Construction of weighted Gene Co-expression modules
In order to identify the function modules in PRAD patients. Weighted gene co-expression analysis (WGCNA) was constructed from the TCGA-PRAD and GSE70768 with the WGCNA R package. The 11 modules were generated in TCGA-PRAD and 11 modules were generated in GSE70768 where the grey module was not assigned into any cluster. Then heatmap was plotted to show the relationship between different gene modules and the two clinical profiles. The results of the module-clinical profile relationship were displayed in figure 2 and figure 3 which can be clearly revealed that the cyan gene module had closeness with clinical profile in TCGA-PRAD, and the thistle2 gene module had the strongest relationship with clinal profile in GSE70768. The correlation coefficients between the cyan gene module and clinical profile were 0.48, and the correlation coefficients between thistle2 gene module and clinical profile was 0.64. then, the results of the Pearson correlation coefficients between cyan/thistle2 gene module and clinical profile were 0.57/0.8(p<0.01). It is clearly certified that the cyan/thistle2 gene module had strongly relationship with the clinical profile.
Identification of hub gene
A total of 6088 genes were differentially expressed between tumor tissues and corresponding normal tissues in the TCGA database (Figure 4A), and 563 genes were differentially expressed in GSE70768 database (Figure 4B). 1230 and 2513 co-expression genes were identified in the cyan and thistle2 modules respectively. The interactive network among this gene including 108 genes, visualized by venn diagram (figure 4C), which indicated that these genes were the hub genes. Then we further explored the relationship between this genes and prostate cancer overall survival (OS). The result revealed that Foxf1 was the target gene in prostate cancer. subsequently, we utilized the CamcAPP to verify the relationship between the expression of Foxf1 and the biochemical recurrence of prostate cancer, which had a meaningful relationship.
Low expression and hypermethylation of Foxf1 in prostate cancer
We explore the expression of Foxf1 in prostate cancer and adjacent tissues by utilizing Ualcan, visualized as a box plot graph (Fig 6D). we further explored the expression level of Foxf1 in prostate cancer based on nodal metastasis status. Foxf1 was lower in cancer tissues than adjacent tissues, and lower in nodal metastasis tissues (Fig 6E). In addition, the analysis from Ualcan demonstrated that Foxf1 were significantly higher methylation in cancer tissues compared with normal tissues (P < 0.05, Fig 6F). Furthermore, the protein expressional levels of the Foxf1 gene were significantly lower in prostate cancer tissues compared with the corresponding normal tissues based on the immunohistochemistry staining (Fig 5).
GO and KEGG pathway analysis of the 108 Genes
GO and KEGG pathway analysis were performed to obtain further insight into the potential function of the identified 108 genes. The analysis results were showed in figure 7. Among the most enriched GO terms were cell-substrate adhesion and focal adhesion. KEGG pathway analysis shows that hub genes are highly correlate with proteoglycans in cancer.
PPI network construction
A protein-protein network among the overlapped genes was constructed by using cytoscape(v3.8.1), with 2672 nodes and 81425 edges (Fig 8A). Then we selected the particular nodes we were interested to construct the ultimate PPI by using the algorithm of Cytohubba plugin, with 21 nodes and 26 edges (Fig 8B). The data suggested GAPVD1, MIR522, MIR937, CPS1, HOXB9, FUBP3, MIR936, SDF4, SATB2, HOXB13, MIR375, PUSL1, SATB1, MIR382, MIR429, HOXD13, PRSS50, FLNC, STRBP, KHSRP were significant associated with the regulation and function of differentially expressed Foxf1 in prostate.