Prognostic Factor For Osteosarcoma Patients Research Articles

A novel aging-associated lncRNA signature for predicting prognosis in osteosarcoma

Osteosarcoma (OS) is one of the most prevalent bone tumors in adolescents, and the correlation between aging and OS remains unclear. Currently, few accurate and reliable biomarkers have been determined for OS prognosis. To address this issue, we carried out a detailed bioinformatics analysis based on OS with data from the Cancer Genome Atlas data portal and Human Aging Genomic Resources database, as well as in vitro experiments. A total of 88 OS samples with gene expression profiles and corresponding clinical characteristics were obtained. Through univariate Cox regression analysis and survival analysis, 10 aging-associated survival lncRNAs (AASRs) were identified to be associated with the overall survival of OS patients. Based on the expression levels of the 10 AASRs, the OS patients were classified into two clusters (Cluster A and Cluster B). Cluster A had a worse prognosis, while Cluster B had a better prognosis. Then, 5 AASRs were ultimately included in the signature through least absolute shrinkage and selection operator-Cox regression analysis. Kaplan‒Meier survival analysis verified that the high-risk group exhibited a worse prognosis than the low-risk group. Furthermore, univariate and multivariate Cox regression analyses confirmed that the riskScore was an independent prognostic factor for OS patients. Subsequently, we discovered that the risk signature was correlated with the properties of the tumor microenvironment and immune cell infiltration. Specifically, there was a positive association between the risk model and naïve B cells, resting dendritic cells and gamma delta T cells, while it was negatively related to CD8+ T cells. Finally, in vitro experiments, we found that UNC5B-AS1 inhibited OS cells from undergoing cellular senescence and apoptosis, thereby promoting OS cells proliferation. In conclusion, we constructed and verified a 5 AASR-based signature, that exhibited excellent performance in evaluating the overall survival of OS patients. In addition, we found that UNC5B-AS1 might inhibit the senescence process, thus leading to the development and progression of OS. Our findings may provide novel insights into the treatment of OS patients.

Novel pyroptosis-related lncRNAs and ceRNAs predict osteosarcoma prognosis and indicate immune microenvironment signatures

ObjectiveTo study pyroptosis-related biomarkers that are associated with the prognosis and immune microenvironment characteristics of osteosarcoma (OS). The goal is to establish a foundation for the prognosis and treatment of OS. MethodsWe retrieved transcriptome and clinical data from The Cancer Genome Atlas (TCGA) database for 88 OS patients. Using this data, we constructed a prognostic model to identify pyroptosis-related genes (PRGs) associated with OS prognosis. To further explore the biological function of these PRGs, we performed enrichment analysis. To identify pyroptosis-related long non-coding RNAs (PRLncs) associated with the prognosis of OS, we performed co-expression analysis. Subsequently, a risk prognostic model was constructed using these PRLncs to generate a risk score, termed as PRLncs-score, thereby obtaining PRLncs associated with the prognosis of OS. The accuracy of the prognostic model was verified through survival analysis, risk curve, independent prognostic analysis, receiver operating characteristic (ROC) curve, difference analysis between high- and low-risk groups, and clinical correlation analysis. And to determine whether PRLncs-score is independent prognostic factor for OS. In addition, we further conducted external and internal validation for the risk prognosis model. Further analyses of immune cell infiltration and tumor microenvironment were performed. A pyroptosis-related competitive endogenous RNA (PRceRNA) network was constructed to obtain PRceRNAs associated with the prognosis of OS and performed gene set enrichment analysis (GSEA) on PRceRNA genes. ResultsWe obtained five PRGs (CHMP4C, BAK1, GSDMA, CASP1, and CASP6) that predicted OS prognosis and seven PRLncs (AC090559.1, AP003119.2, CARD8-AS1, AL390728.4, SATB2-AS1, AL133215.2, and AC009495.3) and one PRceRNA (CARD8-AS1-hsa-miR-21-5p-IL1B) that predicted OS prognosis and indicated characteristics of the OS immune microenvironment. The PRLncs-score, in combination with other clinical features, was established as an independent prognostic factor for OS patients. Subsequent scrutiny of the tumor microenvironment and immune infiltration indicated that patients with low-PRLncs-scores were associated with reduced metastatic risk, improved survival rates, heightened levels of immune cells and stroma, and increased immune activity compared to those with high-PRLncs-scores. ConclusionThe study's findings offer insight into the prognosis of OS and its immune microenvironment, and hold promise for improving early diagnosis and immunotherapy.

