncs. To create the prognostic signature of ARLncs, we performed multivariate Cox regression analysis. We calculated the risk score based on the risk coefficient, dividing OS patients into high- and low-risk subgroups. Additionally, the relationship between the OS immune microenvironment and risk prognostic models was investigated using function enrichment, including Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), single-sample gene set enrichment analysis (ssGSEA), and GSEA analysis. Finally, the potential effective drugs in OS were found by immune checkpoint and drug sensitivity screening. Results: A prognostic signature consisting of four ARLncs (AC079612.1, MEF2C-AS1, SNHG6, and TBX2-AS1) was constructed. To assess the regulation patterns of anoikis-related lncRNA genes, we created a risk score model. According to a survival analysis, high-risk patients have a poor prognosis as they progress. By using immune functional analysis, the lower-risk group demonstrated the opposite effects compared with the higher-risk group. GO and KEGG analysis showed that the ARLncs pathways and immune-related pathways were enriched. Immune checkpoints and drug sensitivity analysis might be used to determine the better effects of the higher group. Conclusion: We identified a novel prognostic model based on a four-ARLncs signature that might serve as potential prognostic indicators that can be used to predict the prognosis of OS patients, and immunotherapy and drugs that may contribute to improving the overall survival of OS patients and advance our understanding of OS.
ABSTRACT
The osteonecrotic area of steroid-induced avascular necrosis of the femoral head (SANFH) is a hypoxic microenvironment that leads to apoptosis of transplanted bone marrow mesenchymal stem cells (BMSCs). However, the underlying mechanism remains unclear. Here, we explore the mechanism of hypoxic-induced apoptosis of BMSCs, and use the mechanism to improve the transplantation efficacy of BMSCs. Our results show that the long non-coding RNA AABR07053481 (LncAABR07053481) is downregulated in BMSCs and closely related to the degree of hypoxia. Overexpression of LncAABR07053481 could increase the survival rate of BMSCs. Further exploration of the downstream target gene indicates that LncAABR07053481 acts as a molecular "sponge" of miR-664-2-5p to relieve the silencing effect of miR-664-2-5p on the target gene Notch1. Importantly, the survival rate of BMSCs overexpressing LncAABR07053481 is significantly improved after transplantation, and the repair effect of BMSCs in the osteonecrotic area is also improved. This study reveal the mechanism by which LncAABR07053481 inhibits hypoxia-induced apoptosis of BMSCs by regulating the miR-664-2-5p/Notch1 pathway and its therapeutic effect on SANFH.
Subject(s)
Femur Head Necrosis , Mesenchymal Stem Cells , MicroRNAs , Humans , Femur Head Necrosis/chemically induced , Femur Head Necrosis/genetics , Femur Head Necrosis/therapy , Mesenchymal Stem Cells/metabolism , Apoptosis/genetics , Hypoxia/metabolism , Steroids/metabolism , MicroRNAs/genetics , MicroRNAs/metabolismABSTRACT
BACKGROUND: Early metastasis is a hallmark of osteosarcoma (OS), a highly common type of malignant tumor. Members of the potassium inwardly rectifying channel family exert oncogenic effects in various cancers. However, the role of the potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) in OS is unclear. METHODS: The expression of KCNJ2 in OS tissues and cell lines was measured using bioinformatic analysis, immunohistochemistry, and western blotting. Wound-healing assays, Transwell assays, and lung metastasis models were used to analyze the effects of KCNJ2 on mobility of OS cells. The molecular mechanisms linking KCNJ2 and HIF1α in OS were explored by mass spectrometry analysis, immunoprecipitation, ubiquitination detection, and chromatin-immunoprecipitation quantitative real-time polymerase chain reaction. RESULTS: KCNJ2 was found to be overexpressed in advanced-stage OS tissues, as well as in cells with high metastatic potential. High expression of KCNJ2 was associated with a shorter survival rate of OS patients. KCNJ2-inhibition repressed the metastasis of OS cells, whereas KCNJ2-elevation induced the opposite effects. Mechanistically, KCNJ2 binds to HIF1α and inhibits its ubiquitination, thus increasing the expression of HIF1α. Interestingly, HIF1α binds directly to the KCNJ2 promoter and increases its transcription under hypoxic conditions. CONCLUSION: Taken together, our results indicated that a KCNJ2/HIF1α positive feedback loop exists in OS tissues, which significantly promotes OS cell metastasis. This evidence may contribute to the diagnosis and treatment of OS. Video Abstract.
