3.1. The Expression Profile of IGF2BPs gene family in HNSCC.
The expression pattern of all members of the IGF2BPs gene family across pan-cancer was examined (Figure 2a). Significant upregulation of IGF2BP1, IGF2BP2, and IGF2BP3 was observed in several malignancies, including BRCA, CESC, CHOL, ESCA, LIHC, COAD, LUSC, HNSC, GBM, PAAD, READ, OV, SKCM, UCEC, STAD, and UCS. The mRNA expression levels of the IGF2BPs gene family were found to be considerably higher in HNSCC samples as compared to healthy oral samples (P < 0.001), as revealed by both unpaired and paired sample analyses (Figure 2c-d).
Furthermore, the 3D protein structures of three genes from the IGF2BPs gene family were visualized (Figure 2b). The protein expression levels of the IGF2BPs gene family in both healthy and cancerous tissues were also assessed (Figure 3). The findings showed that the IGF2BP2 and IGF2BP3 protein levels were elevated in HNSCC tissues but lowered in normal tissues, including oral mucosa, tonsils, salivary gland, and thyroid gland. Nevertheless, IGF2BP1 was not observed to be expressed in either HNSCC or healthy tissues.
Additionally, to evaluate the diagnostic usefulness of the IGF2BPs gene family in HNSCC, the ROC curve was employed. The variables of IGF2BP1, IGF2BP2, and IGF2BP3 revealed a higher accuracy in distinguishing healthy controls from HNSCC samples (AUC = 0.929, 0.910, and 0.811.) (Figure 4).
3.2. Correlation between Clinicopathologic Features and Expression of the IGF2BPs gene family in HNSCC.
The study utilized logistic regression analysis to investigate the association between the clinical characteristics of patients with HNSCC and the mRNA expression of genes in the IGF2BPs gene family (Table 1). The findings revealed a positive association of IGF2BP1 expression with the T stage (P = 0.001). IGF2BP2 expression was also significantly positively correlated with lymph node neck dissection and T stage (P = 0.020 and P = 0.009, respectively). Moreover, there was a positive association of IGF2BP3 expression with the clinical stage (P = 0.039) and T stage (P = 0.043).
3.3. Significance of the IGF2BPs gene family in the Prognosis of HNSCC.
The survival heatmaps (Figure 5a-b) depict the prognostic significance of the IGF2BPs gene family for overall survival and disease-free survival in diverse tumor types.
The Kaplan–Meier curves (Figure 5c) revealed that HNSCC patients with elevated expression levels of IGF2BP2 displayed a lower overall survival(OS) (p = 0.018), with no considerable link to progression-free interval survival (PFI) and disease-specific survival (DSS). The upregulation of IGF2BP2 was related to a poorer HNSCC prognosis, however, the IGF2BP1 and IGF2BP3 expressions did not reveal any links to DSS, PFI, and OS.
The univariate analyses demonstrated that certain clinical characteristics were significant risk factors for mortality in individuals with HNSCC (Figure 6a). The M1 stage (P = 0.002), the lack of radiation therapy (P = 0.002), the SD and PD primary therapy outcomes (P <0.001), and lymphovascular invasion (P = 0.002) were among these. Importantly, the HR value of 1.385 for the high expression level of IGF2BP2 in individuals with HNSCC illustrated that the risk of death increased in this group in comparison to the group with low expression (P = 0.018). The multivariate analyses (Figure 6b) showed that a number of variables, such as high N stage (N1, N2, and N3) (P = 0.047), lack of radiation therapy, and primary therapeutic outcomes of SD and PD (P < 0.001), had an impact on overall survival in individuals with HNSCC. However, the level of IGF2BP2 expression did not have an impact on survival outcomes.
Moreover, to illustrate the connection between the IGF2BPs gene family and survival likelihood, a nomogram was created (Figure 7a). The individuals with higher points exhibited poor survival outcomes. Additionally, the calibration curves demonstrated that the nomogram plot exhibited excellent predictive ability for OS over 1 and 3 years (Figure 7b).
3.4. The Associated and Co-expressed Genes of the IGF2BPs gene family.
A GGI network of the IGF2BPs gene family was generated using the STRING database (Figure 8a). Furthermore, the top 20 co-expressed genes were considered crucial, highly connected hub node genes, i.e., ELAVL4, MOV10, and YBX1, performing a crucial role in some important biological functions, for instance, negative regulation of cellular amide metabolic process, mRNA catabolic process regulation, and RNA stability regulation.