New gene signature from the dominant infiltration immune cell type in osteosarcoma predicts overall survival

The immune microenvironment of osteosarcoma (OS) has been reported to play an important role in disease progression and prognosis. However, owing to tumor heterogeneity, it is not ideal to predict OS prognosis by examining only infiltrating immune cells. This work aimed to build a prognostic gene signature based on similarities in the immune microenvironments of OS patients. Public datasets were used to examine the correlated genes, and the most consistent dominant infiltrating immune cell type was identified. The LASSO Cox regression model was used to establish a multiple-gene risk prediction signature. A nine-gene prognostic signature was generated from the correlated genes for M0 macrophages and then proven to be effective and reliable in validation cohorts. Signature comparison indicated the priority of the signature. Multivariate Cox regression models indicated that the signature risk score is an independent prognostic factor for OS patients regardless of the Huvos grade in all datasets. In addition, the results of the association between the signature risk score and chemotherapy sensitivity also showed that there was no significant difference in the sensitivity of any drugs between the low- and high-risk groups. A GSEA of GO and KEGG pathways found that antigen processing- and presentation-related biological functions and olfactory transduction receptor signaling pathways have important roles in signature functioning. Our findings showed that M0 macrophages were the dominant infiltrating immune cell type in OS and that the new gene signature is a promising prognostic model for OS patients.

Association of survivin positive circulating tumor cell levels with immune escape and prognosis of osteosarcoma.

Function of survivin protein (encoded by BIRC5) in circulating tumor cells (CTCs) of osteosarcoma (OS) has not been investigated. The goal of this study is to determine whether the expression of survivin protein of CTCs is associated with circulating immune cell infiltration and disease prognosis of OS. Blood samples of 20 patients with OS were collected. CanPatrol™ CTC enrichment technology combined with in situ hybridization (ISH) was applied to enrich and test CTCs and survivin protein. Bioinformation analysis combined with data of routine blood test was used to verify the association between survivin and immune cell infiltration in circulatory system. To screen independent prognostic factors, Kaplan-Meier survival curve, univariate and multivariable Cox regression analyses were performed. Bioinformatics analysis showed that BIRC5 was strongly negatively related to lymphocyte, including T cell, NK cell and B cell, which released that BIRC5 played a key role in immune escape via reducing immune cell infiltration in circulatory system. Meanwhile, the number of survivin+ CTCs was significantly negatively connection with lymphocyte count (R = -0.56, p = 0.011), which was consistent with bioinformatics analysis. Kaplan-Meier curve showed that the overall survival rate in high survivin+ CTCs group was significantly lower than low group (88.9% vs 36.4%, p = 0.04). Multivariable Cox regression analyses showed that survivin+ CTCs were an independent prognostic factor (p = 0.019). These findings suggested that survivin protein played a key role in immune escape of CTCs and the presence of survivin+ CTCs might be a promising prognostic factor in OS patients.

The bioinformatic approach identifies PARM1 as a new potential prognostic factor in osteosarcoma.

To explore the key factors affecting the prognosis of osteosarcoma patients. Based on the GEO dataset and differential expression analysis of normal and osteosarcoma tissues, the gene modules related to the prognosis of osteosarcoma patients were screened by WGCNA, and intersecting genes were taken with differential genes, and the risk prognosis model of osteosarcoma patients was constructed by LASSO regression analysis of intersecting genes, and the prognosis-related factors of osteosarcoma patients were obtained by survival analysis, followed by target for validation, and finally, the expression of prognostic factors and their effects on osteosarcoma cell migration were verified by cellular assays and lentiviral transfection experiments. The prognosis-related gene module of osteosarcoma patients were intersected with differential genes to obtain a total of 9 common genes. PARM1 was found to be a prognostic factor in osteosarcoma patients by LASSO regression analysis, followed by cellular assays to verify that PARM1 was lowly expressed in osteosarcoma cells and that overexpression of PARM1 in osteosarcoma cells inhibited cell migration. Pan-cancer analysis showed that PARM1 was lowly expressed in most cancers and that low expression of PARM1 predicted poor prognosis for patients. The data from this study suggest that PARM1 is closely associated with the prognosis of osteosarcoma patients, and PARM1 may serve as a novel potential prognostic target for osteosarcoma, providing a heartfelt direction for the prevention and treatment of osteosarcoma.