Subject(s)
Bone Neoplasms , Osteosarcoma , Potassium Channels, Inwardly Rectifying , Humans , Feedback , Biological Assay , Cell Line , Bone Neoplasms/genetics , Potassium Channels, Inwardly Rectifying/geneticsABSTRACT
OBJECTIVE: Medial opening wedge high tibial osteotomy (HTO) is successful in the treatment of knee osteoarthritis with medial compartment stenosis and tibial varus deformity, but patella infera is the main complication. This study aims to design a new medial tibial open osteotomy scheme, transtibial tuberosity-high tibial osteotomy (TT-HTO), which can fully protect the patellar tendon insertion. In addition, the area of the osteotomy surface and wedge volume were evaluated in TT-HTO, biplanar distal tibial tuberosity osteotomy (biplanar-DTO), and uniplanar-DTO to evaluate the potential advantages of this technology in bone healing. METHODS: The tibial tubercle was divided into four equal sections from proximal to distal, which were defined as zones A, B, C, and D. From September to December 2020, the imaging examinations of 200 patients (95 males and 105 females) with a mean age of 40.6 years (range 19-60 years) were evaluated to observe the zonation of the tibial tubercle where the insertion of the patellar tendon is located. Then, 59 patients (23 males and 36 females) with a mean age 59.6 years (range 43-77 years), for a total of 69 knees (32 right and 37 left), who underwent routine knee surgery were observed and verified. According to the position of the patellar tendon insertion, TT-HTO was designed. Fifteen tibial sawbones were divided equally into three groups: TT-HTO; biplanar-DTO; and uniplanar-DTO. The total area of the osteotomy surface was compared using the graph paper method. The wedge volume at wedge heights of 10 mm was compared among osteotomy types using the plasticine Archimedes principle. One-way repeated-measures analysis of variance was used to compare the total area of the osteotomy surface and the wedge volume. RESULTS: The osteotomy line of TT-HTO passes through the boundary point of zones B and C of the tibial tubercle to fully protect the insertion point of the patellar tendon. The total area of the osteotomy surface in TT-HTO and biplanar-DTO was significantly larger than that in uniplanar-DTO (P < 0.05). The wedge volume in uniplanar-DTO was significantly smaller than that in TT-HTO and biplanar-DTO (P < 0.05). No significant differences in the osteotomy surface and the wedge volume were identified between TT-HTO and biplanar-DTO. CONCLUSION: TT-HTO can protect the patellar tendon insertion and avoid postoperative patella infera. The osteotomy surface is large and located in an area of cancellous bone, which ensures its good healing characteristics.
Subject(s)
Osteoarthritis, Knee , Patellar Ligament , Male , Female , Humans , Young Adult , Adult , Middle Aged , Aged , Patellar Ligament/surgery , Patella/surgery , Osteoarthritis, Knee/surgery , Osteotomy/methods , Tibia/surgery , Knee Joint/surgeryABSTRACT
Background: Cell metabolic reprogramming is a hallmark of tumor prognosis, and fatty acid metabolism (FAM) plays a crucial role in the tumor microenvironment (TME). However, the relationship between FAM, TME, and prognosis of acute myeloid leukemia (AML) patients remains elusive. Methods: We extracted the single-cell RNA sequencing (scRNA-Seq) and bulk transcriptome data of AML patients from the TCGA and GEO databases and assessed the relationship between FAM, TME, and AML patient prognosis. We also performed functional enrichment (FE) assay to evaluate the significance of FAM in anti-AML immunosurveillance. Results: Our scRNA-Seq analysis revealed that the leukemic stem cell (LSC)-enriched population exhibited elevated levels of FAM-related genes. Using these FAM-related genes, we developed a prognostic model that accurately estimated AML patient outcome. FE analysis showed that FAM was strongly related to alterations of TME-based immunosurveillance in AML patients. More importantly, we demonstrated that FAM inhibition via pharmaceutical targeting of PLA2G4A, a highly expressed FAM gene in AML patients with poor prognosis, enhanced the NK cell-mediated immunosurveillance in leukemia cells. Conclusions: Leukemic stem cell (LSC)-enriched population exhibited elevated levels of FAM-related genes. We have successfully established the FAM formula that predicts AML patient prognosis and alterations in the TME-based immunosurveillance. We also found that PLA2G4A was a highly expressed FAM gene in AML patients with poor prognoses. Pharmaceutical targeting of PLA2G4A increased the expression of NKG2DL in leukemia cells in vitro and thus enhanced the NK cell-mediated immunosurveillance.
