Ferroptosis-related genes with post-transcriptional regulation mechanisms in hepatocellular carcinoma determined by bioinformatics and experimental validation.

Abstract

Ferroptosis is a form of iron-dependent cell death with increased free iron and massive lipid peroxidation. The discovery of ferroptosis offers insights into hepatocellular carcinoma (HCC) treatment. However, post-transcriptional regulation mechanisms of ferroptosis in HCC remain to be elucidated. The present study explored ferroptosis-related genes and their post-transcriptional regulation mechanisms in HCC. A ferroptosis score was computed in The Cancer Genome Atlas (TCGA) cohort via gene set variation analysis (GSVA), and ferroptosis-related genes were screened by differential expression and correlation analyses. CircRNA/miRNA-mediated ferroptosis-related genes were predicted, and associations of ferroptosis-related genes with m1A/m5C/m6A regulators were analyzed. Immune cell infiltrations were inferred via CIBERSORT. NUDCD1 expression was examined in L-02, SMMC7721, and HepG2 cells via real time quantitative polymerase chain reaction (RT-qPCR) and western blots. After NUDCD1 was silenced, cell viability, glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) expression, and oxidized glutathione/glutathione (GSSG/GSH) and glutathione (GSH) levels were detected in SMMC7721 and HepG2 cells. The ferroptosis score was linked to poor overall survival (OS) of HCC, which was independent of other clinicopathological parameters. Ten ferroptosis-related genes were determined, namely UGT1A6, ATP6V1C1, MAFG, NUDCD1, PPP1R1A, TSKU, CTSB, AIFM2, CTSA, and CTNND2, which were post-transcriptionally regulated by circRNA/miRNA and m1A/m5C/m6A modifications in HCC. Most were significantly linked with most immune cell compositions within the immune microenvironment, and contributed to undesirable clinical outcomes. NUDCD1 was up-regulated in HCC cells, and its loss facilitated the ferroptosis of HCC cells. Overall, our findings determined ferroptosis-related genes post-transcriptionally regulated by circRNA/miRNA and m1A/m5C/m6A RNA modifications, and experiments demonstrated that loss of NUDCD1 may facilitate the ferroptosis of HCC cells, which provides novel insights into the regulatory mechanisms of ferroptosis in HCC.