N6-methyladenosine methylation analysis of circRNAs in acquired middle ear cholesteatoma

Abstract

Introduction: Middle ear cholesteatoma is a chronic middle ear disease characterized by severe hearing loss and adjacent bone erosion, resulting in numerous complications. This study sought to identify pathways involved in N6-methyladenosine (m6A) modification of circRNA in middle ear cholesteatoma.

Methods: A m6A circRNA epitranscriptomic microarray analysis was performed in middle ear cholesteatoma tissues (n = 5) and normal post-auricular skin samples (n = 5). Bioinformatics analyses subsequently explored the biological functions (Gene Ontology, GO) and signaling pathways (Kyoto Encyclopedia of Genes and Genomes, KEGG) underlying middle ear cholesteatoma pathogenesis. Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR) was performed to verify the presence of circRNAs with m6A modifications in middle ear cholesteatoma and normal skin samples.

Results: Microarray analysis identified 3,755 circRNAs as significantly differentially modified by m6A methylation in middle ear cholesteatoma compared with the normal post-auricular skin. Among these, 3,742 were hypermethylated (FC ≥ 2, FDR < 0.05) and 13 were hypomethylated (FC ≤ 1/2, FDR < 0.05). GO analysis terms with the highest enrichment score were localization, cytoplasm, and ATP-dependent activity for biological processes, cellular components, and molecular functions respectively. Of the eight hypermethylated circRNA pathways, RNA degradation pathway has the highest enrichment score. Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway was hypomethylated. To validate the microarray analysis, we conducted MeRIP-qPCR to assess the methylation levels of five specific m6A-modified circRNAs: hsa_circRNA_061554, hsa_circRNA_001454, hsa_circRNA_031526, hsa_circRNA_100833, and hsa_circRNA_022382. The validation was highly consistent with the findings from the microarray analysis.

Conclusion: Our study firstly presents m6A modification patterns of circRNAs in middle ear cholesteatoma. This finding suggests a direction for circRNA m6A modification research in the etiology of cholesteatoma and provides potential therapeutic targets for the treatment of middle ear cholesteatoma.

Keywords: circRNA-miRNA-mRNA network; circular RNAs (circRNAs); m6A modification; microarray analysis; middle ear cholesteatoma.

FIGURE 1

Expression profiles of significantly altered circRNAs in middle ear cholesteatoma (C) and normal skin (S). (A) The heatmap reveals distinct circRNA methylation patterns between middle ear cholesteatoma and normal skin. The red and blue lines represent middle ear cholesteatoma (C) and normal skin (S), respectively. Each group includes five samples. (B) CircRNAs differentially modified by m6A methylation in middle ear cholesteatoma and normal post-auricular skin, according to the volcano diagram analysis. The red and blue dots represent significantly hyper- and hypomethylated circRNAs (fold change ≥2 or 1/2, FDR < 0.05).

FIGURE 2

GO analysis of circRNAs differentially modified by m6A methylation. (A) Top 10 significantly differentially modified circRNA-enriched GO items. (B) Biological process. (C) Cellular component. (D) Molecular function.

FIGURE 3

Pathway analysis based on the KEGG was conducted to explore the functional implications of circRNAs differentially modified by m6A methylation. (A,B) highlight pathways enriched with significantly hypermethylated circRNAs, while (C,D) depict the pathway enriched with significantly hypomethylated circRNAs.

FIGURE 4

m6A methylation levels of circRNAs verified by MeRIP-qPCR. We selected five differentially modified circRNAs with m6A methylation. Hsa_circRNA_061554, hsa_circRNA_001454, and hsa_circRNA_031526 were significantly hypermethylated in cholesteatoma, while the m6A methylation levels of hsa_circRNA_100833 and hsa_circRNA_022382 were significantly downregulated. Data are presented as mean ± SEM. ***p < 0.001, unpaired t-test.

FIGURE 5

According to the ceRNA hypothesis, a network consisting of circRNA-miRNA-mRNA was established. This network is built upon five validated m6A-modified circRNAs. In the network, microRNAs, protein coding RNAs, noncoding RNAs, and circRNAs are represented by nodes colored in red, light blue, light green, and brown respectively. Directed relationships are depicted by edges with a T-shaped arrow, while undirected relationships are shown by edges without arrows.