Cirsiliol induces autophagy and mitochondrial apoptosis through the AKT/FOXO1 axis and influences methotrexate resistance in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, with poor outcomes for patients with metastatic disease or chemotherapy resistance. Cirsiliol is a recently found flavonoid with anti-tumor effects in various tumors. However, the effects of cirsiliol in the regulation of aggressive behaviors of OS remain unknown. METHODS: The effect of cirsiliol on the proliferation of OS cells was detected using a cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) staining, while cell apoptosis was detected using flow cytometry. Immunofluorescence was applied to visualize the expression level of the mitochondria, lysosomes and microtubule-associated protein light chain 3 (LC3). A computational molecular docking technique was used to predict the interaction between cirsiliol and the AKT protein. The impact of cirsiliol on resistance was investigated by comparing it between a methotrexate (MTX)-sensitive OS cell line, U2OS, and a MTX-resistant OS cell line, U2OS/MTX. Finally, in situ xenogeneic tumor models were used to validate the anti-tumor effect of cirsiliol in OS. RESULTS: Cirsiliol inhibited cell proliferation and induced apoptosis in both U2OS and U2OS/MTX300 OS cells. In addition, treatment with cirsiliol resulted in G2 phase arrest in U2OS/MTX300 and U2OS cells. Cell fluorescence probe staining results showed impaired mitochondria and increased autophagy in OS cells after treatment with cirsiliol. Mechanistically, it was found that cirsiliol targeted AKT by reducing the phosphorylation of AKT, which further activated the transcriptional activity of forkhead Box O transcription factor 1 (FOXO1), ultimately affecting the function of OS cells. Moreover, in situ tumorigenesis experiments showed that cirsiliol inhibited the tumorigenesis and progression of OS in vivo. CONCLUSIONS: Cirsiliol inhibits OS cell growth and induces cell apoptosis by reducing AKT phosphorylation and further promotes FOXO1 expression. These phenomena indicate that cirsiliol is a promising treatment option for OS.

Circular RNA circEMB promotes osteosarcoma progression and metastasis by sponging miR-3184-5p and regulating EGFR expression.

BACKGROUND: Osteosarcoma (OSA) is the most prevalent type of bone cancer with a high rate of metastasis. Circular RNAs (CircRNAs) play an essential role in multiple aspects of tumour biology. This study aimed to elucidate the role of circEMB in OSA. METHODS: circRNAs related to OSA invasion were identified via RNA sequencing and qRT-PCR. The relationship between circEMB levels and clinicopathological features of OSA was examined using the clinical specimens and data of 53 patients with OSA. Several in vivo and in vitro experiments, including intravital imaging, whole-transcriptome sequencing, transwell assay, flow cytometry, dual-luciferase reporter assay, RIP assay, RNA pull-down assay and RNA-FISH, were performed to examine the effects of circEMB on the malignant behaviour of OSA. RESULTS: A novel circRNA, named circEMB (hsa_circ_001310), was identified in this study. circEMB can promote the malignant behaviour of OSA. In vitro experiments revealed that circEMB knockdown decreased cell proliferation, inhibited tumour invasion and metastasis; increased apoptosis and resulted in G1/S phase arrest. In vivo experiments revealed that circEMB knockdown inhibited tumour growth and metastasis in xenograft-bearing mice. Mechanistically, circEMB affects the malignant behaviour of OSA by mediating EGFR as an miR-3184-5p sponge. In addition, the circEMB/miR-3184-5p/EGFR axis modulates methotrexate (MTX) resistance in OSA. CONCLUSIONS: CircEMB plays a critical role in promoting cancer via the miR-3184-5p/EGFR pathway, indicating that circEMB may serve as a therapeutic target for OSA.

The hexosamine biosynthesis pathway-related gene signature correlates with immune infiltration and predicts prognosis of patients with osteosarcoma.

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.

Characterization of the Tumor Microenvironment in Osteosarcoma Identifies Prognostic- and Immunotherapy-Relevant Gene Signatures.

