Table 1.
Details of CRC microarray based expression datasets and the identified hub genes.
Fig 1.
A heatmap of the KEGG pathways to portray role of all the CRC related hub genes (n = 210) identified during present study.
Table 2.
KEGG pathway analysis details of the 210 pooled hub genes, extracted from the various GEO microarray CRC expression datasets.
Fig 2.
(A) A PPI network of all the 210 extracted hub genes. (B) A network of six real hub genes identified on the basis of degree of centrality.
Table 3.
List of the real hub genes identified from the PPI network of the extracted 210 CRC related hub genes.
Table 4.
Details of the KEGG pathway analysis of the identified real hub genes.
Fig 3.
Box plots showing the relative mRNA expression levels of real hub genes in normal and COAD patients (Information retrieved via GEPIA database).
The box plots shows the relative mRNA expression of: (A) CXCL12, (B) CXCL8, (C) AGT, (D) GNB1, (E) GNG4, and (F) CXCL1; in COAD patients and normal samples. A p-value of <0.05 was considered significant.
Fig 4.
Box plots showing the relative expression levels of real hub genes in normal and COAD patients (Information retrieved from GENT2 database).
The box plots shows the relative mRNA expression of: (A) CXCL12, (B) CXCL8, (C) AGT, (D) GNB1, (E) GNG4, and (F) CXCL1; in COAD patients and normal samples. A p-value of <0.05 was considered significant.
Fig 5.
Box plots showing the relative expression levels of CXCL12 in normal and COAD samples of different clinicopathological features via UALCAN database.
Relative mRNA expression of CXCL12: (A) in normal individuals and COAD patients; (B) in normal individuals and COAD patients of different races; (C) in normal individuals and COAD patients of different cancer stages; (D) in normal individuals and COAD patients of different genders; (E) in normal individuals and COAD patients of different age groups; and, (F) in normal individuals and COAD patients of different body weights; (G) Relative protein expression of CXCL12 in normal individuals and COAD patients. A p-value of <0.05 was considered significant. Normal weight = BMI greater than or equal to 18.5 and BMI less than 25, Extereme weight = BMI greater than or equal to 25 and BMI less than 30, Obese = BMI greater than or equal to 30 and BMI less than 40, and Extreme Obese = BMI greater than 40.
Fig 6.
Box plots showing the relative expression levels of CXCL8 in normal and COAD samples of different clinicopathological features via UALCAN database.
Relative mRNA expression of CXCL8: (A) in normal individuals and COAD patients; (B) in normal individuals and COAD patients of different races; (C) in normal individuals and COAD patients of different cancer stages; (D) in normal individuals and COAD patients of different genders; (E) in normal individuals and COAD patients of different age groups; and, (F) in normal individuals and COAD patients of different body weights; (G) Relative protein expression of CXCL8 in normal individuals and COAD patients. A p-value of <0.05 was considered significant. Normal weight = BMI greater than or equal to 18.5 and BMI less than 25, Extereme weight = BMI greater than or equal to 25 and BMI less than 30, Obese = BMI greater than or equal to 30 and BMI less than 40, and Extreme Obese = BMI greater than 40.
Fig 7.
Box plots showing the relative expression levels of AGT in normal and COAD samples of different clinicopathological features via UALCAN database.
Relative mRNA expression of AGT: (A) in normal individuals and COAD patients; (B) in normal individuals and COAD patients of different races; (C) in normal individuals and COAD patients of different cancer stages; (D) in normal individuals and COAD patients of different genders; (E) in normal individuals and COAD patients of different age groups; and, (F) in normal individuals and COAD patients of different body weights; (G) Relative protein expression of AGT in normal individuals and COAD patients. A p-value of <0.05 was considered significant. Normal weight = BMI greater than or equal to 18.5 and BMI less than 25, Extereme weight = BMI greater than or equal to 25 and BMI less than 30, Obese = BMI greater than or equal to 30 and BMI less than 40, and Extreme Obese = BMI greater than 40.
Fig 8.
Box plots showing the relative expression levels of GNB1 in normal and COAD samples of different clinicopathological features via UALCAN database.
Relative mRNA expression of GNB1: (A) in normal individuals and COAD patients; (B) in normal individuals and COAD patients of different races; (C) in normal individuals and COAD patients of different cancer stages; (D) in normal individuals and COAD patients of different genders; (E) in normal individuals and COAD patients of different age groups; and, (F) in normal individuals and COAD patients of different body weights; (G) Relative protein expression of GNB1 in normal individuals and COAD patients. A p-value of <0.05 was considered significant. Normal weight = BMI greater than or equal to 18.5 and BMI less than 25, Extereme weight = BMI greater than or equal to 25 and BMI less than 30, Obese = BMI greater than or equal to 30 and BMI less than 40, and Extreme Obese = BMI greater than 40.
Fig 9.
Box plots showing the relative expression levels of GNG4 in normal and COAD samples of different clinicopathological features via UALCAN database.
