Screening of Potential Key Biomarkers for Ewing Sarcoma: Evidence from Gene Array Analysis

Figures & data

Figure 1 (A) The volcano plot shows the relationship between the fold change and the P value in each group of microarrays. Red is an up-regulated gene, and blue is a down-regulated gene. Adjust P value <0.05. (B) Venn diagram, select DEG fold change> 2, P value <0.01, 3 microarray data sets show that 629 genes overlap. (C) 629DEG’s PPI network.

Table 1 Enrichment Analysis (629DEG’s)

Item	Description or Name	Count in Set	P-value
GO:0005913	Cell-cell adherens junction	24	5.10E-04
GO:0005899	Insulin receptor complex	2	0.096175434
GO:0042383	Sarcolemma	9	0.00713397
GO:0000922	Spindle pole	10	0.010290216
GO:0005925	Focal adhesion	23	0.011379205
GO:0004672	Protein kinase activity	26	6.22E-04
GO:0016491	Oxidoreductase activity	13	4.19E-02
GO:0001047	Core promoter binding	8	0.005995515
GO:0035064	Methylated histone binding	7	0.009061117
GO:0098641	Cadherin binding involved in cell-cell Adhesion	19	0.010937265
GO:0048661	Positive regulation of smooth muscle cell proliferation	7	0.018309355
GO:0048146	Positive regulation of fibroblast proliferation	7	0.011251043
GO:0046777	Protein autophosphorylation	14	0.007695992
GO:0030332	Cyclin binding	4	0.029245751
GO:0004674	Protein serine/threonine kinase activity	21	0.034002412
GO:0001649	Osteoblast differentiation	9	0.029806392
GO:1901796	Regulation of signal transduction by p53 class mediator	14	4.14E-04
GO:0000086	G2/M transition	12	0.008889738
GO:0000082	G1/S transition	15	1.27E-05
GO:0098609	Cell adhesion	18	0.015253207
hsa04110	Cell cycle	17	1.13E-05
hsa05214	Glioma	11	1.18E-04
hsa05200	Pathways in cancer	25	0.010072395
hsa04115	p53 signaling pathway	7	0.036457409

Table 2 Enrichment Analysis (10DEG’s)

Item	Description or Name	Count in Set	FDR
Up
GO:0051301	Cellular division	7	6.26E-09
GO:0007067	Nuclear division	5	5.52E-06
GO:0005515	Protein binding	10	2.79E-03
hsa04114	Oocyte meiosis	4	3.99E-05
hsa04110	Cell cycle	4	5.57E-05
hsa04115	p53 signaling pathway	2	4.78E-02
Down
GO:0004674	Protein threonine/serine kinase activity	4	8.33E-04
GO:0034501	Localization to kinetochore of the protein	2	5.35E-03
GO:0004672	Kinase activity of the protein	3	1.47E-02
GO:0045171	Intercellular bridge	2	2.15E-02
GO:0006468	Protein phosphorylation	3	2.33E-02
GO:0000775	Chromosome, centromeric region	2	2.78E-02
GO:0000777	Condensed chromosome kinetochore	2	4.22E-02

Figure 2 (A) The important module is obtained from a PPI network with 48 nodes and 782 edges. Red is an up-regulated gene; light blue is a down-regulated gene. (B) Using cytohubba’s MCC algorithm to screen out the top ten genes in the module, we can see the connection between them. The weight of these genes in the module network becomes higher as the color darkens.