The osteosarcoma (OS) microenvironment is composed of tumor cells, immune cells, and stromal tissue and is emerging as a pivotal player in OS development and progression. Thus, microenvironment-targeted strategies are urgently needed to improve OS treatment outcomes. Using principal component analysis (PCA), we systematically examined the tumor microenvironment (TME) and immune cell infiltration of 88 OS cases and constructed a TME scoring system based on the TMEscore high and TMEscore low phenotypes. Our analysis revealed that TMEscore high correlates with longer survival in OS patients, elevated immune cell infiltration, increased immune checkpoints, and increased sensitivity to chemotherapy. TMEscore low strongly correlated with immune exclusion. These observations were externally validated using a GEO dataset (GSE21257) from 53 OS patients. Our laboratory data also proved our findings. This finding enhances our understanding of the immunological landscape in OS and may uncover novel targeted therapeutic strategies.

Cordycepin augments the chemosensitivity of osteosarcoma to cisplatin by activating AMPK and suppressing the AKT signaling pathway.

BACKGROUND: Osteosarcoma is the most common primary bone tumor in children and adolescents. However, some patients with osteosarcoma develop resistance to chemotherapy, leading to a poor clinical prognosis. Hence, effective therapeutic agents that can improve the response to chemotherapy drugs to improve the prognosis of patients with osteosarcoma are urgently needed. Cordycepin has recently emerged as a promising antitumor drug candidate. This study aims to explore the effect of cordycepin in suppressing osteosarcoma in vivo and in vitro and the synergistic effect of cordycepin combined with cisplatin and to demonstrate the underlying molecular mechanism. METHODS: CCK-8 assay was performed to investigate the inhibition effect of cordycepin combined with cisplatin in osteosarcoma cell lines. The colony formation and invasion abilities were measured by colony formation assay and Transwell assay. Osteosarcoma cells apoptosis was detected by flow cytometry. Western blot analysis were used to detect the expression of cell apoptosis-related proteins and AMPK and AKT/mTOR signaling pathway-related proteins. Finally, we performed the in vivo animal model to further explore whether cordycepin and cisplatin exert synergistic antitumor effects. RESULTS: Notably, we found that treatment with cordycepin inhibited cell proliferation, invasion, and induced apoptosis in osteosarcoma cells in vitro and in vivo. Moreover, the combination of cordycepin and cisplatin led to marked inhibition of osteosarcoma cell proliferation and invasion and promoted osteosarcoma cell apoptosis in vitro and in vivo. Mechanistically, we demonstrated that cordycepin enhanced the sensitivity of osteosarcoma cells to cisplatin by activating AMPK and inhibiting the AKT/mTOR signaling pathway. CONCLUSIONS: In brief, this study provides comprehensive evidence that cordycepin inhibits osteosarcoma cell growth and invasion and induces osteosarcoma cell apoptosis by activating AMPK and inhibiting the AKT/mTOR signaling pathway and enhances the sensitivity of osteosarcoma cells to cisplatin, suggesting that cordycepin is a promising treatment for osteosarcoma.

Circular RNA circ_001422 promotes the progression and metastasis of osteosarcoma via the miR-195-5p/FGF2/PI3K/Akt axis.

BACKGROUND: Circular RNAs (circRNAs) are involved in diverse processes that drive cancer development. However, the expression landscape and mechanistic function of circRNAs in osteosarcoma (OS) remain to be studied. METHODS: Bioinformatic analysis and high-throughput RNA sequencing tools were employed to identify differentially expressed circRNAs between OS and adjacent noncancerous tissues. The expression level of circ_001422 in clinical specimens and cell lines was measured using qRT-PCR. The association of circ_001422 expression with the clinicopathologic features of 55 recruited patients with OS was analyzed. Loss- and gain-of-function experiments were conducted to explore the role of circ_001422 in OS cells. RNA immunoprecipitation, fluorescence in situ hybridization, bioinformatics database analysis, RNA pulldown assays, dual-luciferase reporter assays, mRNA sequencing, and rescue experiments were conducted to decipher the competitive endogenous RNA regulatory network controlled by circ_001422. RESULTS: We characterized a novel and abundant circRNA, circ_001422, that promoted OS progression. Circ_001422 expression was dramatically increased in OS cell lines and tissues compared with noncancerous samples. Higher circ_001422 expression correlated with more advanced clinical stage, larger tumor size, higher incidence of distant metastases and poorer overall survival in OS patients. Circ_001422 knockdown markedly repressed the proliferation and metastasis and promoted the apoptosis of OS cells in vivo and in vitro, whereas circ_001422 overexpression exerted the opposite effects. Mechanistically, competitive interactions between circ_001422 and miR-195-5p elevated FGF2 expression while also initiating PI3K/Akt signaling. These events enhanced the malignant characteristics of OS cells. CONCLUSIONS: Circ_001422 accelerates OS tumorigenesis and metastasis by modulating the miR-195-5p/FGF2/PI3K/Akt axis, implying that circ_001422 can be therapeutically targeted to treat OS.

