Hypermethylation-mediated downregulation of long non-coding RNA MEG3 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells and promotes pediatric aplastic anemia.

BACKGROUND

The reduced osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is the typical characteristics of pediatric aplastic anemia (AA) pathogenesis. Long non-coding RNA MEG3 is reported to promote osteogenic differentiation of BMSCs via inducing BMP4 expression.

OBJECTIVE

This study aims to investigate the mechanism of DNMT1/MEG3/BMP4 pathway in osteogenic differentiation of BMSCs in pediatric AA.

METHODS

BMSCs were isolated and purified from bone marrows of pediatric AA patients (n = 5) and non-AA patients (n = 5). The expression of DNMT1, MEG3, and BMP4 in isolated BMSCs was detected using quantitative real-time PCR and western blot analysis. Osteogenic differentiation was determined using Alizarin red staining. The methylation of MEG3 promoter and the interaction between DNMT1 and MEG3 promoter were detected using methylation-specific PCR and chromatin immunoprecipitation assay, respectively.

RESULTS

Lowly expressed MEG3 and BMP4 and highly expressed DNMT1 were observed in BMSCs of pediatric AA patients. The overexpression of MEG3 promoted osteogenic differentiation of BMSCs. Luciferase reporter assay showed that MEG3 overexpression increased transcriptional activity of BMP4. The inhibitor of methylation, 5-azacytidine, suppressed DNMT1 expression and reduced methylation of MEG3 promoter. Overexpression of DNMT1 increased the binding between DNMT1 and MEG3 promoter. The simultaneous overexpression of DNMT1 and MEG3 restored the inhibition of osteogenic differentiation caused by DNMT1 overexpression alone.

CONCLUSIONS

Our findings indicated that DNMT1 mediated the hypermethylation of MEG3 promoter in BMSCs, and DNMT1/MEG3/BMP4 pathway modulated osteogenic differentiation of BMSCs in pediatric AA.