CD146 is closely associated with the prognosis and molecular features of osteosarcoma: Guidance for personalized clinical treatment

Background: Osteosarcoma (OSA), a focus for orthopedic surgeons, always results in severe death due to metastasis. CD146 is severely expressed in several tumors, indicating its potential as a biomarker for OSA. Method: Two OSA cohorts were enrolled in this study. A Therapeutically Applicable Research to Generate Effective Treatments-Osteosarcoma (TARGET-OS) cohort was used as a training cohort, and GSE21257 was used as the external validation cohort. The R package "limma" was used to discriminate the differentially expressed genes among CD146-high and CD146-low patients and was further annotated by the enriched signaling pathways. The R package MOVICS was used to evaluate immune infiltration and the response to chemotherapy and immunotherapy. All statistical analyses were performed by R version 4.0.2, and p < 0.05 was considered statistically significant. Result: CD146 plays an important role in promoting the progression, invasion, and metastasis of several tumors. In the current study, we first revealed an integrative unfavorable prognosis in patients with tumors (p < 0.01, HR: 1.10, 95% CI: 1.07-1.14). CD146 is tightly correlated with m5C RNA methylation modification genes in OSA. Furthermore, we revealed that CD146 acts as an oncogene in OSA patients and is linked to poor prognosis in both the TARGET-OS cohort (p = 0.019, HR: 2.61, 95% CI: 1.171-5.834) and the GSE21257 cohort (p = 0.005, HR: 3.61, 95% CI: 1.474-8.855), with a total of 137 patients, regardless of whether they were adjusted for clinical pathological features. Highly-expressed CD146 impacts the signaling pathways of cytokine‒cytokine receptor interactions and is associated with the high infiltration of immunocytes. Moreover, patients with high CD146 expression were more likely to be sensitive to anti-PD-1 immunotherapy, while patients with low expression of CD146 were more likely to be sensitive to cisplatin and doxorubicin chemotherapy. Conclusion: Overall, CD146 is an independent prognostic factor for OSA patients and can help doctors select clinical treatment strategies.

Prognostic significance of modified lung immune prognostic index in osteosarcoma patients.

Purpose: Osteosarcoma is the most common primary malignancy of bone with a dismal prognosis for patients with pulmonary metastases. Evaluation of osteosarcoma prognosis would facilitate the prognosis consultation as well as the development of personalized treatment decisions. However, there is limited effective prognostic predictor at present. Lung Immune Prognostic Index (LIPI) is a novel prognostic factor in pulmonary cancers, whereas, the prognostic significance of LIPI in osteosarcoma has not yet been well clarified. In this study, we firstly explore the prognostic role of LIPI and further modify this predictive model in osteosarcoma. Patients and methods: A retrospectively study was conducted at Musculoskeletal Tumor Center of West China Hospital between January 2016 and January 2021. Hematological factors and clinical features of osteosarcoma patients were collected and analyzed. The area under curve (AUC) and optimal cuff-off of each single hematological factor was calculated. Results: In this study, lactate dehydrogenase (LDH), derived neurtrophil to lymphocyte ratio (dNLR), and Hydroxybutyrate dehydrogenase (HBDH) have higher AUC values. LIPI was composed of LDH and dNLR and was further modified by combing the HBDH, forming the osteosarcoma immune prognostic index (OIPI). OIPI divided 223 osteosarcoma patients divided into four groups, none, light, moderate, and severe (p < 0.0001). OIPI has a higher AUC value than LIPI and other hematological indexes in t-ROC curve. According to the univariate and multivariate analysis, pathological fracture, metastasis, NLR, platelet–lymphocyte ratio (PLR), and OIPI were associated with the prognosis; and metastasis and OIPI were independent prognostic factors of osteosarcoma patients. An OIPI-based nomogram was also established and could predict the 3-year and 5-year overall survival. In addition, OIPI was also revealed correlated with metastasis and pathological fracture in osteosarcoma. Conclusion: This study first explore the prognostic significance of LIPI in osteosarcoma patients. In addition, we developed a modified LIPI, the OIPI, for osteosarcoma patients. Both the LIPI and OIPI could predict the overall survival of osteosarcoma patients well, while OIPI may be more suitable for osteosarcoma patients. In particular, OIPI may have the ability to identify some high-risk patients from clinically low-risk patients.

Identification of a Solute Carrier Family-Based Signature for Predicting Overall Survival in Osteosarcoma.