Identification of prognostic biomarkers associated with metastasis and immune infiltration in osteosarcoma.

Osteosarcoma is the most common primary malignancy of the bones, and is associated with a high rate of metastasis and a poor prognosis. A tight association between the tumor microenvironment (TME) and osteosarcoma metastasis has been established. In the present study, the Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm was applied to calculate the immune and stromal scores of patients with osteosarcoma based on data from The Cancer Genome Atlas database. A metagene approach and deconvolution method were used to reveal distinct TME landscapes in patients with osteosarcoma. Bioinformatics analysis was used to identify differentially expressed genes (DEGs) associated with metastasis and immune infiltration in osteosarcoma, and a risk model was constructed using the DEGs with potential prognostic significance. Subsequently, gene set enrichment and Spearman's correlation analyses were used to delineate the biological processes associated with these prognostic biomarkers. Finally, immunohistochemical (IHC) analysis was performed to evaluate the expression levels of immune infiltrates and prognostic biomarkers in clinical osteosarcoma tissues. The results of the ESTIMATE demonstrated that patients with non-metastatic osteosarcoma presented with higher immune/stromal scores and a more favorable prognosis compared with those with metastatic osteosarcoma. The TME landscapes in patients with osteosarcoma suggested that high levels of tumor-infiltrating immune cells (TIICs) may suppress metastasis. Increased numbers of CD56bright natural killer cells, immature B cells, M1 macrophages and neutrophils, and lower levels of M2 macrophages were observed in the non-metastatic tissues compared with those in the metastatic tissues. A total of 69 DEGs were identified to be associated with metastasis and immune infiltration in osteosarcoma. Of these, GATA3, LPAR5, EVI2B, RIAM and CFH exhibited prognostic potential and were highly expressed in non-metastatic osteosarcoma tissues based on the IHC analysis results. These biomarkers were involved in various immune-related biological processes and were positively associated with multiple TIICs and immune signatures. The risk model constructed using these prognostic biomarkers demonstrated high predictive accuracy for the prognosis of osteosarcoma. In conclusion, the present study proposed a five-biomarker prognostic signature for the prediction of metastasis and immune infiltration in patients with osteosarcoma.

Metastasis-associated gene MAPK15 promotes the migration and invasion of osteosarcoma cells via the c-Jun/MMPs pathway.

Osteosarcoma (OS) is the most common and destructive primary bone malignancy to affect children and adolescents. Metastases remain the primary cause of death in patients with OS. In the present study, weight gene co-expressed network analysis (WGCNA) and differentially-expressed gene analysis were used to identify key genes associated with the metastasis of OS. Reverse transcription-quantitative PCR and immunohistochemical staining were then used to detect the expression levels of these key genes in OS tissues, and to determine the hub genes of interest. Wound-healing and transwell assays, in addition to a lung metastasis model, were used to detect the effects of the hub genes on OS cell proliferation and metastasis in vitro and in vivo. Using WGCNA and differential expression analysis, deleted in lung and esophageal cancer protein 1 (DLEC1), Forkhead box J1 (FOXJ1) and mitogen-activated protein kinase 15 (MAPK15) were predicted to be key metastasis-associated genes, and highly expressed in metastatic OS tissues; among them, the protein and mRNA expression levels of MAPK15 were most significantly increased in our OS tissues from patients who exhibited metastases at diagnosis, and thus MAPK15 was determined to be a metastasis-associated hub gene to further study. Furthermore, inhibiting MAPK15 expression significantly decreased OS cell metastasis in vitro and in vivo, as well as suppressing c-Jun/matrix metalloproteinase (MMP)-associated pathways. Overexpression of MAPK15 activated the c-Jun/MMPs pathway and promoted OS cell metastasis, while inhibition of c-Jun blocked this effect. Taken together, MAPK15 was indicated to be an OS metastasis-associated gene, and was confirmed to promote the migration and invasion of OS cells via the c-Jun/MMP pathway. MAPK15 may therefore be an effective target for the treatment of OS.

