3.1. Expression profiles of TTC21AmRNA and related DEGs
Based on the TCGA database, the expression of TTC21A mRNA was measured in different tumors. As depicted in Figure 1A, among 33 specimens of cancerous, TTC21A mRNA was significantly overexpressed in most of the cancers. Notably, it was observed that the expression of TTC21A in liver cell carcinoma tissues was significantly higher (P<0.001) than that of the adjacent tissues (Figure 1B). Further, 424 LIHC patients were randomly divided into two groups, a high-expression group and a low-expression group, and the expressions of mRNA and lncRNA between the two groups were compared. Finally, 561 mRNAs (537 up-regulated, 24 down-regulated, Figure 1C), 388 lncRNAs (281 up-regulated, 107 down-regulated, Figure S1A) were identified as DEGs (absolute value of fold change > 1.5, P <0.05), illustrated by a heat map (Figure 1D and Figure S1B).
3.2. Functional annotation of TTC21A-related DEGs
Further, Metascape software was used to evaluate the functions of TTC21A-related DEGs in LIHC patients. As presented in Figure 2A-C, it was observed that several pathways related to cancers were enriched, including epidermal development (GO: 00085444, P<0.001), negative regulation of endopeptidase activity (GO: 0010951, P<0.001), and endocrine hormones (GO: 0046879, P<0.001).
3.3. Expression of TTC21A in different clinical subgroups
Notably, the expression of TTC21A in patients with LIHC is often related to clinicopathological characteristics. To demonstrate this aspect, the Wilcoxon rank-sum test was employed to analyze the differences in the expression of TTC21A in liver cancer tumor tissues and normal tissues. The results showcased that there exist significant differences in TTC21A expression and pathological grade (P<0.05), clinical T stage (P<0.001), clinical M stage (P<0.001), histological grade (Figure 3A-F).The baseline characteristics of hepatocellular carcinoma patients from TCGA were listed(Table1).Further, the Univariate analysis showed that the increased expression of TTC21A (based on the median value) was associated with clinicopathological features of poor prognosis (Table 2).
3.4. Immunohistochemistry and PPI network construction
From the immunohistochemical staining of TTC21A in the HPA database, it was observed that the expression of TTC21A in tumor tissues was significantly higher than that of normal tissues（Figure 4）. Further, we employed string tools to construct a protein-protein interaction network of TTC21A and its related potential co-expressed gene network (Figure 5). Among them, TTC21A was considered the central gene related to other 10 genes, including ARMC2, CFAP43, IFT122, IFT140, IFT120, ITF52, IFT74, IFT81, WDR19, and WDR35, with their scores of above 0.7 (Table 4).
3.5. Relationship between TTC21A and immune cell penetration
GSV A package was used to determine the correlation between HSPB1 and other immune cells, displaying that the expression levels of TTC21A in the LIHC microenvironment were positively correlated with the quantitative immune cell infiltration levels of ssGSEA. Specifically, TTC21A expression was positively correlated with macrophages, TFH, and NK CD56 bright cells (Figure 6A). As depicted in Figure 6B-G, macrophages (R = 0.290, P<0.001), Tem (R = 0.210, P<0.001), Th1 (R = 0.220, P<0.001) and Th2 (R = 0.200, P <0.001) were all significantly correlated to the expression of TICRR. Contrarily, B cells (R = +0.061, P>0.001), NK cells (R = 0.074, P>0.001), showed a negative correlation with TTC21A.
In addition, the expression of TTC21A showed a good discrimination ability. The ROC curve of TTC21A expression in normal tissues and tumor tissues had shown an AUC value of 0.778; while the AUC values of stage I, II, and III subgroup analysis were 0.752, 0.810, and 0.814, respectively, which could distinguish tumors and normal tissues (Figure 7). Moreover, the Kaplan-Meier survival analysis showed that the high TTC21A expression group was more correlated with poor prognosis than the low TTC21A expression group (Figure 8). In the univariate COX model, high expression TTC21A levels were associated with high T staging (P<0.001)(Table 3).
3.6. Independent risk and diagnostic value of TTC21A expression
The univariate survival analysis showed that TTC21A expression age (HR=1.205, P=0.295), gender (HR=0.793, P=0.2), T stage (HR=2.598, P<0.001), M stage (HR=2.029, P =0.324), and N stage (HR=4.077, P=0.017) were important factors to be considered in predicting the survival chances in correlation to the TTC21A expression (Figure 9).
3.7. Nomogram chart of survival rate of liver cancer patients
Based on the TCGA training cohort, age, tumor stage, tumor status, and risk score model were also added to construct a prognostic prediction nomogram and predict the probability of OS in 1, 3, and 5 years (Figure 10A). As depicted in Figure 10B, the calibration curve showed that, in all cohorts, the OS predicted by the nomogram was in good agreement with the actual observations in 1, 3, and 5 years.