Given the important role of SLC family in essential physiological processes including nutrient uptake, ion transport, and waste removal, and that their dysregulation was found in distinct forms of cancer, here we identified a novel gene signature of SLC family for patient risk stratification in osteosarcoma. Gene expression data and relevant clinical materials of osteosarcoma samples were retrieved from The Cancer Genome Atlas (TCGA) database. Prognosis-related SLC genes were identified by performing univariate Cox regression analysis and were utilized to construct a four-SLC gene signature in osteosarcoma. It allowed patients to be classified into high- and low-risk groups, and Kaplan-Meier survival analysis in the training, testing, entire, and external GSE21257 cohorts suggested that the overall survival of patients in high-risk group was consistently worse than that in low-risk group, suggesting the promising accuracy and generalizability of the SLC-based signature in predicting the prognosis of patients with osteosarcoma. Moreover, univariate and multivariate Cox regression analyses indicated that the derived risk score was the only independent prognostic factor for osteosarcoma patients in TCGA and GSE21257 cohorts. Besides, a prognostic nomogram comprising the derived risk score and clinical features including gender and age was developed for clinical decision-making. Functional enrichment analyses of the differentially expressed genes between high- and low-risk group revealed that immune-related biological processes and pathways were significantly enriched. Estimation of tumor immune microenvironment using ESTIMATE algorithm revealed that patients with lower risk score had higher stromal, immune, and ESTIMATE score, and lower tumor purity. ssGSEA analyses indicated that the scores of various immune subpopulations including CD8+ T cells, DCs, and TIL were lower in high-risk group than these in low-risk group in both cohorts. As for the related immune functions, the scores of APC co-inhibition, CCR, check-point, T cell co-stimulation, and Type II IFN response were lower in high-risk group than these in low-risk group in both cohorts. In all, we identified a novel prognostic signature based on four SLC family genes that accurately predicted overall survival in osteosarcoma patients. Furthermore, the signature is linked to differences in immunological status and immune cell infiltrations in the tumor microenvironment.

The activity level of follicular helper T cells in the peripheral blood of osteosarcoma patients is associated with poor prognosis

ABSTRACT Osteosarcoma (OS) is solid tumors with high malignancy and incidence starting in the bones. OS pathogenesis has been proved to be closely associated with immune imbalance, and follicular helper T cells (Tfh) significantly affect host humoral immune homeostasis. However, there are few reports on the effect of Tfh cell activation on the prognosis of OS patients. Hence, this investigation on the changes in the proportion of peripheral blood Tfh cells in OS patients, and the relationship between their activity level and OS prognosis. We collected peripheral blood from patients with OS, benign bone tumor (BT group) and healthy subjects (Healthy group), respectively. The number of CD4+CXCR5+ Tfh cell in peripheral blood was measured by flow cytometry and correlation analysis between its activity and OS clinicopathological characteristics was carried out. The data showed that in comparison with the BT and Healthy groups, higher proportion and activation level of peripheral blood CD4+CXCR5+ Tfh cells in CD4+ T cells were found in the OS group. In OS patients, increases of the proportion and activity level of Tfh cells were associated with poorly differentiated OS and tumor metastasis. Additionally, Kaplan-Meier and Cox regression analysis showed a longer overall survival in patients with low proportion of peripheral blood CD4+CXCR5+ Tfh cells in CD4+ T cells, and their activation level may be a prognostic factor in OS patients. In conclusion, peripheral blood CD4+CXCR5+ Tfh cell activation in OS patients was associated with a poor prognosis. This study provided ideas for improving the clinical treatment of OS patients.

SELPLG Expression Was Potentially Correlated With Metastasis and Prognosis of Osteosarcoma.

Background: Osteosarcoma (OS) is the most prevalent malignant primary bone tumor in children. Selectin P ligand gene (SELPLG) has been studied in several cancers. Our research aimed to explore the role of SELPLG in OS. Methods: All OS patient data was obtained from TARGET and GEO databases. Differential expression analyses were conducted in limma package of R. Functional analyses included GO and KEGG enrichment analyses. Immune cell infiltration analysis was done in CIBERSORT software. The overall survival was calculated using survival and survminer package of R. Results: Significantly lower SELPLG expression was observed in metastatic OS samples compared with non-metastatic OS samples, both in TARGET and in GSE21257. Low SELPLG expression was an independent undesirable prognostic factor for OS patients, in both TARGET and GEO datasets. Totally 62 differentially expressed gene (DEG) overlaps were found between high SELPLG vs. low SELPLG and non-metastatic vs. metastatic OS samples, affecting metastases and thereby influencing the prognosis, which were significantly enriched in 40 GO and six KEGG terms. Five types of immune cells were significantly differentially infiltrated between high and low SELPLG expression OS patients. Conclusion: SELPLG is closely correlated with metastases and prognosis of OS patients. The OS patients with low SELPLG expression have relatively poorer prognosis and SELPLG is a potential prognostic biomarker for OS.