Identification of key genes involved in the development and progression of early-onset colorectal cancer by co-expression network analysis.

A number of studies have revealed that there is an increasing incidence of early-onset colorectal cancer (CRC) in young adults (before the age of 50 years) and a progressive decline in CRC among older patients, after the age of 50 years (late-onset CRC). However, the etiology of early-onset CRC is not fully understood. The aim of the present study was to identify key genes associated with the development of early-onset CRC through weighted gene co-expression network analysis (WGCNA). The GSE39582 dataset was downloaded from the Gene Expression Omnibus database, and the data profiles of tissues from patients diagnosed before the age of 50 years were selected. The top 10,000 genes with the highest variability were used to construct the WGCNA. Hub genes were identified from the modules associated with clinical traits using gene significance >0.2 and module membership >0.8 as the cut-off criteria. Gene Ontology and pathway analyses were subsequently performed on the hub genes and a protein-protein interaction network (PPI) was constructed. The diagnostic value of module hub genes with a degree score >5 in the PPI network was verified in samples from patients with CRC diagnosed before the age of 50 years obtained from The Cancer Genome Atlas. Eight co-expressed gene modules were identified in the WGCNA and two modules (blue and turquoise) were associated with the tumor-node-metastasis stage. A total of 140 module hub genes were identified and found to be enriched in 'mitochondrial large ribosomal subunit', 'structural constituent of ribosome', 'poly (A) RNA binding', 'collagen binding', 'protein ubiquitination' and 'ribosome pathway'. Twenty-six module hub genes were found to have a degree score >5 in the PPI network, seven of which [secreted protein acidic and cysteine rich (SPARC), decorin (DCN), fibrillin 1 (FBN1), WW domain containing transcription regulator 1 (WWTR1), transgelin (TAGLN), DEAD-box helicase 28 (DDX28) and cold shock domain containing C2 (CSDC2)], had good prognostic values for patients with early-onset CRC, but not late-onset CRC. Therefore, SPARC, DCN, FBN1, WWTR1, TAGLN, DDX28 and CSDC2 may contribute to the development of early-onset CRC and may serve as potential diagnostic biomarkers.

Tibial tunnel placement in anatomic anterior cruciate ligament reconstruction: a comparison study of outcomes between patient-specific drill template versus conventional arthroscopic techniques.

INTRODUCTION: Accurate anatomic graft tunnel positioning is essential for the successful application of anatomic anterior cruciate ligament (ACL) reconstruction. The accurate insertion of the tibial tunnel (TT) remains challenging. Here, we explored a novel strategy of patient-specific drill template (PDT) for the placement of TT in ACL reconstruction and assessed its efficacy and accuracy. MATERIALS AND METHODS: TT placement was randomized and performed by use of the PDT technique in 40 patients (PDT group) and the conventional arthroscopic technique in 38 patients (Arthroscopic group). After surgery, the deviations at the center point of the ACL tibial attachment area and radiological TT positioning were assessed in both groups. The preoperative and follow-up examinations included pivot-shift testing, KT-1000 arthrometer testing, the Lysholm and International Knee Documentation Committee scales were used to compare the knee stability and the functional state. RESULTS: The ideal center points achieved in the PDT group were more precise than that in the arthroscopic group (p < 0.001). Radiological TT positioning performed by use of the PDT technique was more accurate than that by the arthroscopic technique (p = 0.027). Statistical differences could not be found between the groups in terms of the pivot-shift test, KT-1000 arthrometer laxity measurements, the Lysholm or International Knee Documentation Committee scales. Both groups improved at follow-up compared with the preoperative assessment in terms of the pivot-shift test, the laxity tests, and scoring scales. CONCLUSIONS: The novel PDT strategy could provide more accurate TT positioning than the traditional arthroscopic technique in ACL reconstruction. However, functional scales and stability tests gave similar results in the PDT and the standard techniques. LEVEL OF EVIDENCE: I.