ABSTRACT
Dysregulation of immune cell infiltration in the tumor microenvironment contributes to the progression of osteosarcoma (OS). In the present study, we explored genes related to immune cell infiltration and constructed a risk model to predict the prognosis of and guide therapeutic strategies for OS. The gene expression profile of OS was obtained from TARGET and Gene Expression Omnibus, which were set as the discovery and verification cohorts. CIBERSORT and Kaplan survival analyses were used to analyze the effects of immune cells on the overall survival rates of OS in the discovery cohort. Differentially expressed gene (DEG) analysis and protein-protein interaction (PPI) networks were used to analyze genes associated with immune cell infiltration. Cox regression analysis was used to select key genes to construct a risk model that classified OS tissues into high- and low-risk groups. The prognostic value of the risk model for survival and metastasis was analyzed by Kaplan-Meier survival analyses, receiver operating characteristic curves, and immunohistochemical experiments. Immunological characteristics and response effects of immune checkpoint blockade (ICB) therapy in OS tissues were analyzed using the ESTIMATE and Tumor Immune Dysfunction and Exclusion algorithms, while sensitivity for both targeted and chemotherapy drugs was analyzed using the OncoPredict algorithm. It was demonstrated that the high infiltration of resting dendritic cells in OS tissues was associated with poor prognosis. A total of 225 DEGs were found between the high- and low-infiltration groups of OS tissues, while 94 genes interacted with others. Through COX analyses, among these 94 genes, four genes (including AOC3, CDK6, COL22A1, and RNASE6) were used to construct a risk model. This risk model showed a remarkable prognostic value for survival rates and metastasis in both the discovery and verification cohorts. Even though a high microsatellite instability score was observed in the high-risk group, the ICB response in the high-risk group was poor. Furthermore, using OncoPredict, we found that the high-risk group OS tissues were resistant to seven drugs and sensitive to 25 drugs. Therefore, our study indicates that the resting dendritic cell signature constructed by AOC3, CDK6, COL22A1, and RNASE6 may contribute to predicting osteosarcoma prognosis and thus therapy guidance.
Subject(s)
Bone Neoplasms , Osteosarcoma , Bone Neoplasms/genetics , Bone Neoplasms/therapy , Gene Expression Profiling , Humans , Immune Checkpoint Inhibitors/therapeutic use , Osteosarcoma/genetics , Osteosarcoma/pathology , Osteosarcoma/therapy , Prognosis , Tumor Microenvironment/geneticsABSTRACT
Objectives: Osteosarcoma is a malignant bone tumor with poor outcomes affecting the adolescents and elderly. In this study, we comprehensively assessed the metabolic characteristics of osteosarcoma patients and constructed a hexosamine biosynthesis pathway (HBP)-based risk score model to predict the prognosis and tumor immune infiltration in patients with osteosarcoma. Methods: Gene expression matrices of osteosarcoma were downloaded from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) and Gene Expression Omnibus (GEO) databases. GSVA and univariate Cox regression analysis were performed to screen the metabolic features associated with prognoses. LASSO regression analysis was conducted to construct the metabolism-related risk model. Differentially expressed genes (DEGs) were identified and enrichment analysis was performed based on the risk model. CIBERSORT and ESTIMATE algorithms were executed to evaluate the characteristics of tumor immune infiltration. Comparative analyses for immune checkpoints were performed and the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was used to predict immunotherapeutic response. Finally, hub genes with good prognostic value were comprehensive analyzed including drug sensitivity screening and immunohistochemistry (IHC) experiments. Results: Through GSVA and survival analysis, the HBP pathway was identified as the significant prognostic related metabolism feature. Five genes in the HBP pathway including GPI, PGM3, UAP1, OGT and MGEA5 were used to construct the HBP-related risk model. Subsequent DEGs and enrichment analyses showed a strong correlation with immunity. Further, CIBERSORT and ESTIMATE algorithms showed differential immune infiltration characteristics correlated with the HBP-related risk model. TIDE algorithms and immune checkpoint analyses suggested poor immunotherapeutic responses with low expression of immune checkpoints in the high-risk group. Further analysis revealed that the UAP1 gene can predict metastasis. IHC experiments suggested that UAP1 expression correlated significantly with the prognosis and metastasis of osteosarcoma patients. When screening for drug sensitivity, high UAP1 expression was suggestive of great sensitivity to antineoplastic drugs including cobimetinib and selumetinib. Conclusion: We constructed an HBP-related gene signature containing five key genes (GPI, PGM3, UAP1, OGT, MGEA5) which showed a remarkable prognostic value for predicting prognosis and can guide immunotherapy and targeted therapy for osteosarcoma.
