METTL3-mediated m6A modification of pri-miRNA-31 promotes hypertrophic scar progression

Abstract

Hypertrophic scar (HS) is a pathological scar characterized by excessive dermal fibrosis. Aberrant m6A modification patterns have been identified in HS; however, the expression of the methyltransferase, along with its function and molecular mechanisms in HS, remains unclear. In this study, we find that both the protein level of METTL3 and the level of m6A methylation are upregulated in HS compared with normal skin. To investigate the role of METTL3 in HS, we knock down METTL3 in HS-derived fibroblasts (HSFBs) via shRNA. METTL3 knockdown reduces the expressions of collagen types I and III (COL I/III) and α-SMA, inhibits cell proliferation and migration, and induces cell cycle arrest in the G1 phase. MeRIP-seq analysis reveals m6A modification sites on pri-miR-31. Our data indicate that the expression level of pri-miR-31 is elevated in METTL3-knockdown HSFBs, whereas the level of mature miR-31-5p is reduced. Notably, transfection of a miR-31-5p mimic into HSFBs partially counteracts the inhibitory effects of the m6A methylation inhibitors cycloleucine and STM2457 (a specific inhibitor of METTL3) on fibrosis and cellular proliferation. Additionally, we confirm that ZBTB20 is a downstream target of miR-31-5p and that knockdown of ZBTB20 inhibits fibroblast fibrosis. Collectively, our findings elucidate the epigenetic mechanism of METTL3/m6A/pri-miR-31/ZBTB20 in HS fibrosis, providing a potential therapeutic target for HS.

1. Introduction

Hypertrophic scar (HS) is a pathological scar characterized by abnormal dermal fibrosis [1]. HS can lead to symptoms such as pruritus and joint contracture [2]. The pathogenesis of HS involves excessive proliferation of fibroblasts, increased synthesis and deposition of collagen [3]. However, the molecular mechanisms underlying fibrosis in HS remain inadequately understood.

N6-methyladenosine (m6A) is the most abundant RNA modification in mammals [4]. This methylation is mediated by the methyltransferase complex, which is primarily composed of methyltransferase-like 14 (METTL14), METTL3 and Wilms tumor 1-associated protein (WTAP), and other components [5]. Previous studies have shown that m6A modification plays a crucial role in the development and progression of various diseases [6,7]. M6A modifications are found not only in mRNAs but also in non-coding RNAs, such as microRNAs (miRNAs) [8]. Liu et al. [9] found that alterations in m6A modification patterns during HS formation are associated with fibrosis-related pathways. However, the specific mechanisms by which m6A modifications regulate HS formation remain unclear.

MicroRNAs (miRNAs) are short, single-stranded non-coding RNA molecules consisting of 19 to 25 nucleotides [10]. Generally, miRNAs regulate the post-transcriptional silencing of target genes [10]. The maturation of miRNAs involves the processing of primary miRNAs (pri-miRNAs) by microprocessor complexes [11]. METTL3 participates in pri-miRNA processing and miRNA maturation through m6A modifications [12]. We previously demonstrated higher expression of hsa-miRNA-31-5p in HS tissues than in normal skin [13]. Here, both m6A modification levels and METTL3 expression were elevated in HS tissues compared with normal skin. METTL3 knockdown in HS fibroblasts reduced collagen synthesis and significantly affected cell proliferation and migration. Furthermore, METTL3 promoted the maturation of pri-miR-31 in HS fibroblasts in an m6A-dependent manner. Moreover, miR-31-5p modulated the functions of HS fibroblasts by silencing ZBTB20. Overall, this study reveals the potential role of METTL3 in the pathogenesis of HS and provides evidence that METTL3 promotes the maturation of pri-miR-31 via m6A modification.