Comprehensive Analysis of the Transcriptome-Wide m6A Methylome in Pterygium by MeRIP Sequencing

Abstract

Aim: Pterygium is a common ocular surface disease, which is affected by a variety of factors. Invasion of the cornea can cause severe vision loss. N6-methyladenosine (m6A) is a common post-transcriptional modification of eukaryotic mRNA, which can regulate mRNA splicing, stability, nuclear transport, and translation. To our best knowledge, there is no current research on the mechanism of m6A in pterygium.

Methods: We obtained 24 pterygium tissues and 24 conjunctival tissues from each of 24 pterygium patients recruited from Shanghai Yangpu Hospital, and the level of m6A modification was detected using an m6A RNA Methylation Quantification Kit. Expression and location of METTL3, a key m6A methyltransferase, were identified by immunostaining. Then we used m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq), and bioinformatics analyses to compare the differential expression of m6A methylation in pterygium and normal conjunctival tissue.

Results: We identified 2,949 dysregulated m6A peaks in pterygium tissue, of which 2,145 were significantly upregulated and 804 were significantly downregulated. The altered m6A peak of genes were found to play a key role in the Hippo signaling pathway and endocytosis. Joint analyses of MeRIP-seq and RNA-seq data identified 72 hypermethylated m6A peaks and 15 hypomethylated m6A peaks in mRNA. After analyzing the differentially methylated m6A peaks and synchronously differentially expressed genes, we searched the Gene Expression Omnibus database and identified five genes related to the development of pterygium (DSP, MXRA5, ARHGAP35, TMEM43, and OLFML2A).

Conclusion: Our research shows that m6A modification plays an important role in the development of pterygium and can be used as a potential new target for the treatment of pterygium in the future.

Keywords: GEO; Hippo signaling pathway; MeRIP sequencing; m6A; methyltransferase 3; pterygium.

FIGURE 1

Workflow of the study. (A) Workflow of the experimental process. (B) Workflow of the data analysis.

FIGURE 2

m6A and METTL3 are downregulated in pterygium. (A) Quantification of m6A in total RNA in pterygium and conjunctiva tissues. N = 12 per group. (B) mRNA expression level of the major enzymes of m6A in pterygium and conjunctiva tissues: METTL3, METTL14, WTAP, FTO, ALKBH5, YTHDF1. N = 12 per group. (C,D) Protein expression levels of these enzymes determined via Western blotting (C,D) the results of corresponding densitometric analyses. N = 12 per group. (E) Immunohistochemistry staining of METTL3 in pterygium and conjunctiva tissues. Scale bar: 25 μm. Results are expressed as the mean ± SEM (NS indicates not significant, **P < 0.01, ***P < 0.001, compared to the conjunctiva group).

FIGURE 3

Overview of altered m6A-modified transcripts in pterygium. (A) Volcano plots showing significantly different m6A peaks. (B) Average m6A peaks in conjunctiva and pterygium. (C,D) Pie charts showing the distribution of m6A peaks in conjunctiva (C) and pterygium (D). (E) Top five m6A motifs from the altered m6A peaks in pterygium. (F) Distribution of altered m6A peaks per mRNA. (G) Distribution of altered m6A peaks per gene. (H) Distribution of altered m6A peaks in human chromosomes. Fold change ≥2 and P < 0.05.

FIGURE 4

Systematical analyses of differentially methylated mRNA in pterygium. (A) Gene Ontology (GO) processes analysis of differentially methylated lncRNA. (B) Top three scored networks analysis of differentially methylated lncRNA. (C) Pathway maps analysis of differentially methylated peaks. Left panel represents hypermethylated genes and right panel represents hypomethylated peaks. (D) GO process analysis of differentially methylated peaks. Left panel represents hypermethylated peaks and right panel represents hypomethylated peaks. (E) Top three scored networks analysis of differentially methylated peaks. Left panel represents hypermethylated genes and right panel represents hypomethylated peaks.

FIGURE 5

KEGG enrichment analyses of differentially methylated mRNA in pterygium. Top 10 KEGG pathways of genes with (A) upregulated m6A peaks and (B) downregulated m6A peaks. Squares in left semicircle refer to the upregulated or downregulated mRNAs, the squares in right semicircle refer to the pathways. *P < 0.05, **P < 0.01, ***P < 0.001, genes compared to pathways.

FIGURE 6

Results of significant RNA of pterygium in RNA-seq data. (A) Volcano plots and (B) heatmap plots showing differentially expressed genes in pterygium vs. conjunctiva. Fold change ≥2 and P < 0.05. (C) GO processes and pathway maps analysis of significant lncRNA. (D) Top three scored networks analysis of significant lncRNA. (E) Pathway maps, process networks and GO processes analysis of significant mRNA. Left panel represents pathway maps, middle panel represents process networks and right panel represents GO processes.

FIGURE 7

Systematical analyses of MeRIP-seq and RNA-seq data in pterygium. (A) Significantly m6A-modified RNA identified after conjoint analyses of MeRIP-seq and RNA-seq data. (B) Bubble diagram of GO biological process categories enriched for DEGs with m6A hyper- or hypo-methylated. (C) Pathway maps analysis (left) and Process networks analysis (right) of differentially m6A-modifed genes. (D) Top three scored networks analysis of m6A-modified genes. (E) Key transcription factors and target genes network analysis of m6A-modifed genes. DEGs, differentially expressed genes.

FIGURE 8

Validation gene expression in GEO datasets. (A,B) The expression of genes from GEO datasets (GSE2513, GSE51995, and GSE83627) were normalized to remove batch effects. (C) Wayne chart of the three GEO datasets. (D) mRNA expression level of five hub genes (DSP, MXRA5, TMEM43, OLFML2A, and ARHGAP35) determined via qPCR. N = 6 per group. Results are expressed as the mean ± SEM (NS indicates not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the conjunctiva group).