Differentially Expressed FRGs Were Identified Using RNA-seq
The flow chart of our analysis is presented as Fig. 1. Following the pre-specified thresholds, we obtained a differentially expressed gene set (n = 69; 53 upregulated and 16 downregulated genes) by RNA-seq as well as differential analysis between erastin-treated and untreated groups (Fig. 2A; Table S1). The heatmap of FRGs based on hierarchical clustering indicated that the expression patterns of differentially expressed genes could be well discerned after unsupervised clustering (Fig. 2B).
Functional Enrichment and PPI Analysis of FRGs
Gene ontology enrichment analysis of FRGs between erastin-treated and untreated gastric cancer cells indicated that most of them were enriched in the extracellular exosomes, and participating in the molecular functions of GTPase activity. From the viewpoint of biological process (BP), phylloquinone metabolic process was the most significantly enriched term for FRGs (Fig. 3A). Meanwhile, the differentially expressed FRGs were confirmed to be enriched in several KEGG pathways related to endocytosis, ribosome, and ferroptosis (Fig. 3B).
To determine the interactions among protein products encoded by the differentially expressed FRGs, we performed PPI analysis. The resulted interactions are shown in detail in Fig. 4A-B. Notably, UBB was the node sharing the largest number of interactions across these differentially expressed FRGs.
Identification of FTH1 and Its Expression Pattern
The key gene FTH1 was then obtained by intersecting the differentially expressed gene set with the FRGs extracted from the ferroptosis database. Thereafter, we carried out a set of bioinformatics analyses and biological experiments to validate our screening and prediction results.
Using the HPA database to analyze FTH1 expression pattern in normal cells, we found that it was mainly enriched in blood and immune cell types, including Langerhans cells, monocytes, and proximal enterocytes (Fig. 5A). Furthermore, we examined the expression pattern of FTH1 protein in 44 normal samples. It was expressed in low or undetectable amounts in most tissues, including gastric glandular cells, to a moderate extent in tissues such as pancreas and kidney, while highly expressed in cerebral cortex and bone marrow tissues (Fig. 5B).
Next, we retrieved the differential expression pattern of FTH1 in cancer tissues versus adjacent normal tissues in pan-cancer (n = 33) and observed that the mRNA levels of FTH1 in most solid tumors were generally elevated compared to those in normal controls (Fig. 5C).
Differential Expression of FTH1 in Normal and Gastric Cancer Samples
We compared FTH1 mRNA expression between 414 GC samples and 210 normal tissue samples (36 from TCGA-STAD cohort and 174 from GTEx). As shown in Fig. 6A, FTH1 was significantly upregulated in GC tissues (P < 0.001). To further determine the significance of the biological function of FTH1 at the protein level, we accessed a cohort (containing 354 cases of primary GC paired with non-cancerous tissues) that underwent immunohistochemical staining in HPA. Consistent with the RNA pattern, the FTH1 protein was stained at a low level in normal gastric glandular tissues but at a significantly elevated level in GC tissues. Representative images are shown in Fig. 6B. In addition, we carried out ROC to estimate the discrimination efficacy of FTH1 between GC and corresponding normal tissues. The AUC for FTH1 was 0.619, 95% confidence interval (95%CI) 0.573 to 0.664 (Fig. 6C), suggesting that FTH1 might be an identifying biomarker for GC.
Correlations of FTH1 Expression and Immune Infiltration
Next to investigate the immunomodulatory role of FTH1, we assessed the relationship between FTH1 expression and immune infiltration level in GC samples. The results of correlation analyses of FTH1 with immune cells are shown in Table 1. and Fig. 7, the FTH1 expression level in GC showed a positive correlation with the infiltration of most immune cells. Taken together, these findings suggested that FTH1 might influence immune cell infiltration in GC.
Genetic Alteration and DNA Methylation of FTH1 in Gastric Cancer
Different degrees of genetic alterations in FTH1 were detected in 25 of 32 cancer types, including mutations, amplifications, deep deletions, and multiple alterations (Fig. 8A). Detailed mutations in FTH1 are shown in Fig. 8B, and we found that the genetic alteration rate was 1.36% among the 440 sequenced GC samples.
In addition, we presented the annotation information of DNA methylation probes and samples with a heatmap, including CpG sites, genomic regions, relation to islands, age, body mass index (BMI), ethnicity, survival status, etc. Through visualization of methylation levels and FTH1 expressions, we found that FTH1 methylation level was high in cg11748881 probe (Fig. 9A), and patients with FTH1 hypermethylation had a poor overall survival. Meanwhile, we found that 3 CpG sites (cg24898753, cg24496614, and cg09367425) located in CpG islands indicated a better prognosis (Fig. 9B-D).
Figure 9 The MethSurv obtained the effect of methylation level and FTH1 expression on prognosis in GC. (A) The visualization between the methylation level and the FTH1 expression; (B–D) the Kaplan–Meier survival of the promoter methylation of FTH1
The Mechanism of FTH1 on Erastin-induced Ferroptosis in GC
By suppressing cystine uptake by cells, erastin increases GSH depletion and inactivates GPX4, which leads to the accumulation of lipid peroxidation, resulting in ferroptosis. A study has shown that cellular sensitivity to ferroptosis is related to multiple biological pathways, including lipid, glutathione, and iron metabolism. As shown in Fig. 10, FTH1 was involved in iron storage and autolysosomes and contributed to the maintenance of iron homeostasis and protection against iron overload.
FTH1 Overexpression Promoted Proliferation and Blocked Ferroptosis
To elucidate the role of FTH1 in GC pathogenesis, we transfected MGC803 cells with plasmid vectors to overexpress FTH1 in vitro, and RT-qPCR verified that the transfection efficiency was high (Fig. 11A). FTH1 overexpression promoted the proliferation of MGC803 cells (Fig. 11B). GSH/GPX4-based ROS scavenging indispensably prevents lipid peroxidation during ferroptosis. GSH levels were increased in OE-FTH1 cells (Fig. 11C). FTH1 overexpression inhibited ROS generation and MDA production (Fig. 11D-F).