YTHDF2 介导的 miR27a 通过 m6A 甲基化抑制作为 BMSCs 中激素性骨坏死的保护机制。

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

机构信息

The Fifth Clinical Medical College of Xinjiang Medical University, Xinjiang Uygur Autonomous Region, Urumqi, 830011, China.

Department of Orthopedic Center, The Fifth Affiliated Hospital of Xinjiang Medical University, Xinjiang Uygur Autonomous Region, Urumqi, 830011, China.

出版信息

BMC Musculoskelet Disord. 2024 May 6;25(1):359. doi: 10.1186/s12891-024-07481-3.

DOI:10.1186/s12891-024-07481-3

PMID:38711079

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11071322/

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.

摘要

背景

随着类固醇诱导性股骨头坏死（SNFH）发病率的增加，许多学者对其发病机制进行了研究。目前的证据表明，骨髓间充质干细胞（BMSCs）中脂肪生成和成骨细胞分化之间的失衡是 SNFH 的一个关键病理特征。微小 RNA（miRNA）具有很强的基因调控作用，可以影响细胞分化的方向。N6-甲基腺苷（m6A）是一种普遍存在的表观遗传修饰，参与多种病理生理过程。然而，miRNA 如何调节影响 BMSC 分化的 m6A 相关因子的知识还很有限。

目的

本研究旨在探讨 miR27a 调节 BMSCs 中 YTHDF2 表达的作用。

方法

比较 SNFH 相关 BMSCs 和对照 BMSCs 中 miR27a、YTHDF2 和总 m6A mRNA 水平。CCK-8 和 TUNEL 检测用于评估 BMSC 增殖和凋亡。Western blot 和 qRT-PCR 用于测量成骨（ALP、RUNX2 和 OCN）和脂肪生成（PPARγ 和 C/EBPα）标志物的表达。茜素红和油红 O 染色分别用于定量成骨和脂肪生成分化。敲低或过表达 miR27a 以评估其对 BMSC 分化的影响及其与 YTHDF2 的关系。生物信息学分析鉴定出 YTHDF2 是 SNFH 中差异表达的基因（ROC 分析），并通过 GSEA 揭示了潜在的信号通路。评估 YTHDF2 沉默对 BMSCs 脂肪生成和成骨功能的影响。

结果

在 SNFH BMSCs 中观察到 miR27a 下调和 YTHDF2 上调。miR27a 敲低/过表达调节 YTHDF2 表达，影响 BMSC 分化。miR27a 沉默降低 m6A 甲基化并促进成骨分化，而 YTHDF2 沉默产生类似的效果。GSEA 表明 YTHDF2 在 SNFH 中与潜在信号通路相关。