Hepatocellular carcinoma (HCC) represents a major healthcare issue and continues to be a leading cause of mortality and morbidity worldwide.[12] The occurrence and development of HCC is a multi-step dynamic process involving multiple factors and genes.
Although previous studies have suggested that several ferroptosis-related genes may be associated with overall survival outcomes in HCC patients, these studies have lacked experimental studies that could match to validate their results.[13; 14] With increasing evidence showing growing number of genes or proteins that have been reported to be involved in the process of ferroptosis, this study aimed to explore new potential ferroptosis-related genes to construct predictive models for the prognosis of HCC patients.
In this study, we identify 719 candidate genes that were associated with ferroptosis and liver cancer prognosis by analyzing mRNA-Seq of HCC samples from the TCGA databases. Then, we investigated combined multi-gene biomarkers associated with HCC patient’s prognosis for OS and DFS. Results significantly indicated the feasibility of building a prognostic model with these genes. Our method was based on matrix combination to identify genes with possible synergistic effects to construct prognosis model, compared with single gene, it was more robust, and compared with the prognosis model larger than three genes was more clinically feasible. Finally, three ferroptosis-related genes (GLMP, SLC38A6, and WDR76) that were significantly associated with OS were selected to establish the prognosis model of HCC and verified in ICGC database and experiment. These three ferroptosis-related genes are highly expressed in HCC tissues and work synergistically.
Researchers have recently found that ferroptosis is a lysosome-dependent autophagic cell death process.[5] GLMP has been reported as an important role in maintaining normal lysosomal function and lipid metabolism. Ablation of GLMP in mice had developed marked liver fibrosis associated with hepatic cell death, oxidative stress.[15] In another relative study, they found these mice develop liver tumors at later stages in life.[16] Gao. et al. reported that GLMP was highly expressed in hepatocellular carcinoma and hepatic steatosis patients. GLMP expression is regulated by BRG1 and is involved in the regulation of lipid metabolism in hepatocellular carcinoma.[17] According to our results, HCC showed a remarkably high expression of GLMP as mRNA and protein and higher expression of GLMP was positively correlated with the poor outcomes of HCC patients.
As a member of the SLC38 family, the solute carrier family 38 member 6 (SLC38A6) has been found upregulated in pancreatic adenocarcinoma and gastric cancer.[18; 19] Also, a high degree of specificity for glutamine and glutamate has recently been discovered, which is an essential nutrient for many cancers.[20] Li Huang et al. reported that SLC38A6 expression was higher in HCC compared with expression in normal tissue. Knockdown SLC38A6 inhibited cell viability, colony formation, cell cycle progression, mitochondrial respiration and MMP and decreased glutamine concentration in HCC cells while overexpressing SLC38A6 induced the opposite effects.[21] Glutamine inhibition could cause the accumulation of reactive oxygen species (ROS) following ferroptosis. In line with our findings, we found that SLC3A6 was highly expressed in HCC tissues and that higher SLC38A6 expression was associated with a lower overall survival rate in HCC patients.
Ro EJ et al. reported a higher proliferation ratio and increased colony formation in WDR76 overexpressed cancer cells and a reverse trend in WDR76 ablated cells.[22] Importantly, they found the regulation of LSH stability by WDR76 decreased DNA oxidation and overproduction of ROS, indicating that the WDR76 may be a critical epigenetic modulator of ferroptosis. Furthermore, higher WDR76 expression was associated with poorer overall survival in lung cancers, thus functionally explaining the relationship of WDR76 with cancer prognosis and ferroptosis.[23] In contrast, Jeong WJ et al. found that overexpression of WDR76 increased lipid accumulation in 3T3-L1 cells. WDR76 deficiency in mice enhanced the development of DEN-induced HCC formation and increment in liver weight and liver/body weight ratios.[24] The findings above indicate that WDR76 may have a bidirectional regulatory effect on tumors. In our study, we found the WDR76 mRNA overexpressed in tumors but not at the protein level, which may be due to gene post-transcriptional regulation. However, survival analysis still showed that high expression of WDR76 leads to worse overall survival in patients with HCC.
Our study has several strengths. First, we established a comprehensive prediction model selective combinations of genes with experimental validation. Seconds, the results are robust due to employment of big data analysis. Third, we selected ferroptosis-related candidate genes, which have not yet been reported to relate to HCC patient’s prognosis. However, there are limitations to our analysis. The bioinformatics analysis identified potential prognostic genes based on the RNA-Seq of tissue samples which do not necessarily represent the mRNA and protein expression of tumor cells, further examine the molecular mechanisms involved in ferroptosis–related genes that affect the prognosis of HCC patients should be conducted.