Construction and validation of a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis and immune infiltration

Abstract

Background: Apoptosis played vital roles in the formation and progression of osteosarcoma. However, no studies elucidated the prognostic relationships between apoptosis-associated genes (AAGs) and osteosarcoma.

Methods: The differentially expressed genes associated with osteosarcoma metastasis and apoptosis were identified from GEO and MSigDB databases. The apoptosis-associated prognostic signature was established through univariate and multivariate cox regression analyses. The Kaplan-Meier (KM) survival curve, ROC curve and nomogram were constructed to investigate the predictive value of this signature. CIBERSORT algorithm and ssGSEA were used to explore the relationships between immune infiltration and AAG signature. The above results were validated in another GEO dataset and the expression of AAGs was also validated in osteosarcoma patient samples by immunohistochemistry.

Results: HSPB1 and IER3 were involved in AAG signature. In training and validation datasets, apoptosis-associated risk scores were negatively related to patient survival rates and the AAG signature was regarded as the independent prognostic factor. ROC and calibration curves demonstrated the signature and nomogram were reliable. GSEA revealed the signature related to immune-associated pathways. ssGSEA indicated that one immune cell and three immune functions were significantly dysregulated. The immunohistochemistry analyses of patients' samples revealed that AAGs were significantly differently expressed between metastasis and non-metastasis osteosarcomas.

Conclusions: The present study identified and validated a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis. It could serve as the potential biomarker and therapeutic targets for osteosarcoma in the future.

Keywords: Apoptosis; Immune; Metastasis; Osteosarcoma; Prognosis.

Fig. 1

The flowchart of the study. (A). The apoptosis-associated metastatic DEmRNAs were identified in GSE21257 and MSigDB database. (B). Univariate and multivariate cox regression analyses were conducted to construct the apoptosis-associated gene signature. (C). The appilication of apoptosis-associated gene signature in clinical and validation in GSE39055. (D). The immune infiltration features related to apoptosis risk. DEmRNA: differentially expressed messenger RNA. GEO: Gene Expression Omnibus.

Fig. 2

Identification of apoptosis-associated DEmRNAs related to osteosarcoma metastasis. (A) DEmRNAs related to osteosarcoma metastasis in GSE21257 database. Red indicated up-regulated DEmRNAs (P〈 0.05, FC 〉 1.5) and green indicated down-regulated DEmRNAs (P > 0.05, FC < 0.67). (B) Nine apoptosis-associated DEmRNAs were identified in GSE21257 and MSigDB databases. DEmRNA, differentially expressed messenger RNA; FC, fold change; MSigDB:Molecular Signature Database.

Fig. 3

Establishment of the apoptosis-associated prognostic signature in GSE21257. (A) Univariate cox regression analysis of apoptosis-associated DEmRNAs revealed four prognostic genes. (B)The distribution and median value of risk score. (C) The heatmap of HSPB1 and IER3 in high- and low-risk groups revealed that HSPB1 was positively correlated with risk score while IER3 showed the opposite trend. (D) The scatter plot of patients’ risk scores and survival status. (E) Kaplan-Meier survival plot of patients in high- and low-risk groups. (F) Time-dependent ROC curve at 1, 3, and 5 years of apoptosis-associated prognostic signature. DEmiRNA, differentially expressed microRNA; ROC, receiver operating characteristic;IER3:Immediate Early Response 3, HSPB1:Heat Shock Protein Family B Member 1.

Fig. 4

Relationships between apoptosis-associated gene signature and clinical parameters in GSE21257 and GSE39055. (A and B). Univariate and multivariate cox regression analyses revealed the risk score was the independent prognostic factor in GSE21257. (C and D). Univariate and multivariate cox regression analyses validated risk score and recurrence were two independent prognostic factors in GSE39055. (E). The correlation between apoptosis-associated risk score and osteosarcoma metastasis in GSE21257. (F). The relationship between apoptosis-associated risk score and osteosarcoma recurrence in GSE39055.

Fig. 5

Establishment of the nomogram in the GSE21257. (A) The nomogram to predict the 3- and 5-year survival risk of osteosarcoma patients. (B) The calibration curve of the 3-year survival. (C) The calibration curve of the 5-year survival.

Fig. 6

The relationships between apoptosis and immune infiltration in GSE21257. (A) The infiltrative proportion of 22 immune cells in osteosarcoma with statistically significant. (B) The heatmap of correlations between different immune cells in osteosarcoma. The number and circles in each tiny box represented the corresponding correlation value between two cells. The blue color indicated positively related and the red indicated negatively related. (C) The correlations between risk score and different immune cells. (D) The association between risk score and different immune features. (E) The overlapped immune cells and functions among GSE21257 and GSE39055. The red color implied the opposite trend in two datasets. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.