Environmental cadmium-induced circRNA-miRNA-mRNA network regulatory mechanism in human normal liver cell model

Abstract

At present, hundreds of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been confirmed to be related to the toxicity of cadmium (Cd). However, the role of circular RNAs (circRNAs) in the toxicity of Cd and the underlying regulatory mechanisms remain unclear. In this study, we chose human normal liver cells (L-02) as a model to investigate changes in transcriptome expression levels following exposure to Cd. Total RNA of each sample was extracted by Trizol method, and the expression profiles of circRNAs, miRNAs and mRNAs of each sample were determined by microarray hybridization and scanning. After standardizing the data, differential circRNAs, miRNAs, and mRNAs associated with the toxic effects of Cd were identified. By screening the predicted circRNAs, miRNAs, and mRNAs, we constructed a competing endogenous RNA (ceRNA) network, and predicted the main biological functions and metabolic pathways influenced by Cd toxicity. Our comprehensive screening strategy led to the identification of 266 different circRNAs, 223 different miRNAs and 519 different mRNAs exhibiting differential expression. Following further screening, even circRNAs, 10 miRNAs and 97 mRNAs were incorporated into the ceRNA network. After performing GO enrichment and KEGG pathway analyses on the 97 mRNAs within the ceRNA network, which indicated that the circRNAs in the ceRNA network are poised to modulate key cellular processes, including cell proliferation, apoptosis, oxidative stress and inflammatory responses under the toxic effects of Cd-induced damage in L-02 cells.

Keywords: Cadmium; CeRNA; CircRNA; Environmental factor; L-02 cell.

Graphical abstract

Fig. 1

The relative fluorescence intensity when L-02 cell exposing to CdCl₂ at different concentrations and time points.

Fig. 2

Differentially expressed volcano map and cluster heatmap. (A) circRNA differentially expressed volcano map; (B) circRNA cluster heatmap; (C) miRNA differentially expressed volcano map; (D) miRNA cluster heatmap; (E) mRNA differentially expressed volcano map; (F) mRNA cluster heatmap.

Fig. 3

GO enrichment analysis and KEGG pathway analysis of differentially expressed mRNAs. (A) GO enrichment analysis of upregulated mRNA entries bar chart; (B) Histogram of up-regulated mRNA KEGG pathway analysis; (C) Down-regulated mRNA GO enrichment analysis entry bar chart; (D) Down-regulated mRNA KEGG pathway analysis histogram.

Fig. 4

Construction of circRNA-miRNA-mRNA regulatory network. (A) circRNA-miRNA-mRNA regulatory network associated with the toxic effects of Cd; (B) ceRNA pairs of hsa_circ_0004662, hsa_circ_0000745, hsa_circ_0082546 and hsa_circ_0082547; (C) ceRNA pairs of hsa_circ_0033388, hsa_circ_0002082 and hsa_circ_0017037.

Fig. 5

Verification of circRNA expression and circular structure. (A) Hsa_circ_0004662 produced by back-splicing of exon 3 and 5 from SOD2, and transcript variant 2 as the best transcript; (B) Hsa_circ_0000745 formed through the independent circularization of exon 2 from SPECC1, and transcript variant 4 as the best transcript; (C) and (D): Effects of different concentrations of CdCl₂ on the expression of circ_0000745 and hsa_circ_0004662; (E) and (F): Effect of CdCl₂ exposure time on the expression of circ_0000745 and hsa_circ_0004662.