Int J Stem Cells.  2022 Aug;15(3):324-333. 10.15283/ijsc21147.

MiR-29a-3p Inhibits Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells via Targeting FOXO3 and Repressing Wnt/β-Catenin Signaling in Steroid-Associated Osteonecrosis

Affiliations
  • 1Department of Emergency and Trauma Surgery, People’s Hospital of Pingxiang, Pingxiang, China

Abstract

Background and Objectives
This study was to investigate the role of microRNA-29a-3p (miR-29a-3p) in human bone marrow mesenchymal stem cells (hBMSCs), and its relationship with steroid-associated osteonecrosis.
Methods and Results
The online tool GEO2R was used to screen out the differentially expressed genes (DEGs) in GSE123568 dataset. Quantitative real time-polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-29a-3p, forkhead box O3 (FOXO3), alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (OCN) and RUNX family transcription factor 2 (Runx2) in the hBMSCs isolated from the patients with steroid-associated osteonecrosis. CCK-8 assay was executed to measure cell viability; western blot assay was utilized to detect FOXO3, ALP, Runx2, OCN and β-catenin expression. Cell apoptosis and cell cycle were detected by flow cytometry. Immunofluorescence assay was used to detect the sub-cellular localization of β-catenin. Bioinformatics analysis and luciferase reporter gene assay were performed to confirm whether miR-29a-3p can combine with FOXO3 3’UTR. MiR-29a-3p was markedly up-regulated in the hBMSCs of patients with steroid-associated osteonecrosis, while FOXO3 mRNA was significantly down-regulated. Transfection of miR-29a-3p mimics significantly inhibited the hBMSCs’ proliferation, osteogenic differentiation markers’ expressions, including ALP, Runx2, OCN, and repressed the ALP activity, as well as promoted cell apoptosis and cell-cycle arrest. FOXO3 was identified as a target gene of miR-29a-3p, and miR-29a-3p can inhibit the expression of FOXO3 and β-catenin, and inhibition of miR-29a-3p promoted translocation of β-catenin to the nucleus.
Conclusions
MiR-29a-3p can modulate FOXO3 expression and Wnt/β-catenin signaling to inhibit viability and osteogenic differentiation of hBMSCs, thereby promoting the development of steroid-associated osteonecrosis.

Keyword

Steroid-associated osteonecrosis; miR-29a-3p; FOXO3; Wnt/β-catenin

Figure

  • Fig. 1 miR-29a-3p is significantly up-regulated and FOXO3 is significantly down-regulated in the hBMSCs isolated from SAON patients. (A) The volcano plot was used to show the DEGs in 30 SAON patients and 10 non-SAON individuals from the GSE123568. Genes whose expressions were significantly increased were marked in red, and those whose expressions were significantly decreased were marked in blue. (B, C) GO and KEGG enrichment analysis of the down-regulated DEGs in the GSE123568. (D) Venn diagram was used to screen the common miRNAs which could target FOXO3 among miRDB, miRTarase, miRwalk and Targetscan databases. (E) FOXO3 mRNA expression was detected by qRT-PCR in the hBMSCs from SAON patients and patients with fracture. (F) miR-29a-3p expression was measured by qRT-PCR in the hBMSCs from SAON patients and patients with fracture. (G) Correlation analysis of miR-29a-3p and FOXO3 mRNA in the hBMSCs from SAON patients. *p<0.05, ***p<0.001.

  • Fig. 2 miR-29a-3p inhibits cell proliferation and osteogenic differentiation of hBMSCs. (A) The expression of miR-29a-3p in hBMSCs transfected with miR-29a-3p mimics and miR-29a-3p inhibitors was detected by qRT-PCR. (B∼E) qRT-PCR and Western blot assay was used to detect Runx2, ALP and OCN mRNA and protein expression, markers of osteogenic differentiation, in hBMSCs transfected with miR-29a-3p mimics or miR-29a-3p inhibitors. (F) The ALP activity of hBMSCs transfected with miR-29a-3p mimics or miR-29a-3p inhibitors was measured. (G, H) Flow cytometry was used to detect the apoptosis of hBMSCs transfected with miR-29a-3p mimics or miR-29a-3p inhibitors. (I) CCK-8 assay was used to detect the viability of hBMSCs transfected with miR-29a-3p mimics or miR-29a-3p inhibitors. (J, K) Flow cytometry was used to detect the cell cycle of hBMSCs transfected with miR-29a-3p mimics or miR-29a-3p inhibitors. *p<0.05, **p<0.01, ***p<0.001.

  • Fig. 3 In hBMSCs, miR-29a-3p negatively regulates the expression of FOXO3. (A) Bioinformatics analysis revealed that the FOXO3 3’UTR contained a potential binding site complementary to miR-29a-3p. (B) Dual-luciferase reporter gene assay confirmed that miR-29a-3p could directly bind to FOXO3 3’UTR. (C, D) qRT-PCR and western blot assays were performed to detect the expression of FOXO3 mRNA and protein in hBMSCs transfected with miR-29a-3p mimics or miR-29a-3p inhibitors. *p<0.01, **p<0.001.

  • Fig. 4 FOXO3 overexpression plasmid reverses the inhibitory effect of miR-29a-3p on cell proliferation and osteogenic differentiation of hBMSCs. (A∼D) qRT-PCR and Western blot assay was performed to detect Runx2, ALP and OCN mRNA and protein expression, markers of osteogenic differentiation, in hBMSCs co-transfected with miR-29a-3p mimics and FOXO3 overexpression plasmids. (E) CCK-8 assay was used to detect the viability of hBMSCs after co-transfection with miR-29a-3p mimics and FOXO3 overexpression plasmids. (F) ALP activity in hBMSCs was measured after co-transfection with miR-29a-3p mimics and FOXO3 overexpression plasmids. (G, H) Flow cytometry was used to detect cell apoptosis and cell cycle in hBMSCs transfected with miR-29a-3p mimics and FOXO3 overexpression plasmids. *p<0.05, **p<0.01.

  • Fig. 5 miR-29a-3p regulates cell proliferation and osteogenic differentiation of hBMSCs through Wnt/β-catenin signaling. (A, B) Western blot assay was used to detect β-catenin protein expression in hBMSCs transfected with miR-29a-3p mimics or miR-29a-3p inhibitors. (C) Immunofluorescence assay was used to detect the subcellular expression of β-catenin. (D∼G) qRT-PCR and Western blot assay was used to detect Runx2, ALP and OCN mRNA and protein expression, markers of osteogenic differentiation, in hBMSCs transfected with miR-29a-3p inhibitors and treated with XAV939. (H) CCK-8 assay was used to detect proliferation in hBMSCs transfected with miR-29a-3p inhibitors and treated with XAV939. (I) The ALP activity of hBMSCs transfected with miR-29a-3p inhibitors and treated with XAV939 was detected. (J, K) Flow cytometry was used to detect the apoptosis and cell cycle of hBMSCs transfected with miR-29a-3p inhibitors and treated with XAV939. *p<0.05, **p<0.01, **p<0.001.


Reference

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