3.1 Aberrantly overexpression of SNAI1 in human colorectal cancer.
The results of Oncomine analysis showed that gene expression profiles of zinc-finger transcription factor family were dysregulated in multiple cancer types (Fig. 1A). In addition, expression profile of SNAI1 was obviously elevated in CRC tissues compared to normal tissues across multiple CRC datasets (Fig. 2). Subsequently, mRNA expression levels analyzed by UALCAN database further confirmed that SNAI1 was significantly overexpressed in CRC tissues compared with normal tissues (Fig. 1B). More importantly, survival analysis revealed that aberrantly expression of SNAI1, but not of SNAI2, SNAI3 and ZEB1, was significantly associated with poor overall survival in patients with CRC (Fig. 1C).
3.2 Associations between SNAI1 expression and clinical parameters in patients with colorectal cancer.
Clinical parameters of CRC patients, including survival status, survival time, age, gender, tumor stage, tumor, node and metastasis classification (TNM) were obtained from TCGA database. As shown in Fig. 3A-H, expression of SNAI1 was notably elevated in CRC tissues compared to normal tissues. In addition, SNAI1 expression was also significantly correlated with clinical stage (P = 0.027), N classification (P = 0.003) and M classification (P = 0.02). However, no significant association was found between SNAI1 expression and other clinical variables including gender (P = 0.585), age (P = 0.777) and T classification (P = 0.092). Subsequently, the prognostic value of SNAI1 in patients with CRC was evaluated by Cox proportional-hazards model. As shown in Fig. 4A&B, the univariate analysis revealed that overexpression of SNAI1 was significantly correlated with poor overall survival in human CRC [hazard ratio (HR): 1.133; 95%CI: 1.053–1.219; P < 0.001]. Other clinical parameters, including age, stage, T, N, and M classification, are also closely related to poor survival. Multivariate analysis indicated that overexpression of SNAI1 remains as an independent prognostic factor for overall survival [HR: 1.110; 95%CI: 1.017–1.212; P = 0.020]. Other clinical parameters remained as an independent prognostic factor for overall survival including age and T classification. The results above suggested that over expression of SNAI11 was significantly correlated with invasive tumor phenotype and worse prognosis in CRC patients.
3.3 Mutations of SNAI1 in colorectal cancer are rare.
We applied cBioPortal database to analysis the copy number variations (CNVs) and mutations of SNAI1 in human CRC. As shown in Fig. 5A&B, the frequency of genetic alterations is only 8%, most of which is gene amplification, and mutations of SNAI1 in human CRC is very low, only 0.8%. Subsequently, we used COSMIC database to evaluate the mutations of SNAI1in human CRC. As shown in Fig. 5C&D, the types of mutations in SNAI1 include nonsense mutations, missense mutations, and synonymous mutations, the major type of which is missense mutations, up to 53.21%. The types of nucleotide alterations include C > T, G > A, C > A, C > G and G > C mutations, the largest proportion of which is C > T, up to 38.81%.
3.4 Protein expression of SNAI1 in cancer tissues compared to normal tissues.
We used the immunohistochemistry data from the HPA database to further examine the protein expression of SNAI1 in human CRC. As shown in Fig. 6A-D, protein expression of SNAI1 is mainly localized in the nucleus. More importantly, the staining of SNAI1 was significantly higher in CRC tissues in relative to normal tissues, confirming our findings at the mRNA expression level.
3.5 KEGG and GO enrichment analysis revealing functional role of SNAI1 in colorectal carcinogenesis.
The top 200 co-expressed genes of SNAI1 in human CRC were obtained by using GEPIA and cBioPortal database, respectively (Fig. 7A&B). A total of 84 common co-expressed genes were identified through the intersect function of Venn Diagram (Fig. 7C). Subsequently, we applied DAVID online database to analysis the biological function of these co-expressed genes. KEGG pathway enrichment analysis revealed that the co-expressed genes of SNAI1 were mainly enriched in Focal adhesion, PI3K-Akt signaling pathway, ECM-receptor interaction, and MicroRNAs in cancer (Fig. 7D). GO function analysis showed that the co-expressed genes were mainly enriched in extracellular matrix organization, cell adhesion, collagen catabolic process, and collagen fibril organization in BP group. In CC group, co-expressed genes were mainly enriched in extracellular space, extracellular exosome, extracellular region, and extracellular matrix. In MF group, co-expressed genes were mainly enriched in protein binding, platelet-derived growth factor, and collagen binding. The top 10 GO terms according to P-value in each group are shown in Fig. 7E.
3.6 BGN was positively co-expressed with SNAI1 in human colorectal cancer.
A PPI network consisted of co-expressed genes were constructed by using the STRING online database, and the network was subsequently analyzed by using Cytoscape software (Fig. 8A). The top 10 genes of the PPI network were identified according to different algorithm generated by CytoHubba plug-in, and 5 genes (COL1A1, MMP2, BGN, LOX, and PDGFRB) were identified as core genes (Fig. 8B&C). Hierarchical clustering of the core co-expressed genes was performed through the use of UCSC Cancer Genomics Browser, revealing that these core genes were differentially expressed in most CRC samples (Fig. 8D). Ultimately, prognostic values of these core genes and the correlations between SNAI1 and core genes were evaluated by GEPIA database. Co-expression analysis demonstrated that all of these core genes were positively co-expressed with SNAI1 (Fig. 8E). However, only one of them (BGN) was significantly correlated with both poorer overall survival (HR = 2, P = 0.0017) and disease free survival (HR = 2, P = 0.0017) in patients with CRC (Fig. 8F). Therefore, BGN was identified as a hub gene which positively co-expressed with SNAI1 in human CRC.
3.7 Associations between BGN expression and clinical parameters in colorectal cancer patients.
Based on information of clinical samples obtained from TCGA database, the relationships between BGN expression pattern and clinical parameters of CRC patients were analyzed and visualized by using R software. As shown in Fig. 9A-H, expression of BGN was significantly elevated in CRC tissues compared to normal tissues. In addition, BGN expression was also remarkably associated with clinical stage (P = 0.042), T classification(P = 0.007) and N classification (P = 0.001). However, no significant association was found between BGN expression and other clinical variables including M classification (P = 0.28), gender (P = 0.443), and age (P = 0.092).