Risk Factors, Prognostic Factors, and Nomograms for Distant Metastasis in Patients With Newly Diagnosed Osteosarcoma: A Population-Based Study.

BackgroundOsteosarcoma is the most common bone cancer, mainly occurring in children and adolescents, among which distant metastasis (DM) still leads to a poor prognosis. Although nomogram has recently been used in tumor areas, there are no studies focused on diagnostic and prognostic evaluation of DM in primary osteosarcoma patients.MethodsThe data of osteosarcoma patients diagnosed between 2004 and 2015 were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. Univariate and multivariate logistic regression analyses were used to identify independent risk factors for DM in osteosarcoma patients, and univariate and multivariate Cox proportional hazards regression analyses were used to determine independent prognostic factors of osteosarcoma patients with DM. We then established two novel nomograms and the results were evaluated by receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA).ResultA total of 1,657 patients with osteosarcoma were included, and 267 patients (16.11%) had DM at the time of diagnosis. The independent risk factors for DM in patients with osteosarcoma include age, grade, T stage, and N stage. The independent prognostic factors for osteosarcoma patients with DM are age, chemotherapy and surgery. The results of ROC curves, calibration, DCA, and Kaplan–Meier (K-M) survival curves in the training, validation, and expanded testing sets, confirmed that two nomograms can precisely predict occurrence and prognosis of DM in osteosarcoma patients.ConclusionTwo nomograms are expected to be effective tools for predicting the risk of DM for osteosarcoma patients and personalized prognosis prediction for patients with DM, which may benefit clinical decision-making.

Construction of immune-related gene pairs signature to predict the overall survival of osteosarcoma patients.

The purpose of this study is to establish the prognosis of osteosarcoma patients based on the characteristics of immune-related gene pairs. We used the lasso Cox regression model to construct and verify the signature consisting of 14 immune-related gene pairs. This signature can accurately predict the overall survival of osteosarcoma patients and is an independent prognostic factor for osteosarcoma patients. For this we constructed a signature-based nomogram. The results of the nomogram show that our signature can bring clinical net benefits. We then assessed the abundance of infiltrating immune cells in each sample, and combine the results of the gene set enrichment analysis of a single sample to explore the differences in the immune microenvironment between IRPG signature groups. The result of gene set enrichment analysis shows the strong relationship between signature and immune system. Finally, we evaluated the relationship between signature and immunotherapy efficiency using algorithms such as TIMI and SubMap to explore patients who might benefit from immunotherapy. In conclusion, our signature can predict the overall survival rate of osteosarcoma patients and provide potential guidance for exploring patients who may benefit from immunotherapy.

The Long Noncoding RNA ZEB2-AS1 Contributes to Proliferation and Epithelial-to-Mesenchymal Transition of Osteosarcoma.

Background: Long non-coding RNA Zinc finger E-box binding homeobox 2 (ZEB2) antisense RNA 1 (ZEB2-AS1) has been shown to promote tumor progression. However, the clinical significance and fundamental function role of ZEB2-AS1 in osteosarcoma (OS) has been poorly understood. Methods: The expression of ZEB2-AS1 was determined in tumor tissues and matched normal tissues from 67 OS patients using quantitative reverse transcriptase PCR analysis. Clinical value of ZEB2-AS1 was evaluated by χ2 test and Kaplan-Meier method. Cell proliferation was analyzed using CCK-8 assay, colony formation. Cell apoptosis status was determined by caspase-3 activity assay. Cell migration, invasion and epithelial-mesenchymal transition (EMT) were investigated by scratch wound healing, transwell invasion assays and Western blotting. Results: Clinical association analysis revealed that high ZEB2-AS1 expression correlated with tumor size, distant metastasis and poor prognosis of OS patients. Moreover, ZEB2-AS1 expression was identified as an independent prognostic factor for OS patients. Loss-of-function assays demonstrated that ZEB2-AS1 knockdown suppressed the proliferation and induced apoptosis in OS cells. In addition, ZEB2-AS1 knockdown inhibited cell migration, invasion, EMT of OS cells in vitro. Conclusions: Taken together, our data demonstrate that ZEB2-AS1 serves a putative oncogenic role and associates with unfavorable prognosis in OS.

Four genes predict the survival of osteosarcoma patients based on TARGET database.

