YTHDF2-Mediated m6A methylation inhibition by miR27a as a protective mechanism against hormonal osteonecrosis in BMSCs

Abstract

Abstract Background With the increasing incidence of steroid-induced necrosis of the femoral head (SNFH), numerous scholars have investigated its pathogenesis. Current evidence suggests that the imbalance between lipogenesis and osteoblast differentiation in bone marrow mesenchymal stem cells (BMSCs) is a key pathological feature of SNFH. MicroRNAs (miRNAs) have strong gene regulatory effects and can influence the direction of cell differentiation. N6-methyladenosine (m6A) is a prevalent epigenetic modification involved in diverse pathophysiological processes. However, knowledge of how miRNAs regulate m6A-related factors that affect BMSC differentiation is limited. Objective We aimed to investigate the role of miR27a in regulating the expression of YTHDF2 in BMSCs. Methods We compared miR27a, YTHDF2, and total m6A mRNA levels in SNFH-affected and control BMSCs. CCK-8 and TUNEL assays were used to assess BMSC proliferation and apoptosis. Western blotting and qRT‒PCR were used to measure the expression of osteogenic (ALP, RUNX2, and OCN) and lipogenic (PPARγ and C/EBPα) markers. Alizarin Red and Oil Red O staining were used to quantify osteogenic and lipogenic differentiation, respectively. miR27a was knocked down or overexpressed to evaluate its impact on BMSC differentiation and its relationship with YTHDF2. Bioinformatics analyses identified YTHDF2 as a differentially expressed gene in SNFH (ROC analysis) and revealed potential signaling pathways through GSEA. The effects of YTHDF2 silencing on the lipogenic and osteogenic functions of BMSCs were assessed. Results miR27a downregulation and YTHDF2 upregulation were observed in the SNFH BMSCs. miR27a knockdown/overexpression modulated YTHDF2 expression, impacting BMSC differentiation. miR27a silencing decreased m6A methylation and promoted osteogenic differentiation, while YTHDF2 silencing exerted similar effects. GSEA suggested potential signaling pathways associated with YTHDF2 in SNFH. Conclusion miR27a regulates BMSC differentiation through YTHDF2, affecting m6A methylation and promoting osteogenesis. This finding suggests a potential therapeutic target for SNFH.

Keywords

• Steroid-induced necrosis of the femoral head
• Bone marrow mesenchymal stem cells
• miRNA
• N6-methyladenosine