Relative mRNA expression of GNG4: (A) in normal individuals and COAD patients; (B) in normal individuals and COAD patients of different races; (C) in normal individuals and COAD patients of different cancer stages; (D) in normal individuals and COAD patients of different genders; (E) in normal individuals and COAD patients of different age groups; and, (F) in normal individuals and COAD patients of different body weights; (G) Relative protein expression of GNG4 in normal individuals and COAD patients. A p-value of <0.05 was considered significant. Normal weight = BMI greater than or equal to 18.5 and BMI less than 25, Extereme weight = BMI greater than or equal to 25 and BMI less than 30, Obese = BMI greater than or equal to 30 and BMI less than 40, and Extreme Obese = BMI greater than 40.
Fig 10.
Box plots showing the relative expression levels of CXCL1 in normal and COAD samples of different clinicopathological features via UALCAN database.
Relative mRNA expression of CXCL1: (A) in normal individuals and COAD patients; (B) in normal individuals and COAD patients of different races; (C) in normal individuals and COAD patients of different cancer stages; (D) in normal individuals and COAD patients of different genders; (E) in normal individuals and COAD patients of different age groups; and, (F) in normal individuals and COAD patients of different body weights; (G) Relative protein expression of CXCL1 in normal individuals and COAD patients. A p-value of <0.05 was considered significant. Normal weight = BMI greater than or equal to 18.5 and BMI less than 25, Extereme weight = BMI greater than or equal to 25 and BMI less than 30, Obese = BMI greater than or equal to 30 and BMI less than 40, and Extreme Obese = BMI greater than 40.
Fig 11.
Box plot showing real hub genes promoter methylation levels in normal individuals and COAD patients via UALCAN database.
The box plot shows the promoter methylation levels of: (A) CXCL12 (B) CXCL8 (C) AGT (D) GNB1 (E) GNG4 (F) CXCL1 in normal individuals and COAD patients. A p-value of <0.05 was considered significant.
Fig 12.
Frequency of the genetic alterations and CNVs of the real hub genes in COAD patients.
Fig 13.
The COAD related prognostic information of the real hub genes obtained via GEPIA database.
The calculated prognostic value of: (A) CXCL12, (B) CXCL8, (C) AGT, (D) GNB1, (E) GNG4, (F) CXCL1 gene. Blue color indicates this low expression while red color indicates the high expression of a gene.
Fig 14.
TIMER based Spearman correlational analysis between the expression of real hub genes and CD8+ T immune cell infiltration in COAD.
TIMER based Spearman correlational analysis between: (A) CXCL12 expression and CD8+ T immune cell infiltration; (B) CXCL8 expression and CD8+ T immune cells’ infiltration; (C) AGT expression and CD8+ T immune cells’ infiltration; (D) GNB1 expression and CD8+ T immune cells’ infiltration; (E) GNG4 expression and CD8+ T immune cells’ infiltration; (F) CXCL1 expression and CD8+ T immune cells’ infiltration. Red color box represents the positive correlation; blue color represents the negative correlation while white color represents no correlation. A p-value of <0.05 was considered as significant.
Fig 15.
The miRNA–real hub gene interaction network.
The pink circular node represents the miRNA. The blue v shape node represents the hub gene while pink and red v shape node represents the mir-1-3p/CXCL8 or CXCL12, CXCL1, and GNB1 axis. The arrow shape represents the interaction between the miRNAs and real hub genes.
Fig 16.
Real hub gene-drug interaction network of the identified real hub genes.
Panels A–F indicates the available chemotherapeutic drugs in CTD that decrease or increase the expression levels of the real hub genes. (A) Real hub gene-drug network of CXCL12; (B) Real hub gene-drug network of CXCL8; (C) Real hub gene-drug network of AGT; (D) Real hub gene-drug network of GNB1; (E) Real hub gene-drug network of GNG4; (F) Real hub gene-drug network of CXCL1. Red arrows indicate the chemotherapeutic drugs that could increase the expression levels of the real hub genes, while green arrows indicate the chemotherapeutic drugs that could decrease the expression levels of the real hub genes. The numbers of arrows between chemotherapeutic drugs and real hub genes in the network represent the supported numbers of previous studies reported in literature.
Fig 17.
Crosstalk between the real hub genes involved pathways.
At the cellular level, CXCL1, CXCL8 or CXCL12 binds to the GPCRs (CXCR1, CXCR2, or CXCR4) and activates the G protein. In this study, the up-regulation of GPCRs ligands (CXCL1 and CXCL8) and G protein subunits (β and γ) collectively are supposed to up-regulate the various downstream pathways. For example, the Heterotrimeric Gα subunit up-regulation in this study further up-regulates its main effectors PLC and PI3K to induce hyper phosphorylation of PKC and Akt, respectively. The oncogenic roles of these two signaling pathways have already been reported to activate various respective transcription factors associated with angiogenesis, cell survival, and migration of tumor cells. On the other hand, β and γ G protein subunits up-regulation in this study further up-regulates the Rho-GTPase family and Raf-1/MAP/Erk signaling cascade which has been earlier reported to contribute in the cell invasion, cell survival, and cell proliferation [78].