Osteosarcoma represents one of the most aggressive tumors of bone among adolescents and young adults. Despite improvements in treatment, osteosarcoma has a grave prognosis. The identification of prognostic factors is still in its infancy. Weighted gene correlation network analysis (WGCNA) was conducted on mRNA-sequencing and clinical information (gender, survival and metastasis) of osteosarcoma patients from the TARGET database to obtain genes in modules associated with metastasis of osteosarcoma. The Cox regression analysis was then performed on the gene expression profile from TARGET to screen genes associated with patients' survival. Known genes related to osteosarcoma were obtained by intersecting osteosarcoma-related genes from DisGeNET and DiGSeE, followed by the construction of PPI network of osteosarcoma-related genes and survival-related genes in modules. The screened key genes were subject to multi-factor Cox proportional hazards model, and osteosarcoma patients were classified into high- and low- risk groups according to the risk score to evaluate the potential of key genes to predict the survival of osteosarcoma patients. The WGCNA showed that 4 genes in tan and 19 genes in pink modules were related to the survival of osteosarcoma patients. Osteosarcoma-related known genes (9) were obtained in intersection of DisGeNET and DiGSeE. PPI network identified 4 key genes (KRT5, HIPK2, MAP3K5 and CD5) closely associated with survival of osteosarcoma patients. HIPK2, MAP3K5 and CD5 expression was inversely correlated with survival risk, while KRT5 expression was positively correlated with survival risk. These results show KRT5, HIPK2, MAP3K5 and CD5 serve as prognostic factors of osteosarcoma patients.

A comprehensive analysis for associations between multiple microRNAs and prognosis of osteosarcoma patients.

BackgroundOsteosarcoma (OS) is the most common malignant primary bone tumor occurring in children and young adults, which occupies the second important cause of tumor-associated deaths among children and young adults. Recent studies have demonstrated that many microRNAs (miRNAs) have abnormal expression in OS, and can function as prognostic factors of OS patients. However, no previous studies have comprehensively analyzed the relationship between multiple miRNAs and prognosis of OS patients.MethodsA total of 63 OS patients were retrospectively enrolled. The clinical characteristics were collected, and the expression levels of miRNA-21, miRNA-30c, miRNA-34a, miRNA-101, miRNA-133a, miRNA-214, miRNA-218, miRNA-433 and miRNA-539 in tumor tissues were measured through quantitative real-time polymerasechain reaction. Kaplan–Meier analysis was used to perform univariate survival analysis, and Cox regression model was used to perform multivariate survival analysis which included the variables with P < 0.1 in univariate survival analysis.ResultsThe cumulative survival for 1, 2 and 5 years was 90.48%, 68.25% and 38.10%, respectively, and mean survival time was (45.39 ± 3.60) months (95% CI [38.34–52.45]). Kaplan–Meier analysis demonstrated that TNM stage, metastasis or recurrence, miRNA-21, miRNA-214, miRNA-34a, miRNA-133a and miRNA-539 were correlated with cum survival, but gender, age, tumor diameter, differentiation, miRNA-30c, miRNA-433, miRNA-101 and miRNA-218 were not. Multivariate survival analysis demonstrated that miRNA-21 (hazard ratio (HR): 3.457, 95% CI [2.165–11.518]), miRNA (HR: 3.138, 95% CI [2.014–10.259]), miRNA-34a (HR: 0.452, 95% CI [0.202–0.915]), miRNA-133a (HR: 0.307, 95% CI [0.113–0.874]) and miRNA-539 (HR: 0.358, 95% CI [0.155–0.896]) were independent prognostic markers of OS patients after adjusting for TNM stage (HR: 2.893, 95% CI [1.496–8.125]), metastasis or recurrence (HR: 3.628, 95% CI [2.217–12.316]) and miRNA-30c (HR: 0.689, 95% CI [0.445–1.828]).ConclusionsHigh expression of miRNA-21 and miRNA-214 and low expression of miRNA-34a, miRNA-133a and miRNA-539 were associated with poor prognosis of OS patients after adjusting for TNM stage, metastasis or recurrence and miRNA-30c.

The lncRNAs RP1-261G23.7, RP11-69E11.4 and SATB2-AS1 are a novel clinical signature for predicting recurrent osteosarcoma.

