Integrated Analysis of m6A Methylome in Cisplatin-Induced Acute Kidney Injury and Berberine Alleviation in Mouse

Abstract

Background: N6-methyladenosine (m6A) is the most abundant modification known in mRNAs. It participates in a variety of physiological and pathological processes, such as metabolism, inflammation, and apoptosis.

Aims: To explore the mechanism of m6A in cisplatin-induced acute kidney injury (AKI) and berberine alleviation in mouse.

Methods: This study investigated the N6-methyladenosine (m6A) methylome of kidneys from three mouse groups: C57 mice (controls), those with CI-AKI (injury group, IG), and those pretreated with berberine (treatment group, TG). Methylated RNA Immunoprecipitation Next Generation Sequencing (MeRIP-seq) and RNA-seq were performed to identify the differences between the injury group and the control group (IvC) and between the treatment group and the injury group (TvI). Western blotting was performed to identify the protein levels of candidate genes.

Results: In IvC, differentially methylated genes (DMGs) were enriched in metabolic processes and cell death. In TvI, DMGs were enriched in tissue development. Several genes involved in important pathways related to CI-AKI showed opposite methylation and expression trends in the IvC and TvI comparisons.

Conclusion: m6A plays an important role in cisplatin induced AKI and berberine may alleviate this process.

Keywords: FGA; Havcr1; M6A; SLC12A1; berberine; cisplatin induced nephrotoxicity.

FIGURE 1

Establishment of cisplatin-induced acute kidney injury model in C57 mouse. (A) Analysis of Scr level in mice following different treatments. Error bars represent the standard deviation. ***p < 0.001, Student’s t-test. (B) Analysis of BUN level in mice following different treatments. Error bars represent the standard deviation. *p < 0.05, ***p < 0.001, Student’s t-test. (C) Represents the image of hematoxylin and eosin staining in kidney (black arrows indicating the injury). (D) Score for characteristic histologic signs of renal injury. **p < 0.01, ***p < 0.001, Student’s t-test.

FIGURE 2

Overview of N6-methyladenosine methylation within mRNAs in the control, injury, and treatment groups. (A) Venn diagram showing the overlap of m6A peaks within mRNAs between IvC (left) and TvI (right). (B) The top motif enriched across m6A peaks identified from three groups. (C) Proportion of genes harboring different numbers of m6A peaks in three groups. The majority of genes harboring more than one m6A peak. (D) Pie charts showing the percentage of m6A peaks in five segments of transcripts. m6A peaks were most enriched in the coding sequence segment. (E) Distributions of fold enrichment of m6A peaks in five segments. The mean fold enrichment in the stop codon segments was the highest in injury group, while that value in CDS was the largest in treatment group. (F) Analysis of m6A peaks in different groups. Error bars represent the standard deviation. Student’s t-test.

FIGURE 3

Distribution of differentially methylated N6-methyladenosine sites. (A) Relative occupancy of differentially methylated m6A sites in each chromosome normalized by length in IvC. (B) Pie chart showing the percentage of DMM peaks in five non-overlapping segments in IvC. (C) Statistics of fold change of DMM peaks in five segments. The histogram shows the mean of the fold change in IvC. Error bars represent the standard error of the mean. (D) Relative occupancy of differentially methylated m6A sites in each chromosome normalized by length in TvI. (E) Pie chart showing the percentage of DMM peaks in five non-overlapping segments in TvI. (F) Statistics of fold change of DMM peaks in five segments. The histogram shows the mean of the fold change in TvI. Error bars represent the standard error of the mean. (G) Relative occupancy of differentially methylated m6A sites in each chromosome normalized by length. (H) Pie chart showing the percentage of DMM peaks in five non-overlapping segments.

FIGURE 4

Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses of coding genes containing DMMSs in IvC. (A) Major gene ontology terms were enriched for the genes containing up-regulated m6A sites in IvC. (B) Major gene ontology terms were enriched for the genes containing down-regulated m6A sites in IvC. (C) Major enriched pathways for the genes containing up-regulated m6A sites in IvC. (D) Major enriched pathways for the genes containing down-regulated m6A sites in IvC.

FIGURE 5

Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses of coding genes containing DMMSs in TvI. (A) Major gene ontology terms were enriched for the genes containing up-regulated m6A sites in TvI. (B) Major gene ontology terms were enriched for the genes containing down-regulated m6A sites in TvI. (C) Major enriched pathways for the genes containing up-regulated m6A sites in TvI. (D) Major enriched pathways for the genes containing down-regulated m6A sites in TvI.

FIGURE 6

Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses of coding genes containing DMMSs. (A) Major gene ontology terms were enriched for the genes containing m6A sites with reverse methylation trend between IvC and TvI. (B) Major enriched pathways for the genes containing m6A sites with reverse trend between IvC and TvI.

FIGURE 7

Conjoint analysis of differentially methylated genes and differentially expressed genes. (A) Four-quadrant graph exhibiting the DEGs containing differentially methylated m6A peaks in IvC. (B) Four-quadrant graph exhibiting the DEGs containing differentially methylated m6A peaks in TvI. (C) Major gene ontology terms were significantly enriched for the genes containing m6A sites with reverse methylation and expression trends between IvC and TvI. (D) Major enriched pathways for the genes containing m6A sites with reverse methylation and expression trends between IvC and TvI.

FIGURE 8

Representative genes with opposite m6A methylation and expression trends between IvC and TvI. (A) Visualization of m6A-modified genes FGA in the control group, injury group, and treatment group. (B) Visualization of m6A modified genes Slc12a1 in the control group, injury group, and treatment group. (C) Visualization of m6A-modified genes Havcr1 in the control group, injury group, and treatment group. (D) Western blot images of FGA, Slc12a1, and Havcr1 in control group, injury group, and Havcr1 group. Error bars represent the standard deviation. *p < 0.05, ***p < 0.001, Student’s t-test.