Integrated analysis of transcriptomic and metabolomic profiling reveal the p53 associated pathways underlying the response to ionizing radiation in HBE cells

Abstract

Background: Radiation damage to normal tissues is a serious concern. P53 is a well-known transcription factor which is closely associated with radiation-induced cell damage. Increasing evidence has indicated that regulation of metabolism by p53 represents a reviving mechanism vital to protect cell survival. We aimed to explore the interactions of radiation-induced transcripts with the cellular metabolism regulated by p53.

Methods: Human bronchial epithelial (HBE) cell line was used to knockout p53 using CRISPR/cas9. Transcriptomic analysis was conducted by microarray and metabolomic analysis was conducted by GC-MS. Integrative omics was performed using MetaboAnalyst.

Results: 326 mRNAs showed significantly altered expression in HBE p53-/- cells post-radiation, of which 269 were upregulated and 57 were downregulated. A total of 147 metabolites were altered, including 45 that increased and 102 that decreased. By integrated analysis of both omic data, we found that in response to radiation insult, nitrogen metabolism, glutathione metabolism, arachidonic acid metabolism, and glycolysis or gluconeogenesis may be dysregulated due to p53.

Conclusions: Our study provided a pilot comprehensive view of the metabolism regulated by p53 in response to radiation exposure. Detailed evaluation of these important p53-regulated metabolic pathways, including their roles in the response to radiation of cells, is essential to elucidate the molecular mechanisms of radiation-induced damage.

Fig. 1

Bioinformatic analysis of the mRNA expression alteration based on HBE cells with or without CRISPR/Cas9-mediated p53-knockout. a p53 knockout efficiency was determined using western blotting. b Quantitative measurement of differential expressed mRNA in HBE cells with (p53-/-) or without CRISPR/Cas9-mediated p53-knockout (p53-wt) after 4 Gy IR. c. A heatmap was illustrated to show the differential expressed mRNA in HBE cells with (p53-/-) or without CRISPR/Cas9-mediated p53-knockout (p53-wt) after 4 Gy IR. Pink represents upregulated mRNA, and purple represents down-regulated mRNA. d A volcano map was illustrated to show the differential expressed mRNA in HBE cells with (p53-/-) or without CRISPR/Cas9-mediated p53-knockout (p53-wt) after 4 Gy IR. Red represents upregulated mRNA, green represents down-regulated mRNA and grey represents the mRNA without expression change

Fig. 2

GO analysis of significantly differential expressed mRNA based on HBE cells with or without CRISPR/Cas9-mediated p53-knockout. a Enrichment of mRNA significantly differential expressed in the biological process. b Enrichment of mRNA significantly differential expressed in the molecular function. c Enrichment of mRNA significantly differential expressed in the cellular component

Fig. 3

Verification of selected mRNA. a KEGG analysis of significantly differential expressed mRNA in HBE cells with (p53-/-) or without CRISPR/Cas9-mediated p53-knockout (p53-wt) after 4 Gy IR. b five upregualted mRNA and five downregulated mRNA were selected to verify whether the expression trend consistence with microarray assay by qRT-PCR on HBE cells with CRISPR/Cas9-mediated p53-knockout

Fig. 4

Bioinformatic analysis of metabolomics. a A heatmap was used to visually represent upregulated and downregulated metabolites. b A volcano was used to visually represent upregulated and downregulated metabolites. c The interactions among altered metabolites were analyzed by pearson correlation analysis

Fig. 5

Integrated transcriptomic and metabolomic analyses of p53-dependent metabolic pathways. a KEGG analysis of significantly altered metabolites. b The mRNA enrichment and metabolic pathway analyses was based on joint pathways using MetaboAnalyst 4.0. c The arachidonic acid metabolism pathway with altered significantly mRNA (red) in HBE cells with CRISPR/Cas9-mediated p53-knockout. d The nitrogen metabolism pathway with altered significantly mRNA (red) in HBE cells with CRISPR/Cas9-mediated p53-knockout

Fig. 6

Integrated transcriptomic and metabolomic analyses of p53-dependent metabolic pathways. a The glutathione metabolism pathway with altered significantly mRNA (red) in HBE cells with CRISPR/Cas9-mediated p53-knockout. b The glycolysis or gluconeogenes metabolism pathway with altered significantly mRNA (red) in HBE cells with CRISPR/Cas9-mediated p53-knockout. c GGT1, PLA2G, PTGS, GPX6, ALDOA, ACSS2, ALDH3A1, GGT6 expression levels were determined using qRT-PCR in HBE cells of p53-wt, p53-wt treated with 4 Gy radiation (p53-wt –IR), p53-/-, and p53-/- treated with 4 Gy radiation (p53-/- IR), respectively. The data are presented as the means ± SDs from three independent experiments; * p < 0.05 between different groups