Background: Osteosarcoma is the most common primary bone malignancy in children and adolescents. In order to find factors related to its recurrence, and thus improve recovery prospects, a powerful clinical signature is needed. Long noncoding RNAs (lncRNAs) are essential in osteosarcoma processes and development, and here we report significant lncRNAs to aid in earlier diagnosis of osteosarcoma.Methods: A univariate Cox proportional hazards regression analysis and a multivariate Cox regression analysis were used to analyze osteosarcoma patients’ lncRNA expression data from the Therapeutically Applicable Research To Generate Effective Treatments (TARGET), a public database.Results: A lncRNA signature consisting of three lncRNAs (RP1-261G23.7, RP11-69E11.4 and SATB2-AS1) was selected. The signature was used to sort patients into high-risk and low-risk groups with meaningful recurrence rates (median recurrence time 16.80 vs. >128.22 months, log-rank test, P<0.001) in the training group, and predictive ability was validated in a test dataset (median 16.32 vs. >143.80 months, log-rank test, P=0.006). A multivariate Cox regression analysis showed that the significant lncRNA was an independent prognostic factor for osteosarcoma patients. Functional analysis suggests that these lncRNAs were related to the PI3K-Akt signaling pathway, the Wnt signaling pathway, and the G-protein coupled receptor signaling pathway, all of which have various, important roles in osteosarcoma development. The significant 3-lncRNA set could be a novel prediction biomarker that could aid in treatment and also predict the likelihood of recurrence of osteosarcoma in patients.

WTAP Promotes Osteosarcoma Progression &lt;i&gt;via&lt;/i&gt; Regulating the m6A Methylation of HMBOX1

Background: N6-methyladenosine (m6A) regulators are involved in the progression of various cancers via regulating m6A modification. However, the potential role and mechanism of the m6A modification in osteosarcoma (OS) remains obscure. Methods: The expression of m6A regulators were detected using qPCR and western blot. Cell proliferation, colony formation, wound healing and transwell invasion assays were used to reveal the role of WTAP in the progression of OS. The mechanism of WTAP-mediated m6A modification was explored using M6A dot blot, RNA-seq and MeRIP-seq, MeRIP-qRT-PCR and luciferase reporter assays. Results: Here, WTAP was found to be high expression in OS tissue and it was an independent prognostic factor for OS patients . Functionally, WTAP, as an oncogene, was involved in the proliferation and metastasis of OS. Mechanistically, M6A dot blot, RNA-seq and MeRIP-seq, MeRIP-qRT-PCR and luciferase reporter assays showed that HMBOX1 is the target gene of WTAP, which regulated HMBOX1 stability in an m6A-dependent manner by targeting to the 3’UTR of HMBOX1 mRNA. In addition, HMBOX1 expression was downregulated in OS and negatively linked to WTAP expression in OS patients. Silenced HMBOX1 evidently attenuated shWTAP-mediated suppression on OS growth and metastasis in vivo and vitro. Finally, WTAP/HMBOX1 regulated OS growth and metastasis via PI3K/AKT pathway. Interpretation: The study demonstrates the critical role of the WTAP-mediated m6A modification in the progression of OS, which could provide novel insights into therapy of OS. Funding Statement: This study was supported by National Natural Science Foundation of China (Grant Nos. 81772866); National Natural Science Foundation of China (Grant Nos. 81502331); Natural Science Foundation of Hunan Province (Grant Nos. 2018JJ2617); Natural Science Foundation of Hunan Province (Grant Nos. 2016JJ3176); National Key Research and Development Program of China (No. 2016YFC1201800) ; Natural Science Foundation of Hunan Province (No. 2018SK2090). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Informed consent was obtained from each patient or their guardians, and the study were approved by the Ethics Committee of The Third Xiangya Hospital of Central South University. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of The Third Xiangya Hospital of Central South University, Changsha, China.

Expression Changes, Prognostic Analysis and Risk Factors of miR-625-3p and miR-449a in Osteosarcoma Patients after Surgery

