2. Academic Validation
  3. METTL3-mediated long non-coding RNA MIR99AHG methylation targets miR-4660 to promote bone marrow mesenchymal stem cell osteogenic differentiation

METTL3-mediated long non-coding RNA MIR99AHG methylation targets miR-4660 to promote bone marrow mesenchymal stem cell osteogenic differentiation

  • Cell Cycle. 2022 Nov 12;1-18. doi: 10.1080/15384101.2022.2125751.
Lintao Li 1,  Beiyue Wang 1,  Xing Zhou 1,  Hao Ding 1,  Chang Sun 1,  Yicun Wang 1,  Fan Zhang 2,  Jianning Zhao 1
Affiliations

Affiliations

  • 1 Department of Orthopedic, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing, China.
  • 2 Department of Orthopaedic, Changzheng Hospital, Navy Military Medical University, Shanghai, China.
PMID: 36369887 DOI: 10.1080/15384101.2022.2125751
Abstract

Whether long non-coding RNA Mir-99a-Let-7c Cluster Host Gene (LncRNA MIR99AHG) is involved in osteoporosis (OP) remains vague, so we hereby center on its implication. Old C57BL/6J mice were injected with the silencing lentivirus of MIR99AHG and subjected to microCT analysis and immunohistochemistry on osteogenic cells. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) with or without transfection was determined by Alkaline Phosphatase (ALP) and Alizarin Red S staining. Total N(6)-methyladenosine (m6A) on the bone marrow mesenchymal stem cells (BMSCs) was quantified. The potential methylation site and the complementary binding sites with candidate MicroRNA (miR) were predicted via bioinformatic analyses, with the latter being confirmed via dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Quantitative Real-Time PCR and Western blot were used for quantification assays. MIR99AHG was decreased during the osteogenic differentiation of BMSCs, where increased Osterix (OSX), Collagen, Type I, Alpha 1 (Col1A1), Osteocalcin (OCN) and RUNX Family Transcription Factor 2 (RUNX2) as well as more color-stained areas were found. Also, silencing MIR99AHG relieved the OP in mice and reduced the loss of osteogenic cells. M6A methylation in undifferentiated BMSCs was low and MIR99AHG overexpression abolished the effects of overexpressed METTL3 on promoting osteogenic differentiation. MiR-4660, which was downregulated in BMSCs without differentiation but increased during osteogenic differentiation, could bind with MIR99AHG. Furthermore, miR-4660 promoted osteogenic differentiation and reversed the effects of overexpressed MIR99AHG. The present study demonstrated that METTL3-mediated LncRNA MIR99AHG methylation enhanced the osteogenic differentiation of BMSCs via targeting miR-4660.

Keywords

Mir-99a-Let-7c cluster host gene; Osteoporosis; bone marrow mesenchymal stem cells; microRNA-4660; osteogenic differentiation.

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