Alterations of the m6A Methylation Induced by TGF-β2 in ARPE-19 Cells

Background: N ${}^{6}$ -methyladenosine (m ${}^{6}$ A) participates in diverse physiological processes and contributes to many pathological conditions. Epithelial-mesenchymal transition (EMT) of retinal pigmental epithelial (RPE) cells plays an essential role in retinal-related diseases, and transforming growth factor $\beta{}$ 2 (TGF- $\beta{}$ 2) is known to induce EMT in vitro. However, the effect of TGF- $\beta{}$ 2 on m ${}^{6}$ A methylation in RPE cells is not yet known. Methods: RNA-seq and MeRIP-seq were performed to analyze changes at the mRNA and m ${}^{6}$ A levels after TGF- $\beta{}$ 2 treatment of human ARPE-19 cells. mRNA levels and total m ${}^{6}$ A levels were subsequently validated. Results: Sequencing revealed 929 differentially expressed genes and 7328 differentially methylated genes after TGF- $\beta{}$ 2 treatment. Conjoint analysis identified 290 genes related to microtubule cytoskeleton, focal adhesion, ECM-receptor interaction, cell division, cell cycle, AGE-RAGE, PI3K-Akt and cGMP-PKG pathways. Further analysis revealed that 12 EMT-related genes were altered at the mRNA and m ${}^{6}$ A levels after TGF- $\beta{}$ 2 treatment (CALD1, CDH2, FN1, MMP2, SPARC, KRT7, CLDN3, ELF3, FGF1, LOXL2, SHROOM3 and TGFBI). Moreover, the total m ${}^{6}$ A level was also reduced. Conclusions: This study revealed the transcriptional profiling of m ${}^{6}$ A modification induced by TGF- $\beta{}$ 2 in RPE cells. Novel connections were discovered between m ${}^{6}$ A modification and TGF- $\beta{}$ 2-induced EMT, suggesting that m ${}^{6}$ A may play crucial roles in the EMT process.

Keywords

m⁶A methylation

RPE cells

EMT

TGF-β2

transcriptomics

MeRIP-seq

Funding

81770952/National Natural Science Foundation of China

81873681/National Natural Science Foundation of China

20JCQN005/Basic Research Program of Henan Eye Hospital

222300420194/Natural Science Foundation of Henan Province

LHGJ20210075/Henan Provincial Medical Science and Technology Research Joint Co-construction Project

Figures

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Fig. 1.

Analysis of DEGs induced by TGF- $\beta{}$ 2 in ARPE-19 cells. (A) PCA clustering map of the gene expression profile in each group. (B) Volcano plot analysis of DEGs. Blue dots represent down-regulated genes, grey dots represent genes with no difference in expression, and red dots represent up-regulated genes. (C) Number of DEGs with increased or decreased expression. (D) Heatmap analysis of DEGs. Red indicates hyper-expressed genes and blue indicates hypo-expressed genes. (E) GO enrichment analysis of DEGs. (F) KEGG enrichment analysis of DEGs. The dot size indicates the number of genes enriched, and the dot color indicates significant enrichment.

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Front. Biosci. (Landmark Ed) Print ISSN 2768-6701 Electronic ISSN 2768-6698