The expression changes of miR-625-3p and miR-449a in osteosarcoma patients after surgery is the critical study in this paper. Analysis of their prognosis and risk factors can be helpful in understanding the prognostic value for clinical purposes. Fifty-eight patients with osteosarcoma diagnosed in our hospital were considered as the research group (RG), and 52 health subjects at the same time were collected as the control group (CG). Fluorescence quantitative PCR (RTPCR) was employed to test the expression levels of miR-625-3p and miR-449a in the serum of subjects in both groups before surgery and patients in the RG after surgery. The survival conditions of both groups with high and low miR-625-3p and miR-449a expression levels were compared after 5-year follow-up of patients in the RG. The prognostic factors of osteosarcoma patients were assessed through Cox regression analysis. The miR-625-3p expression level in the RG was dramatically higher than that in the CG (p < 0.05), and the miR-449a expression level was dramatically lower than that in the CG (p < 0.05). The miR-625-3p expression level in the RG after surgery was markedly lower than that before surgery (p < 0.05), and the miR-449a expression level was markedly higher than that before surgery (p < 0.05). After they were followed up for 5 years, the overall survival rate of those in the RG was 60.34%, the rate of miR-625-3p low expression group (LEG) was dramatically higher than that of high expression group (HEG) (p < 0.05), and the rate of miR-449a HEG was markedly higher than that of miR-449a LEG (p < 0.05). The relative expression of miR-625-3p and miR- 449a was remarkably different from tumor size, lymph node metastasis, and Enneking stage (p < 0.05). Univariate Cox regression analysis identified that tumor size, lymph node metastasis, Enneking stage, miR-625-3p and miR-449a were risk factors affecting the prognosis of patients; multivariate Cox regression analysis found that the five were independent prognostic factors of them. miR-625-3p and miR-449a are differentially expressed in the postoperative serum of osteosarcoma patients, and their differential expression is an independent risk factor affecting their prognosis, which has prognostic value and can provide a reference value for clinical practice.

Expression profiling of long noncoding RNAs associated with vasculogenic mimicry in osteosarcoma.

Osteosarcoma (OS) is the most common highly malignant bone tumor in teens. Vasculogenic mimicry (VM) is defined as de novo extracellular matrix-rich vascular-like networks formed by highly aggressive tumor cells. We previously reported the presence of VM and it is an unfavorable prognostic factor in OS patients. Long noncoding RNAs (lncRNAs) are aberrantly expressed in OS and involved in cancer cell VM. However, lncRNAs in VM formation of OS have not been investigated. We, therefore, profiled the expression of lncRNAs in highly aggressive OS cell line 143B compared with its parental poorly aggressive cell line HOS. The differentially expressed (DE) lncRNAs and messenger RNA (mRNAs) were subjected to constructed lncRNA-mRNA coexpressed network. The top-ranked hub gene lncRNA n340532 knockdown 143B cells were used for in vitro and in vivo VM assays. The annotation of DE lncRNAs was performed according to the coexpressed mRNAs by Gene Ontology and pathway analysis. A total of 1360 DE lncRNAs and 1353 DE mRNAs were screened out. lncRNA MALAT1 and FTX, which have known functions related to VM formation and tumorigenesis were identified in our data. The coexpression network composed of 226 lncRNAs and 118 mRNAs in which lncRNA n340532 had the highest degree number. lncRNA n340532 knockdown reduced VM formation in vitro. The suppression of n340532 also exhibited potent anti-VM and antimetastasis effect in vivo, suggesting its potential role in OS VM and metastasis. Furthermore, n340532 coexpressed with 10 upregulation mRNAs and 3 downregulation mRNAs. The enriched transforming growth factor-β signaling pathway, angiogenesis and so forth were targeted by those coexpressed mRNAs, implying n340532 may facilitate VM formation in OS through these pathways and gene functions. Our findings provide evidence for the potential role of lncRNAs in VM formation of OS that could be used in the clinic for anti-VM therapy in OS.

SP1-mediated upregulation of lncRNA ILF3-AS1 functions a ceRNA for miR-212 to contribute to osteosarcoma progression via modulation of SOX5

Long noncoding RNA ILF3-AS1 (ILF3-AS1) has been reported to be abnormally expressed in several tumors. However, its expression pattern and function in osteosarcoma have not been investigated. In this study, we showed that ILF3-AS1 expression was significantly up-regulated in both osteosarcoma tissues and cell lines. We first reported that ILF3-AS1 upregulation was induced by nuclear transcription factor SP1. Clinical assays revealed that higher expression of ILF3-AS1 was associated with advanced clinical stage, distant metastasis and shorter overall survival. in multivariate analysis, ILF3-AS1 expression level was found to be an independent prognostic factor for osteosarcoma patients. Functional investigations showed that knockdown of ILF3-AS1 suppressed the proliferation, migration and invasion of osteosarcoma cells, and promoted apoptosis. Bioinformatic software predicted that miR-212 both targeted the 3′-UTR of ILF3-AS1 and SOX5, which was confirmed using luciferase reporter assay, RT-PCR and Western blot. Taken together, ILF3-AS1 displayed its tumor-promotive roles in the progression of osteosarcoma through miR-212/SOX5 axis. Our findings help to elucidate the tumorigenesis of osteosarcoma, and future study will provide a novel therapeutic target for osteosarcoma.