N6-methyladenosine (m6A) is a methylation modification of the nitrogen atom at position 6 of adenine, which is widely found in eukaryotes (Zhong et al., 2020). This dynamic, reversible biomolecule is regulated mainly by methyltransferases (writers), demethylases (erasers), and methylated binding proteins (readers) (Wei et al., 2022). The core proteins of methyltransferases catalyze m6A modification and include methyltransferase 3 (METTL3), METTL14, WT1-associated protein (WTAP), and vir like m6A methyltransferase associated (KIAA1492) (An and Duan, 2022). Demethylases include fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) (Petri and Klinge, 2023). Methylation binding proteins include the YT521-B homology (YTH) and heterogeneous nuclear ribonucleoprotein (hnRNP) families which recognize m6A methylation binding regions and regulate specific mRNA functions (Qin et al., 2020). m6A is implicated in the nuclear export, splicing, translation, and degradation of mRNA (Zhang et al., 2021). It also plays important roles in diseases of the immune system (Li et al., 2021), metabolism (Wang et al., 2022), cardiovascular system (Li et al., 2022), intestines (Nie et al., 2021), and eyes (Ni et al., 2023) and in tumorigenesis (Wu et al., 2022). m6A is also associated with the development of epithelial-mesenchymal transition (EMT). In chronic obstructive pulmonary disease (COPD), METTL3 knockdown upregulates SOCS3 protein in an m6A methylation-dependent manner and inhibits EMT via the STAT3/SNAI1 pathway (Zhang et al., 2023b). YTH domain-containing family protein 1 (YTHDF1) promotes protein synthesis and facilitates translation mechanisms (Wang et al., 2015) and is a potential prognostic marker for hepatocellular carcinoma (HCC). YTHDF1 downregulation inhibits HCC cell proliferation, migration, and invasion (Bian et al., 2020).

EMT is the loss of polarity in epithelial cells and their transformation into motile mesenchymal cells. EMT promotes proliferation, differentiation, migration, and invasion (Alsaab, 2023) and is involved in the onset and progression of fibrosis. The regulatory mechanism of the EMT network involves transforming growth factor-beta (TGF-β), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF), cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), and IL-6, extracellular matrix (ECM) including collagen and fibronectin (FN), transcription factors (TFs) such as Snail, Slug, and TWIST, endoplasmic reticulum (ER) stress, cellular autophagy (P62), epigenetic regulators including acetylation and phosphorylation, and non-coding RNAs such as microRNA (miRNA) and long noncoding RNA (lncRNA) (Zhao et al., 2022). m6A methylation in epigenetic regulators may partially explain the EMT mechanism. As EMT may be induced in cells in vitro, its relationship with m6A modification may be elucidated at the cellular level. TGF-β induces EMT and fibrosis, upregulates alpha-smooth muscle actin (α-SMA), and differentiates fibroblasts into myofibroblasts (Desmoulière et al., 1993). It also activates the ECM and upregulates collagen and FN (Lee and Massagué, 2022).

EMT plays an important role in the onset and progression of eye diseases. Proliferative vitreoretinopathy (PVR) is characterized by retinal fibrosis and, in severe cases, blindness (Idrees et al., 2019). Retinal pigment epithelium (RPE) cells undergoing EMT help form proliferative membranes. TGF-β induces EMT and promotes fibrosis in RPE cells (Li et al., 2020). The ARPE-19 and human telomerase reverse transcriptase (hTERT) RPE-1 cell lines have been used to study RPE-related retinal diseases. ARPE-19 cells were derived from the eyes of normal 19-year-old males whereas hTERT RPE-1 cells are human retinal pigment epithelium cells that were immortalized by editing hTERT. Proteomics studies revealed that ARPE-19 and hTERT RPE-1 significantly differ in terms of their expression levels of proteins involved in the biosynthesis of other proteins as well as cytoskeletal remodeling (Alge et al., 2006). Moreover, m6A methyltransferases (METTL3/METTL14) and demethylases (FTO) affect ARPE-19 cell proliferation, apoptosis, and EMT (Ma et al., 2021; Tang et al., 2022; Yin et al., 2022). m6A is associated with TGF-β-mediated EMT in ARPE-19 cells (Zhao et al., 2023a). However, it is uncertain whether m6A is implicated in TGF-β-induced EMT in hTERT RPE-1 cells.

Here, we extracted the RNA from RPE cells with or without TGF-β2 treatment and subjected them to m6A-Seq and RNA-Seq to identify their transcript-level differences in m6A modification and mRNA expression. We selected the EMT-related genes with altered m6A and mRNA levels. We then knocked down METTL3 and YTHDF1 and measured the EMT marker expression levels to disclose the mechanisms by which m6A methylation regulates TGF-β2-induced EMT in RPE cells.