Extraction and functional enrichment analysis of differential genes associated with ferroptosis.
First, we started analyzing the BCa transcriptome expression data obtained from TCGA. By differential enrichment analysis by DAVID database, we found 47 DEGs associated with ferroptosis. Of these genes, 22 were downregulated, and 25 were upregulated (Table 3). BP functional enrichment analysis revealed that these genes are involved in the following pathways: intrinsic apoptotic signaling pathway, multicellular organismal homeostasis, and response to toxic substances. Among the MF specific terms, we found “regulate iron ion binding”, “oxidoreductase activity”, “acting on single donors with incorporation of molecular oxygen”, and “protein kinase”. Among the CC terms, we mainly found “chromatin”, “receptor complex”, and “endoplasmic reticulum lumen”. kEGG-based analysis showed that overexpression of genes mainly involved the p53 signaling pathway, ferroptosis, Kaposi sarcoma-associated herpesvirus infection, IL-17 signaling pathway, MicroRNAs in cancer, the TNF signaling pathway, PI3K-Akt signaling pathway, and HIF-1 signaling pathway (Figure 1A-D ).
Prognostic features and survival analysis based on ferroptosis-associated lncRNAs
We identified 270 lncRNAs associated with ferroptosis (Table 4). In addition, univariate Cox analysis identified 140 important lncRNAs associated with ferroptosis, which were included in the multivariate Cox analysis. Overall, 11 differentially expressed lncRNAs (Table 5) were found to be independent prognostic indicators for BCa.
Therefore, we proceeded to calculate risk scores and construct a BCa prognostic model using lncRNAs. Kaplan-Meier analysis showed that patients with high-risk lncRNAs expression had poorer survival than low fractional risk lncRNAs (P < 0.001, Figure 2A). This risk model (AUC = 0.720) showed superior performance and predictive power than traditional clinicopathological features (Figure 2B). Using patients' risk survival status maps, we found that patients' risk scores were inversely correlated with the survival of BCa patients. Interestingly, the heat map also demonstrated that most of the novel lncRNAs identified in this study were negatively correlated with our risk model (Figure 2C). The AUC predictive values of these new IncRNA models for 1-year, 3-year, and 5-year survival were 0.720, 0.697, and 0.706, respectively (Figure 2D). Univariate and multifactorial Cox analyses showed that lncRNAs model (HR: 1.05, 95CI: 1.02-1.07) and tumor stage (HR: 1.66, 95CI: 1.37-2.02) were independent prognostic factors for OS in BCa patients (Figure 3A-B). lncRNA-mRNA relationships are shown in Figure 3D. A heatmap of the association between prognostic models and clinicopathological manifestations of lncRNAs associated with ferroptosis was also analyzed (Figure 4A). A hybrid column line plot combining clinicopathological characteristics and the novel ferroptosis-related lncRNAs prognostic signature (Figure 4B) was stable and accurate, thus may be applied in clinical management of BCa patients.
Gene set enrichment analysis
The gene set enrichment analysis (GSEA) revealed various immune and tumor-related pathways that are prognostic signature regulators of most novel lncRNAs associated with ferroptosis, such as Adhesion junction, ECM receptor interaction, Chemokine signaling pathway, B cell receptor signaling pathway, TGF-β signaling pathway, MAPK receptor signaling pathway, Notch signaling pathway, and Bladder cancer. (Figure 5 ).
Expression of immune-related genes
Next, we created a heat map of the immune response based on CIBERSORT, ESTIMATE, MCPcounter, single sample gene set enrichment analysis (ssGSEA), and TIMER algorithm (Figure 6A). ssGSEA based on the TCGA-BLCA data, correlation analysis between immune cell subsets and related functions showed that T cell functions include checkpoint (suppression), lysis, HLA, inflammatory regulation, co-stimulation, co-inhibition, and type II INF response. Significant differences between low-risk and high-risk patients (Figure 6B). As checkpoint inhibitor-based immunotherapy strategies are emerging as one of the best cancer treatment tools for some drug-resistant tumors, we further explored the differences in immune checkpoint expression between the two groups and found significant differences in PDCD-1 (PD-1), CTLA4, LAG3, and BTLA expression (Figure 6C). In addition, the comparison of m6A-related mRNA expression and the expression of METTL3, KIAA1429, RBM15, ZC3H13, YTHDC1, YTHDF1, YTHDF2, HNRNPC, and FTO in the high-risk and low-risk groups were significant (Figure 6D).
Validation of the identified Diff-miRNAs
Figure 7 shows the results of the qRT-PCR. It is shown that the expression of LncRNAs(AL031775.1, AC018653.3, AC011468.1, AL583785.1, AC021321.1, AP003352.1, `ETV7-AS1`, U47924.1, AC010326.3) is significantly decreased in the SV-HUC cell line as compared with that in the T24 cell line, while LINC02762 expression did not show significant differences in the two cells line.Similarly, the expression of LncRNAs (AL031775.1, AC018653.3, AC011468.1, AL583785.1, AC021321.1, `ETV7-AS1`, U47924.1, AC010326.3) was significantly upregulated in EJ cell lines, differing from SV-HUC cells. However, the expression of AP003352.1 and LINC02762 did not show significant differences in the two cell lines.The result show us that eight LncRNAs, AL031775.1, AC018653.3, AC011468.1, AL583785.1, AC021321.1, `ETV7-AS1`, U47924.1, AC010326.3, could be used as ferroptosis-related biomarkers to predict the prognosis of BCa patients. provide new targets for their treatment and research.（The specific primer sequences are listed in Table 6）