当前位置: X-MOL 学术Stem Cell Rev and Rep. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
METTL3-Mediated lncRNA m6A Modification in the Osteogenic Differentiation of Human Adipose-Derived Stem Cells Induced by NEL-Like 1 Protein
Stem Cell Reviews and Reports ( IF 4.8 ) Pub Date : 2021-09-10 , DOI: 10.1007/s12015-021-10245-4
Yidan Song 1 , Yihua Pan 1 , Mengsong Wu 1 , Wentian Sun 1 , Liangyu Luo 1 , Zhihe Zhao 1 , Jun Liu 1
Affiliation  

Objectives

This study aimed to explore the regulatory mechanism of methyltransferase3 (METTL3) -mediated long non-coding RNA (lncRNA) N6-methyladenosine (m6A) modification in the osteogenic differentiation of human adipose-derived stem cells (hASCs) induced by NEL-like 1 protein (NELL-1).

Materials and Methods

Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and high- throughput sequencing for RNA (RNA-seq) were performed on hASCs. Osteogenic ability was detected by alkaline phosphatase (ALP) staining, Alizarin Red S(ARS) staining, ALP quantification and Quantitative real-time polymerase chain reaction analysis (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis predicted the osteogenesis-related pathways enriched for the lncRNAs and identified the target lncRNAs. After overexpression and knockdown of METTL3, methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) and qRT-PCR were used to detect the levels of m6A modification and the expression of the target lncRNA, and the binding of both was confirmed by RNA binding protein immunoprecipitation (RIP) assay. The effects of lncRNA and METTL3 on phosphorylation of the key proteins of the pathway were detected by western blot analysis.

Results

In vitro experiments showed that METTL3 can promote osteogenic differentiation and that its expression level is upregulated. KEGG pathway analysis predicted that lncRNAs with differentially upregulated methylated peaks were enriched mostly in the mitogen-activated protein kinase (MAPK) signaling pathway, in which Serine/threonine protein kinase 3 (STK3) was the predicted target gene of the lncRNA RP11-44 N12.5. The m6A modification and expression of RP11-44 N12.5 were both regulated by METTL3. Subsequently, lncRNA RP11-44 N12.5 and METTL3 were found to regulate the phosphorylation levels of three key proteins in the MAPK signaling pathway, ERK, JNK and p38.

Conclusions

This study shows, for the first time, that METTL3 can activate the MAPK signaling pathway by regulating the m6A modification and expression of a lncRNA, thereby enhancing the osteogenic differentiation of hASCs.

Graphical abstract



中文翻译:

METTL3 介导的 lncRNA m6A 修饰在 NEL 样 1 蛋白诱导的人脂肪源性干细胞成骨分化中的作用

目标

本研究旨在探讨甲基转移酶3(METTL3)介导的长链非编码RNA(lncRNA)N6-甲基腺苷(m 6 A)修饰在NEL-诱导的人脂肪干细胞(hASCs)成骨分化中的调控机制。像 1 种蛋白质 (NELL-1)。

材料和方法

在 hASC 上进行甲基化 RNA 免疫沉淀测序 (MeRIP-seq) 和 RNA 高通量测序 (RNA-seq)。通过碱性磷酸酶 (ALP) 染色、茜素红 S(ARS) 染色、ALP 定量和定量实时聚合酶链反应分析 (qRT-PCR) 检测成骨能力。京都基因和基因组百科全书 (KEGG) 通路分析预测了富含 lncRNA 的成骨相关通路并确定了目标 lncRNA。在 METTL3 过表达和敲低后,使用甲基化 RNA 免疫沉淀-qPCR (MeRIP-qPCR) 和 qRT-PCR 检测 m 6的水平通过 RNA 结合蛋白免疫沉淀 (RIP) 测定证实了靶 lncRNA 的修饰和表达,以及两者的结合。通过蛋白质印迹分析检测lncRNA和METTL3对通路关键蛋白磷酸化的影响。

结果

体外实验表明,METTL3可以促进成骨分化,并且其表达水平上调。KEGG通路分析预测,甲基化峰差异上调的lncRNA主要富集在丝裂原活化蛋白激酶(MAPK)信号通路中,其中丝氨酸/苏氨酸蛋白激酶3(STK3)是lncRNA RP11-44 N12的预测靶基因.5. RP11-44 N12.5的m 6 A修饰和表达均受METTL3调控。随后,发现lncRNA RP11-44 N12.5和METTL3调节MAPK信号通路中三种关键蛋白ERK、JNK和p38的磷酸化水平。

结论

本研究首次表明METTL3可以通过调节m 6 A修饰和lncRNA的表达来激活MAPK信号通路,从而增强hASCs的成骨分化。

图形概要

更新日期:2021-09-10
down
wechat
bug