Subject(s)
Bone Neoplasms , Osteosarcoma , Adolescent , Humans , Aged , Hexosamines , Osteosarcoma/pathology , Prognosis , Bone Neoplasms/genetics , Survival AnalysisABSTRACT
Bone marrow mesenchymal stem cells (BMSCs) have strong regenerative potential and show good application prospects for treating clinical diseases. However, in the process of BMSC transplantation for treating ischemic and hypoxic diseases, BMSCs have high rates of apoptosis in the hypoxic microenvironment of transplantation, which significantly affects the transplantation efficacy. Our previous studies have confirmed the key role of long non-coding RNA Tmem235 (LncRNA Tmem235) in the process of hypoxia-induced BMSC apoptosis and its downstream regulatory mechanism, but the upstream mechanism by which hypoxia regulates LncRNA Tmem235 expression to induce BMSC apoptosis is still unclear. Under hypoxic conditions, we found that the level of LncRNA Tmem235 promoter histone H3 lysine 27 trimethylation modification (H3K27me3) was significantly increased by CHIP-qPCR. Moreover, H3K27me3 cooperated with LncRNA Tmem235 promoter DNA methylation to inhibit the expression of LncRNA Tmem235 and promote apoptosis of BMSCs. To study the mechanism of hypoxia-induced modification of LncRNA Tmem235 promoter H3K27me3 in the hypoxia model of BMSCs, we detected the expression of H3K27 methylase and histone demethylase and found that only histone methylase enhancer of zeste homolog 2 (EZH2) expression was significantly upregulated. Knockdown of EZH2 significantly decreased the level of H3K27me3 modification in the LncRNA Tmem235 promoter. The EZH2 promoter region contains a hypoxia-responsive element (HRE) that interacts with hypoxia-inducible factor-1alpha (HIF-1α), which is overexpressed under hypoxic conditions, thereby promoting its overexpression. In summary, hypoxia promotes the modification of the LncRNA Tmem235 promoter H3K27me3 through the HIF-1α/EZH2 signaling axis, inhibits the expression of LncRNA Tmem235, and leads to hypoxic apoptosis of BMSCs. Our findings improve the regulatory mechanism of LncRNA Tmem235 during hypoxic apoptosis of BMSCs and provide a more complete theoretical pathway for targeting LncRNA to inhibit hypoxic apoptosis of BMSCs.
Subject(s)
Mesenchymal Stem Cells , RNA, Long Noncoding , Apoptosis/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Histone Demethylases/genetics , Histone Demethylases/metabolism , Histones/genetics , Histones/metabolism , Humans , Hypoxia/genetics , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lysine/genetics , Lysine/metabolism , Mesenchymal Stem Cells/metabolism , Promoter Regions, Genetic , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolismABSTRACT
Hematopoietic stem cells (HSCs) have reduced capacities to properly maintain and replenish the hematopoietic system during myelosuppressive injury or aging. Expanding and rejuvenating HSCs for therapeutic purposes has been a long-sought goal with limited progress. Here, we show that the enzyme Sphk2 (sphingosine kinase 2), which generates the lipid metabolite sphingosine-1-phosphate, is highly expressed in HSCs. The deletion of Sphk2 markedly promotes self-renewal and increases the regenerative potential of HSCs. More importantly, Sphk2 deletion globally preserves the young HSC gene expression pattern, improves the function, and sustains the multilineage potential of HSCs during aging. Mechanistically, Sphk2 interacts with prolyl hydroxylase 2 and the Von Hippel-Lindau protein to facilitate HIF1α ubiquitination in the nucleus independent of the Sphk2 catalytic activity. Deletion of Sphk2 increases hypoxic responses by stabilizing the HIF1α protein to upregulate PDK3, a glycolysis checkpoint protein for HSC quiescence, which subsequently enhances the function of HSCs by improving their metabolic fitness; specifically, it enhances anaerobic glycolysis but suppresses mitochondrial oxidative phosphorylation and generation of reactive oxygen species. Overall, targeting Sphk2 to enhance the metabolic fitness of HSCs is a promising strategy to expand and rejuvenate functional HSCs.
Subject(s)
Hematopoietic Stem Cells , Sphingosine , Glycolysis/genetics , Hematopoietic Stem Cells/metabolism , Phosphotransferases (Alcohol Group Acceptor) , Prolyl Hydroxylases/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Hematopoietic stem cells (HSCs) adapt their metabolism to maintenance and proliferation; however, the mechanism remains incompletely understood. Here, we demonstrated that homeostatic HSCs exhibited high amino acid (AA) catabolism to reduce cellular AA levels, which activated the GCN2-eIF2α axis, a protein synthesis inhibitory checkpoint to restrain protein synthesis for maintenance. Furthermore, upon proliferation conditions, HSCs enhanced mitochondrial oxidative phosphorylation (OXPHOS) for higher energy production but decreased AA catabolism to accumulate cellular AAs, which inactivated the GCN2-eIF2α axis to increase protein synthesis and coupled with proteotoxic stress. Importantly, GCN2 deletion impaired HSC function in repopulation and regeneration. Mechanistically, GCN2 maintained proteostasis and inhibited Src-mediated AKT activation to repress mitochondrial OXPHOS in HSCs. Moreover, the glycolytic metabolite, NAD+ precursor nicotinamide riboside (NR), accelerated AA catabolism to activate GCN2 and sustain the long-term function of HSCs. Overall, our study uncovered direct links between metabolic alterations and translation control in HSCs during homeostasis and proliferation.