Comprehensive Analysis of Aerobic Exercise-Related Genes Identifies CDCA4 That Promotes the Progression of Osteosarcoma

Abstract

Background: Exercise has a positive impact on patients with osteosarcoma, improving function, reducing disability, maintaining independence and quality of life. Exercise may also directly affect the effectiveness of cancer treatment. Cell division cycle-associated protein 4 (CDCA4) is reported to function importantly during numerous human cancers development. Nevertheless, the details toward CDCA4 function are still to be investigated.

Methods: This study comprehensively analyzed the GSE74194 database and obtained aerobic exercise-related genes. Protein-protein interaction network (PPI) and Gene Ontology (GO) analysis were performed on the differentially expressed genes (DEGs). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and tumor genome atlas (TCGA) data mining were applied to measure aerobic exercise-related gene CDCA4 level in osteosarcoma tissue. We conducted lots of functional experiments to uncover CDCA4 function and its corresponding mechanism in osteosarcoma.

Results: We screened a total of 547 DEGs related to aerobic exercise, of which 373 were up-regulated and 174 were down-regulated. PPI analysis revealed 90 genes that might play key roles. GO analysis showed that aerobic exercise-related DEGs were significantly enriched during the mitotic cell cycle, cell division, mitotic nuclear division and sister chromatid segregation, nuclear division, microtubule cytoskeleton organization involved protein, microtubule-based process, spindle organization, G2/M transition of mitotic cell cycle. Our results indicated that CDCA4 was increased in osteosarcoma tissues and cell lines, and its level had association with high mortality of osteosarcoma patients. Further studies revealed that absence of CDCA4 largely hindered osteosarcoma cancer cell proliferation, invasion, and migration.

Conclusion: Comprehensive bioinformatics analysis improves our understanding of the underlying molecular mechanisms of aerobic exercise on osteosarcoma. This provides evidence for the effect of aerobic exercise on CDCA4 expression. Our data suggested that CDCA4 could facilitate osteosarcoma development, and gave a hint that CDCA4 was a candidate target in the treatment of osteosarcoma, aerobic exercise might help the treatment and prognosis of patients with osteosarcoma.

Keywords: CDCA4; aerobic exercise; comprehensive analysis; osteosarcoma; progression.

FIGURE 1

DEGs hierarchical cluster analysis between aerobic and anaerobic exercise groups in GSE74194 database. Three hundred seventy-three up-regulated DEGs related to aerobic exercise and 174 down-regulated DEGs related to aerobic exercise were identified. Red, up-regulated genes, blue, down-regulated genes.

FIGURE 2

Totally, 90 aerobic exercise-related DEGs were included in PPI network. The nodes indicated proteins. The edges represented proteins’ interaction.

FIGURE 3

GO enrichment analysis of biological process of aerobic exercise-related DEGs with fold change >2.

FIGURE 4

Kaplan Meier plotter online tool was conducted to identify the association between CDCA4 expression and the OS time (A), disease-free survival (B), and metastasis-free survival (C) of osteosarcoma patients. Patients with high levels of CDCA4 glioma had lower survival rates, and high levels of CDCA4 were negatively correlated with non-metastatic survival.

FIGURE 5

(A) According to the TCGA database, CDCA4 was highly expressed in SARC tissues. (B) CDCA4 expression level had correlation with the gender of SARC patients, and the expression was higher in female patients. (C) CDCA4 expression level displayed correlation with the age of SARC patients.

FIGURE 6

(A) CDCA4 was more highly expressed in osteosarcoma tumor tissues than normal tissues (n = 12). (B) CDCA4 was more highly expressed in cancer cells than normal cell, *P < 0.05, **P < 0.01.

FIGURE 7

(A) CDCA4 expression was decreased in SW1353 after transfection of si-CDCA4. (B) CDCA4 expression was decreased in U2OS after transfection of si-CDCA4. (C) Reduced CDCA4 resulted in inhibited SW1353 cell proliferation. (D) Reduced CDCA4 resulted in inhibited U2OS cell proliferation,*P < 0.05, **P < 0.01.

FIGURE 8

(A) Ablated CDCA4 obviously suppressed osteosarcoma cell invasion. (B) Ablated CDCA4 obviously suppressed osteosarcoma cell migration. (C) Detection of invasion and migration of osteosarcoma cells by Transwell assay, **P < 0.01, ***P < 0.001.