Screening and evaluation of autophagy-related hub genes in cadmium neurotoxicity based on transcriptomics and network toxicology

Autophagy dysfunction plays a significant role in the neurotoxicity of cadmium. This study aims to screen autophagy-related hub genes in cadmium-exposed HT22 cells and evaluate the role of the hub gene-related regulatory networks in cadmium neurotoxicity.

CCK-8 assay was used to detect the survival rate of HT22 cells exposed to CdCl₂ at concentrations of 0, 5, 10, 15, 20 and 25 μmol/L for 24 h, and the half-maximal inhibitory concentration (IC₅₀) was calculated. The control group (n=4) was not treated with cadmium, while the cadmium exposure group (n=4) was treated with an IC₅₀ of CdCl₂ for 24 h. RNA samples from the control group and the cadmium exposure group were extracted for transcriptome sequencing. Autophagy-related differentially expressed genes (DEGs) were screened based on transcriptomic results of cadmium-exposed HT22 cells and public databases, and GO and KEGG analyses were performed to identify the main enriched pathways. Further screening of autophagy-related hub genes was carried out by protein-protein interaction (PPI) network, and the relative expression levels of these hub genes were validated by qPCR. Multiple regulatory networks related to hub genes (miRNAs, drugs, transcription factors, RNA binding proteins) were further constructed.

Cadmium exposure caused to a dose-dependent decrease in the survival rate of HT22 cells (P<0.01), with an IC₅₀ value of about 10 μmol/L. Compared with the control group, the transcriptomic expression profile of the cadmium exposure group underwent significant global changes. A total of 36 autophagy-related DEGs and 13 hub genes (Atf3, Csf2, Cdkn1a, Cd68, Cryab, Hbegf, Adam8, Areg, Gadd45a, Plk3, Aldh3a1, Adm, Dusp4) were identified, mainly enriched in oxidative stress and signal transduction-related pathways. Among them, the expression of 11 hub genes (Atf3, Csf2, Cdkn1a, Cd68, Cryab, Hbegf, Adam8, Areg, Gadd45a, Plk3, Dusp4) was upregulated significantly (P<0.05). The hub genes were involved in the multi-regulatory networks of 271 miRNAs, 86 drugs, 20 transcription factors, and 17 RNA-binding proteins.

Cadmium exposure induces alterations in the expression of 11 autophagy-related hub genes in HT22 cells, and the multiple regulatory networks involving autophagy-related hub genes mediate cadmium-induced neurotoxicity in neuronal cells.