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METTL3-Mediated lncRNA m6A Modification in the Osteogenic Differentiation of Human Adipose-Derived Stem Cells Induced by NEL-Like 1 Protein

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An Author Correction to this article was published on 16 October 2021

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Abstract

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.

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Data Availability

All data generated or analysed during this study are included in this article and its supplementary materials.

Change history

Abbreviations

lncRNA:

long non-coding RNA

m6A:

N6-methyladenosine

hASCs:

human adipose-derived stem cells

NELL-1:

NEL-like 1 protein

BMSCs:

bone marrow-derived mesenchymal stem cells

TGF:

transforming growth factor

RUNX2:

Runt-related transcription factor 2

BSP:

bone sialoprotein

METTL14:

methyltransferase 14

METTL3:

methyltransferase 3

FTO:

fat-mass and obesity-associated protein

ALKBH5:

ALKB homologue 5

ERK:

extracellular signal-regulated kinase

MAPK:

mitogen-activated protein kinase

JNK:

c-Jun N-terminal kinase

STK3:

Serine/threonine protein kinase 3

GCK II:

germinal centre kinase II

ALP:

alkaline phosphatase

ARS:

alizarin red

qRT-PCR:

quantitative real-time polymerase chain reaction analysis

KEGG:

Kyoto Encyclopedia of Genes and Genomes

RIP:

RNA binding protein immunoprecipitation

NFIC:

nuclear factor I-C

MeRIP-seq:

methylated RNA immunoprecipitation sequencing

MeRIP-qPCR:

methylated RNA immunoprecipitation-qPCR

RNA-seq:

high- throughput sequencing for RNA

ncRNAs:

noncoding RNAs

dhASC:

differentiated hASC

NELL1-dhASC:

rhNELL-1-induced osteogenic differentiated hASC

IPP:

immunoprecipitation

DREME:

Discriminative Regular Expression Motif Elicitation

PBS:

phosphate buffered saline

sodium dodecyl sulfate-polyacrylamide gel electrophoresis:

SDS-PAGE

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Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (81870743, 82170934 and 81771048). And we appreciated Cloud-seq Company (Shanghai, China) that provided MeRIP-Seq and lncRNA sequencing service.

Code Availability

Not applicable.

Funding

National Natural Science Foundation of China (81870743, 82170934 and 81771048).

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Authors and Affiliations

  1. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, Sichuan, China

    Yidan Song, Yihua Pan, Mengsong Wu, Wentian Sun, Liangyu Luo, Zhihe Zhao & Jun Liu

Authors
  1. Yidan Song
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  2. Yihua Pan
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  3. Mengsong Wu
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  4. Wentian Sun
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  7. Jun Liu
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Contributions

Yidan Song and Yihua Pan mainly carried out the experimental operations. Yidan Song, Mengsong Wu, Wentian Sun and Liangyu Luo analyzed and interpreted the data. Yidan Song wrote the manuscript. Jun Liu and Zhihe Zhao designed the experiments and revised the manuscript.

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Correspondence to Jun Liu.

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Song, Y., Pan, Y., Wu, M. et al. METTL3-Mediated lncRNA m6A Modification in the Osteogenic Differentiation of Human Adipose-Derived Stem Cells Induced by NEL-Like 1 Protein. Stem Cell Rev and Rep 17, 2276–2290 (2021). https://doi.org/10.1007/s12015-021-10245-4

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  • DOI: https://doi.org/10.1007/s12015-021-10245-4

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