Table 3 Annotation (10DEG’s)

Number	Gene Symbol	Name	Partial Function Description
1	BUB1	BUB1 mitotic checkpoint serine/threonine kinase	Important for correct chromosome alignment and spindle assembly inspection
2	CDK1	Cyclin dependent kinase 1	It plays a key role in controlling the eukaryotic cell cycle (such as regulating the initiation of mitosis and the centrosome cycle).
3	CCNB2	Cyclin B2	It is necessary to control the cell cycle during the G2/M transition period.
4	AURKB	Aurora kinase B	It is the threonine/serine-protein kinase component of CPC (chromosomal passenger complex).
5	CDCA8	Cell division cycle associated 8	It is an integral part of CPC (CPC is the key regulator of mitosis).
6	MAD2L1	Mitotic arrest deficient 2 like 1	It is an integral part of the spindle assembly checkpoint. Its function is to make all chromosomes align correctly in the metaphase plate and prevent the early start of the later period.
7	PLK4	Polo like kinase 4	Play a key role in centriole replication.
8	CENPF	Centromere protein F	It is necessary for the function of kinetochore and the segregation of chromosomes in mitosis.
9	FANCI	FA complementation group I	It plays a vital role in the repair of DNA double-strand breaks.
10	NCAPH	Non-SMC condensin I complex subunit H	Regulatory subunit of the condensin complex, a complex required for conversion of interphase chromatin into mitotic-like condense chromosomes.

Figure 3 PPI-network and Bioanalysis chart. (A) Co-expression of hub genes. The blue nodes are co-expressed genes, and the red nodes are hub genes. (B) Some biological process diagrams of hub genes. The size of the node refers to the ontology of the gene. The color depth of the node refers to the adjusted P value.

Figure 4 (A) Hierarchical clustering: down-regulated genes are marked in blue, and up-regulated genes are marked in red. Sample type: Different colors in the picture correspond to different sample types. The sarcoma sample data (n=271) comes from the TCGA database. P<0.05 is considered statistically significant. (B) The correlation between gene expression and clinical outcome. (SARC, n=260).

Figure 5 The relationship between gene expression and immune infiltration.

Figure 6 Survival analysis. CDCA8, NACPH, CDK1, MAD2L1, CCNB2 and FANCI have a relatively high overall survival rate (>30%). P<0.05 was considered statistically significant.

Figure 7 Comparison of genes in multiple analyses. FANCI: Cancer Type: Sarcoma. 1. Neale Multi-cancer. 2. Neale Multi-cancer 2; MAD2L1: Cancer Type: Sarcoma. 1. Barretina CellLine 2. 2. Neale Multi-cancer. 3. Rothenberg CellLine; CDCA8: Cancer Type: Sarcoma. 1. Barretina CellLine 2. 2. Neale Multi-cancer. 3. Neale Multi-cancer 2. 4. Rothenberg CellLine; NCAPH: Sarcoma Type: Ewing’s Sarcoma. 1. Baird Sarcoma. Cancer Type: Sarcoma. 2. Neale Multi-cancer. 3. Neale Multi-cancer 2; AURKB: Cancer Type: Sarcoma. 1. Barretina CellLine 2. 2. Bittner Multi-cancer. 3. Neale Multi-cancer. 4. Wooster CellLine; CCNB2: Sarcoma Type: Ewing’s Sarcoma. 1. Bittner Sarcoma. Cancer Type: Sarcoma. 2. Neale Multi-cancer. 3. Rothenberg CellLine; BUB1: Cancer Type: Sarcoma. 1. Bittner Multi-cancer. Sarcoma Type: Ewing’s Sarcoma. 2. Henderson Sarcoma; CDK1: Sarcoma Type: Ewing’s Sarcoma. 1. Baird Sarcoma. Cancer Type: Sarcoma. 2. Barretina CellLine 2. The rank for a gene is the median rank for that gene across each of the analyses. The p-value for a gene is its p-value for the median-ranked analysis. In the heat map of 8 genes, CDCA8, MAD2L1 and FANCI have more or higher expressions.

Figure 8 The relationship between the expression of genes (CDCA8, MAD2L1 and FANCI) and clinical Ewing’s sarcoma grade, staging, EWSR1-FLI1 pathway and tp53 mutation pathway. MAD2L1 has higher expression in the four cases, while CDCA8 and FANCI have higher expression in the grades of ES.