The ten leading genes of the IGF2BPs gene family with the highest levels of positive correlation, including PLAG1, HMGA2, and DDX18, and the top ten genes with the lowest levels of expression, including S100A8, TNFRSF13B, and CST3, were illustrated on a heatmap (Figure 8b).
3.5. Biological function enrichment analysis of the IGF2BPs gene family in HNSCC.
The 1169 correlated genes of the IGF2BPs gene family that were selected (Figure 9a) were subjected to GO analysis, KEGG analysis, and gene set enrichment analysis. Bubble charts (Figure 9b-e) were generated to represent the analysis results of GO terms, including cellular components (CC), biological processes (BP), and molecular functions (MF), along with KEGG pathway analysis results. Additionally, the mountain plot (Figure 9f) demonstrated the findings of gene set enrichment analysis (GSEA).
Furthermore, the substantially linked genes of the IGF2BPs gene family were predominantly enriched in some specific biological processes, for instance, proteasomal protein catabolic process, regulation of neuron projection development, and process utilizing autophagic mechanism (Figure 9b). The substantially associated genes of the IGF2BPs gene family were predominantly enriched in certain cellular components (Figure 9c), such as the cell leading edge, cell−substrate junction, and focal adhesion. The considerably related genes of the IGF2BPs gene family were mainly enriched in certain molecular functions, i.e., cell adhesion molecule binding, serine/threonine protein kinase activity, and cadherin binding (Figure 9d). The associated genes were primarily linked to the regulation of several cellular processes, including the actin cytoskeleton, endocytosis, focal adhesion, protein processing in the endoplasmic reticulum, and signaling pathways like the Hippo and Wnt pathways, as well as pathogenic Escherichia coli infection, according to the KEGG enrichment analysis (Figure 9e).
In addition, the results of GSEA illustrated that substantial enrichment of these associated genes was observed in various signaling pathways, i.e., focal adhesion, PI3K-AKT and MAPK signaling pathways, and epithelial-mesenchymal transition (Figure 9f).
3.6. The correlation between the IGF2BPs gene family and tumor Immunity in HNSCC.
The estimate-immune-stromal score was used to assess the relationship between the expression of the IGF2BPs gene family and the tumor immune microenvironment (Figure 10). The scatter plot revealed a considerable negative association between the immune score and the expression of IGF2BP1 (r = -0.176; p<0.001), IGF2BP2 (r = -0.335; p<0.001), and IGF2BP3 (r = -0.242; p<0.001). Furthermore, the estimate score was found to be significantly negatively associated with the expression of IGF2BP1 (r = -0.092; p = 0.040), IGF2BP2 (r = -0.235; p<0.001)), and IGF2BP3 (r = -0.151; p<0.001). However, no significant link was found between the expression of the IGF2BPs gene family and stromal score.
Moreover, as per the Pearson correlation study, tumor immune infiltration cells (TIICs) displayed a negative association with the expression of the IGF2BPs gene family, such as T cells, Th17 cells, and Treg (Figure 11a).
The study also examined the relationship between the IGF2BPs gene family and genes that promote or suppress the immune system in HNSCC. Immunoinhibitory genes such as NECTIN2, TGFBR1, and VTCN1 were found to correlate significantly with the expression of the IGF2BPs gene family. Immunostimulatory genes such as TMIGD2, TNFRSF13B, and TNFRSF4 were significantly negatively associated with the expression of the IGF2BPs gene family (Figure 11b).
3.7. Knockdown of IGF2BP2 gene expression inhibited SCC4 cell line proliferation, migration, and invasion.
To gain further insights into the potential involvement of the IGF2BPs gene family in the malignant progression of HNSCC, the IGF2BP2 gene was subjected to in vitro experiments. To achieve knockdown of IGF2BP2 expression in the SCC4 cell line, siRNA was employed, and the knockdown efficiency was validated by RT-qPCR and western blot assays (Figure 12a–b). The si-IGF2BP2-3 effectively attenuated the expression of the IGF2BP2 gene in the SCC4 cell line when compared to si-NC. Thus, si-IGF2BP2-3 was selected for further experiments. The proliferation ability of the SCC4 cell line was evaluated using CCK8 and EDU assays (Figure 12c-d). The knockdown of the IGF2BP2 gene through siRNA significantly suppressed the DNA replication and proliferation capabilities of the SCC4 cell line when compared to the si-NC treatment. Additionally, the invasion and migration capabilities of the SCC4 cell line were evaluated through a transwell assay (Figure 12e), which showed that the knockdown of the IGF2BP2 gene could effectively suppress the cell migration and invasion abilities.