METTL3-Mediated lncRNA m6A Modification in the Osteogenic Differentiation of Human Adipose-Derived Stem Cells Induced by NEL-Like 1 Protein
METTL3-Mediated lncRNA m6A Modification in the Osteogenic Differentiation of Human Adipose-Derived Stem Cells Induced by NEL-Like 1 Protein
复制标题
METTL3介导的lncRNA m6A修饰对NEL-like 1蛋白诱导的人脂肪干细胞成骨分化的影响
DOI：
10.1007/s12015-021-10245-4
复制
发表时间：
2021-09-10
影响因子：
4.8
通讯作者：
Liu, Jun
中科院分区：
文献类型：
--
作者：
Song, Yidan;Pan, Yihua;Liu, Jun
目的探讨甲基转移酶3（Methyltransferase 3，L3）介导的长链非编码RNA（longnoncodingRNA，lncRNA）N6-甲基腺苷（m6 A）修饰在NEL样1蛋白（NELL-1）诱导人脂肪干细胞（humanadipose-derivedstemcells，hASCs）成骨分化中的调控机制。对hASC进行MeRIP-seq测序和RNA的高通量测序（RNA-seq）。通过碱性磷酸酶（ALP）染色、茜素红S（ARS）染色、ALP定量和实时定量聚合酶链反应（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，并鉴定了靶lncRNA。在过表达和敲低胃L3后，使用甲基化RNA免疫沉淀-qPCR（MeRIP-qPCR）和qRT-PCR检测m6 A修饰的水平和靶lncRNA的表达，并通过RNA结合蛋白免疫沉淀（RIP）测定证实两者的结合。结果体外实验表明，胃L3能促进成骨分化，其表达水平上调。KEGG通路分析预测，具有差异上调的甲基化峰的lncRNA主要富集在促分裂原活化蛋白激酶（MAPK）信号通路中，其中丝氨酸/苏氨酸蛋白激酶3（STK 3）是lncRNA RP 11 -44 N12.5的预测靶基因。m6 A修饰和RP 11 -44 N12.5的表达均受胃L3调节。随后发现lncRNA RP 11 -44 N12.5和胃L3可调节MAPK信号通路中三个关键蛋白ERK、JNK和p38的磷酸化水平。结论本研究首次发现胃L3可通过调节m6 A修饰和lncRNA的表达激活MAPK信号通路，从而促进hASCs的成骨分化。
ObjectivesThis 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 MethodsMethylated 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.ResultsIn 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.ConclusionsThis 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