Identification of the hub modules in PCa.
Although PCa is a tumor with high occurrence and good prognosis, its molecular mechanism of tumorigenesis is still unclear. To explore the key molecules in the pathogenesis of PCa, WGCNA was used to analysis the GEO dataset GSE6919 (supplementary Figure 1). Fourteen modules were screened and shown in Figure 2A with the trait normal, adjacent and tumor. The turquoise and magenta modules were picked out as the key modules and shown in Cytoscape (Figure 2B and Figure 2C). Obviously, the magenta module was enrichment in ribosome pathway.
The focal adhesion pathway was the key dysregulated pathway in PCa.
To further clarify the molecular mechanism of PCa, genes from turquoise module were analyzed by enrichGO or enrichKEGG function of clusterProfiler packages in R software, respectively[20]. GO analysis results were enriched in the extracellular structure organization (Figure 3A). The focal adhesion pathway was found to be the foremost pathway by KEGG analysis (Figure 3B). In order to investigate the proteins interaction in turquoise module, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was applied for revealing the core protein-protein interaction network. The focal adhesion pathway was also found to be the foremost enriched pathway (Figure 3C). Furthermore, GESA results shown that the focal adhesion pathway ranked the top enriched pathway (Figure 3D and supplementary table 1).
FERMT2 was Screened out as the key gene of PCa.
Genes in key module were candidate for screening the key gene. The turquoise module contained 1450 genes. These genes were sorted by its module membership to turqupise module (MMturqupise), and the top 10 genes were listed in supplementary table 2. FERMT2 was found to be most correlated to turquoise module (R = 0.928), also FERMT2 was positive correlated to normal tissue (GS=0.287, P=0.001) and negative correlated to tumor (GS=-0.184, p=0.028). Take the results from Figure 2C together, FERMT2 was considered as the key gene in PCa.
Validation the main disorder pathway and key gene in TCGA PRAD dataset.
The possible main disorder pathway (Focal adhesion pathway) and key gene (FERMT2) was screened from GSE6919 dataset by using WGCNA, we wanted to see if we could get the same results in other datasets. TCGA prostate adenocarcinoma (PRAD) dataset was performed analysis as the same with GSE6919. Eleven modules were obtained from WGCNA analysis. The green module was the most positive relevant module (r=0.29, p=5e-12) and the brown module was the most negative relevant module (r=-0.5, p=7e-36) in tumor (Figure 4A). The genes in green module were enriched in the ribosome pathway. The brown module was shown in Figure 4B and mainly enrichment in the focal adhesion pathway (supplementary table 3). FERMT2 was also found to be one the hub genes in TCGA PRAD dataset (Figure 4B), which is consistent with the results from GSE6919 dataset.
FERMT2 was downregulated in pan-cancers and related to focal adhesion pathway.
The expression of FERMT2 in GSE6919 and TCGA were plotted by boxplot in Figure 5A and Figure 5B. FERMT2 was found to be downregulated in PCa tumor tissues compared with controls. To overview the profile expression of FERMT2, expression of FERMT2 in the TCGA cancers with tumor and normal samples was shown in Figure 5C. FERMT2 was found to be downregulated in 19 out of 24 cancers indicating that it is a common feature of tumors. To investigate the relationship of the key gene FERMT2 and the main disorder pathway focal adhesion pathway, genes were sorted according its correlation with FERMT2 and analysis by GSEA. The focal adhesion pathway was found to be shown in top 10 pathways (supplementary table 4).
FERMT2 inhibited the proliferation and migration of PC3 cells.
In order to determine the effect of FERMT2 on the proliferation and migration of PCa cells, Flag-FERMT2 plasmid was constructed and transferred into PC3 cells (Figure 6A). Overexpression of FERMT2 inhibited PC3 cell proliferation (Figure 6B) and Clonogenesis (Figure 6C). We also found that FERMT2 can inhibit PC3 cells migration.