Mechanism and Role of the Neuropeptide LGI1 Receptor ADAM23 in Regulating Biomarkers of Ferroptosis and Progression of Esophageal Cancer

Abstract

Background: According to recent studies, ferroptosis is closely related to the efficacy and prognosis of tumour treatment. However, the role of ferroptosis in esophageal squamous cell carcinoma (ESCC) has not been explored comprehensively.

Materials and methods: The esophageal cancer (EC) transcriptome data was downloaded from The Cancer Genome Atlas (TCGA), then analyzed, to obtain the differentially expressed messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA) between groups with the low and high Ferroptosis Potential Index (FPI) and construct a ferroptosis-associated ceRNA network. In addition, the expression of ARHGEF26-AS1 and miR-372-3p in ESCC cell lines was assessed, and the appropriate cell lines were selected. The interaction between ARHGEF26-AS1, miR-372-3p, and ADAM23 was also determined through a dual-luciferase reporter assay. Moreover, the Western blot, Cell Counting Kit-8 (CCK-8), wound healing, cell viability, and cell death assays were conducted to establish the biological functions of the ARHGEF26-AS1/miR-372-3p/ADAM23 pathway in ESCCs.

Results: An FPI scoring model reflecting the activity of the ferroptosis pathway was constructed, and a ferroptosis-associated ceRNA network was established. The findings revealed that low expression of ADAM23 and ARHGEF26-AS1 as well as high expression of miR-372-3p was associated with poor prognosis and a lower FPI score in EC patients. Functionally, overexpression of ADAM23 and ARHGEF26-AS1 and the miR-372-3p inhibitor not only promoted ferroptosis in ESCC cells in vitro but also inhibited the proliferation and migration of cells. Mechanistically, ARHGEF26-AS1 upregulated the expression of ADAM23 by competitively binding to miR-372-3p.

Conclusions: The study showed that the lncRNA, ARHGEF26-AS1 acts as a miR-372-3p sponge that regulates the neuropeptide LGI1 receptor ADAM23 expression. This in turn not only inhibits the proliferation and migration of ESCC cells but also upregulates the ferroptosis pathway. A neuropeptide-related ferroptosis regulatory pathway was identified in this study.

Figure 1

A flow chart of the study.

Figure 2

Establishment of the ferroptosis index model and identification of FPI-associated differentially expressed genes. (a–c) Volcano plots showing the (a) DE-lncRNA, (b) DE-mRNA, and (c) DE-miRNA. (d) A heatmap of the key DE-lncRNA, DE-mRNA, and DE-miRNA. (e) KEGG pathway enrichment analysis and (f) GO function analysis of the DE-mRNA.

Figure 3

Clinical correlation analysis. (a) A ceRNA network of esophageal cancer and ferroptosis. (b) Correlations among ADAM23, miR-372, and ARHGEF26-AS1. (c) The correlation between ADAM23 and ARHGEF26-AS1 (p < 0.0001). (d) The correlation between ADAM23 and hsa-miR-372 (p < 0.001). (e) Differential expression of ADAM23 in different clinical stages. (f) Association of ADAM23, miR-372, and ARHGEF26-AS1 with FPI. (g) Distribution of ADAM23 expression in human tissues.

Figure 4

Overexpression of ADAM23 suppressed tumorigenesis and promoted ferroptosis in esophageal carcinoma cells. (a) Relative expression of ARHGEF26-AS1, miR-372-3p, and ADAM23 in ESCC cells; ^∗p < 0.05; (b) Western blot results showing the expression of the ADAM23, GPX4, SLC7A11, and SLC3A2 proteins in TE-1 cells from the NC, EV, ADAM23-OE, shADMA23-1, and shADAM23-2 groups; ^∗p < 0.05; (c) real-time PCR results showing the mRNA expression levels of ADAM23, GPX4, SLC3A2, and SLC7A11 in TE-1 cells following the overexpression and silencing of ADAM23. ^∗p < 0.05; (d) the effect ADAM23 on cell proliferation, assessed through the CCK-8 assay. ^∗p < 0.05; (e) cell viability and (f) cell death measured in subgroups with different levels of ADAM23 expression ^∗p < 0.05; (g) proliferation and migration of TE-1 cells assessed through the wound healing assay. ^∗p < 0.05.

Figure 5

Upregulation of ADAM23 enhanced ferroptosis and inhibited cell growth in esophageal carcinoma cells through the downregulation of miR-372-3p. (a) The binding of miR-372-3p on ADAM23 as predicted by RNAhybrid 2.12. (b) Regulation of ADAM23 by miR-372-3p, assessed through luciferase reporter assays. ^∗p < 0.05 vs. miR-NC. (c) The effect of up- and downregulation of miR-372-3p on the expression level of ADAM23, assessed through the WB assay; ^∗p < 0.05; (d) real-time PCR results showing the mRNA expression levels of GPX4, SLC3A2, and SLC7A11 in TE-1 cells cotransfected with the miR-372-3p mimic or miR-372-3p inhibitor after the overexpression of ADAM23; ^∗p < 0.05; (e) the effect of the miR-372-3p mimic and miR-372-3p inhibitor on cell proliferation after the overexpression of ADAM23, assessed through the CCK-8 assay. ^∗p < 0.05; (g) the effect of the miR-372-3p mimic and miR-372-3p inhibitor on (f) cell proliferation and (g) cell death after the overexpression of ADAM23; ^∗p < 0.05; (h) the proliferation and migration of TE-1 cells assessed through the wound healing assay. ^∗p < 0.05.

Figure 6

The ARHGEF26-AS1 lncRNA promoted ferroptosis, inhibited cell growth and upregulated the expression of ADAM23 by sponging miR-372-3p in esophageal carcinoma cells. (a) The binding of miR-372-3p to ARHGEF26-AS1, predicted with RNAhybrid 2.12. (b) Regulation of miR-372-3p by ARHGEF26-AS1, assessed through luciferase reporter assays. ^∗p < 0.05 vs. miR-NC.; (c) the relative mRNA levels of ADAM23, GPX4, SLC3A2, and SLC7A11 in TE-1 cells cotransfected with the miR-372-3p mimic or shADAM23-1, following the overexpression of ARHGEF26-AS1. Data is presented as the mean ± SD. ^∗p < 0.05; (d) the effect of the miR-372-3p mimic and shADAM23-1 on cell proliferation after the overexpression of ARHGEF26-AS1, assessed through the CCK-8 assay. ^∗p < 0.05; (e, f) the effect of ARHGEF26-AS1 overexpression on (e) cell proliferation and (f) cell death after treatment with either the miR-372-3p mimic or shADAM23-1. ^∗p < 0.05; (g) verification of ARHGEF26-AS1 overexpression through Western blotting following the overexpression of miR-372-3p and the effect of shADAM23-1 on the expression of ADAM23; ^∗p < 0.05; (h) proliferation and migration of TE-1 cells, assessed through the wound healing assay; (i) effect of the ARHGEF26-AS1/miR-372-3p/ADAM23 pathway on ferroptosis. ^∗p < 0.05.

Figure 7

Graphical abstract image.