3.1 Aberrant expression and prognostic value of CDH23 in patients with AML
As shown in Figure 1A, we utilized the GEPIA dataset to analyze CDH23 expression level in AML. CDH23 mRNA expression was significantly higher in 173 AML tissues compared to the corresponding normal samples from TCGA and GTEx data. We then investigated whether CDH23 level was predictive of survival of patients with AML. Survival analysis performed using the GEPIA database demonstrated that high CDH23 mRNA levels were associated with shorter OS of patients with AML (HR=1.9, P=0.0019, Figure 1B, C). Analysis of the LinkedOmics database indicated that overexpression of CDH23 corresponded to poor prognosis (P=0.01). Notably, these results showed that high CDH23 expression was significantly related to poor OS in AML.
3.2 Correlated gene analysis of CDH23 in patients with AML
To illustrate the potential mechanisms and functions of CDH23 in AML, we used the LinkedOmics database for correlation analysis between CDH23 and various genes. The results in Figure 2A-C are presented as heat and volcano maps of the top 50 genes that are either positively or negatively correlated with CDH23 expression. CDH23 was positively and significantly correlated with PPM1M, C10orf105, TFEB, FGD2, and IGF2R. On the other hand, CDH23 had a significant negative correlation with CASP6, KDM5B, KCNQ5, and SCCPDH. We chose the most frequently altered neighboring genes (cor>0.5), including ITGAM (Spearman correlation: 0.6822, P=1.765e-24), TFEB (Spearman correlation: 07165, P=0), PPM1M (Spearman correlation: 0.7524, P=4.274e-32), and SLC8A1 (Spearman correlation: 0.6635, P=0), to conduct a correlation analysis via LinkedOmics (Figure 2D-G). As shown in Figure 3, we further analyzed the prognostic value of the four genes in AML. The results demonstrated that overexpression of ITGAM was highly associated with poor prognosis. Similarly, high TFEB, PPM1M, and SLC8A1 mRNA expression corresponded to poor OS.
3.3 Impact of CDH23 expression on immune cell infiltration and tumor microenvironment (TME) in patients with AML
Tumor-infiltrating immune cells are a major component of the TME and are involved in the occurrence, progression, and metastasis of cancer. Tumor-infiltrating lymphocyte grade is a powerful independent predictor of sentinel lymph node status and clinical survival in some cancer types.[26, 27] To explore the relationship between CDH23 expression and immune cell infiltration, we performed analysis using the TIMER database based on sequencing data from AML cases in the TCGA database. The online database stratified patients with AML into high and low CDH23 expression groups. Subsequently, we used several computational algorithms to estimate the abundance of various tumor-infiltrating immune cell types (B cells, CD4+ T cells, CD8+ T cells, macrophages, monocytes, etc.) among the high and low CDH23 expression groups. As shown in Figure 4A, CDH23 expression was significantly related to the infiltration of various immune populations in AML including B cells (P<0.01), CD4+ T cells (P<0.01), CD8+ T cells (P<0.05), mast cells (P<0.01), monocytes (P<0.001), etc. Additionally, we used the TIMER database to further investigate the role of CDH23 in the TME. The analysis revealed that the ESTIMATE score (P<0.001) of the high CDH23 expression group was significantly higher than that of the low CDH23 expression group, and there was a statistically significant positive correlation between CDH23 expression and the immune and stromal scores (Figure 4B-D, P<0.001). All these results suggested that the expression level of CDH23 was highly correlated with tumor immune infiltration and the TME.
3.4 Functional enrichment analysis of CDH23 and related genes in patients with AML
To further study the molecular functions and biological processes of CDH23, we employed the LinkFinder module of the LinkedOmics database to analyze the mRNA sequencing data of patients with AML in the TCGA. The GO enrichment analysis data indicated that the expression of CDH23 and most of the related genes were correlated to pathways or biological processes of immune response and cytokine production such as neutrophil activation involved in immune response, neutrophil degranulation, positive regulation of cytokine production, phagocytosis, cellular response to biotic stimulus, cellular response to molecules of bacterial origin, cellular response to lipopolysaccharide, and so on (Figure 5A, B). Moreover, the significant transcription factor targets of CDH23 included PEA3, ELF1, IRF, PU.1, etc (Figure 5C). In addition, the top 10 pathways related to the functions of CDH23 expression level in AML were illustrated through KEGG analysis and included osteoclast differentiation, phagosome, lysosome, tuberculosis, chemokine signaling pathway, endocytosis, NOD-like receptor signaling apthway, regulation of actin cytoskeleton and others (Figure 5D). In conclusion, CDH23 had an extensive influence on the regulation of several pathways and processes involved in tumor immunity.
3.5 Protein-protein interaction network (PPI) analysis of CDH23 expression level via GeneMANIA
To further elucidate the mechanism of CDH23 in tumorigenesis, we constructed an integrated PPI network using GeneMANIA to screen for CDH23-binding proteins. As described in Figure 6, the interaction network showed that CDH23 is highly linked with USH1C, ABI1, NCKAP1, CYFIP1, WASF1, PCDH15, and other vital proteins. Among the interacting proteins, we observed enrichement of biological processes associated with actin-based cell projection, extrinsic components of the plasma membrane, neuromuscular processes, clusters of actin-based cell projections, retina homeostasis, stereocilium bundles, and extrinsic components of membranes. These results strongly support the hypothesis that CDH23 is involved in the tumorigenesis and pathogenesis of AML.