Hmga2 knockdown enhances osteogenic differentiation of adipose-derived mesenchymal stem cells and accelerates bone defect healing in mice

doi:10.12122/j.issn.1673-4254.2024.07.02

Abstract

Abstract:

Objective To investigate the role of high-mobility group AT-hook 2 (HMGA2) in osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) and the effect of Hmga2 knockdown for promoting bone defect repair. Methods Bioinformatics studies using the GEO database and Rstudio software identified HMGA2 as a key factor in adipogenic-osteogenic differentiation balance of ADSCs. The protein-protein interaction network of HMGA2 in osteogenic differentiation was mapped using String and visualized with Cytoscape to predict the downstream targets of HMGA2. Primary mouse ADSCs (mADSCs) were transfected with Hmga2 siRNA, and the changes in osteogenic differentiation of the cells were evaluated using alkaline phosphatase staining and Alizarin red S staining. The expressions of osteogenic markers Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcein (OCN) in the transfected cells were detected using RT-qPCR and Western blotting. In a mouse model of critical-sized calvarial defects, mADSCs with Hmga2-knockdown were transplanted into the defect, and bone repair was evaluated 6 weeks later using micro-CT scanning and histological staining. Results GEO database analysis showed that HMGA2 expression was upregulated during adipogenic differentiation of ADSCs. Protein-protein interaction network analysis suggested that the potential HMGA2 targets in osteogenic differentiation of ADSCs included SMAD7, CDH1, CDH2, SNAI1, SMAD9, IGF2BP3, and ALDH1A1. In mADSCs, Hmga2 knockdown significantly upregulated the expressions of RUNX2, OPN, and OCN and increased cellular alkaline phosphatase activity and calcium deposition. In a critical-sized calvarial defect model, transplantation of mADSCswith Hmga2 knockdown significantly promoted new bone formation. Conclusion HMGA2 is a crucial regulator of osteogenic differentiation in ADSCs, and Hmga2 knockdown significantly promotes osteogenic differentiation of ADSCs and accelerates ADSCs-mediated bone defect repair in mice.

Key words: adipose-derived mesenchymal stem cells, high-mobility group AT-hook 2, osteogenic differentiation, bone defect healing

Zhiyong KE, Zicheng HUANG, Ruolin HE, Qian ZHANG, Sixu CHEN, Zhong-Kai CUI, Jing DING. Hmga2 knockdown enhances osteogenic differentiation of adipose-derived mesenchymal stem cells and accelerates bone defect healing in mice[J]. Journal of Southern Medical University, 2024, 44(7): 1227-1235.

Figures/Tables 9

Tab.1 Primer sequences for RT-qPCR

Gene		Sequence
Hmga2	Forward	CCGGTAGAGGCAGTGGTAGC
Hmga2	Reverse	GGTTGTTCCCTGGGCTGATGT
Alp	Forward	GAGCAGGAACAGAAGTTTGC
Alp	Reverse	GTTGCAGGGTCTGGAGAGTA
Osx	Forward	GCCGCTTTGTGCCTTTGAAATG
Osx	Reverse	CGTTATGCTCTTCCCAGACTCC
Runx2	Forward	CCGCACGACAACCGCACCAT
Runx2	Reverse	CGCTCCGGCCCACAAATCTC
Opn	Forward	CCCTCGATGTCATCCCTGTT
Opn	Reverse	CCCTTTCCGTTGTTGTCCTG
Ocn	Forward	AGCTCAACCCCAATTGTGAC
Ocn	Reverse	AGCTGTGCCGTCCATACTTT
Dlk1	Forward	GCGGGAACGCAACAACATC
Dlk1	Reverse	GTCACTGGTCAACTCCAGCAC
Pparγ	Forward	GTGATGGAAGACCACTCGCATT
Pparγ	Reverse	CCATGAGGGAGTTAGAAGGTTC

Fig.1 Differential gene expression profile during adipogenic differentiation of human ADSCs. A: Schematic illustration of the balance between adipogenic and osteogenic differentiation. B: Venn plot of up-regulated genes in the GSE175624 and GSE125331 datasets. C: Bubble chart of GO pathway enrichment analysis of the differentially expressed genes. D: Heatmap of the differentially expressed genes under the "fat cell differentiation" term. E: Expression of HMGA2 in GSE175614 dataset.

Fig.2 Hmga2 knockdown promotes osteogenic differentiation of mouse ADSCs in vitro. A: si-Hmga2 inhibits Hmga2 mRNA expression in ADSCs. B-E: ALP staining and activity assay of mouse ADSCs on day 3 (B, C) and 7 (D, E) of osteogenic induction. F, G: ARS staining and quantification of calcium deposition in ADSCs on day 14 of osteogenic induction. *P<0.05, ***P<0.001 vs si-NC group. Scale bar: 500 μm.

Fig.3 Effects of Hmga2 knockdown on expressions of osteogenic and adipogenic markers of ADSCs. A-C: Relative expression levels of Osx, Pparγ, and Dlk1 mRNAs in ADSCs on day 3 of osteogenic induction. D-F:Expressions of Runx2 and Opn mRNA and proteins on day 7. G: Expressions of OCN protein on day 7. *P<0.05, **P<0.01, ***P<0.001, ****P <0.0001 vs si-NC group.

Fig.4 Mechanistic illustration of the role of HMGA2 in osteogenic differentiation of ADSCs.

Fig.5 Three-dimensional micro-CT reconstruction and histomorphometric analysis bone defect repair in mice at 6 weeks after surgery. A: Reconstructed micro-CT images. B: Morphometric analyses of bone regeneration in calvarial defects by assessing relative bone growth surface area, bone volume/tissue volume (BV/TV%), trabecular number (Tb.N) and trabecular thickness (Tb.Th). **P<0.01, ***P<0.001, ****P<0.0001 vs Blank group.

Fig.6 HE and Masson's trichrome staining of bone regeneration in the calvarial defects at 6 weeks after surgery. A, C: HE staining overview image (scale bar: 500 μm) and magnified image (scale bar: 100 μm). B, D: Masson's trichrome staining overview image (scale bar: 500 μm) and magnified image (scale bar: 100 μm).

Fig.7 Protein-protein interaction (PPI) network analysis of HMGA2. A: PPI networks after cluster analyses. B: Predicted HMGA2 direct interacting proteins.

Tab.2 HMGA2-interacting protein possibility score

Gene	Combined score	Experimentally determined interaction	Coexpression
IGF2BP3 0.909 0.292 0.257
SMAD9	0.816	0.294	0.067
SNAI1	0.728	0	0.042
SMAD7	0.68	0.045	0.055
CDH1	0.593	0.059	0
ALDH1A1	0.569	0	0
CDH2	0.538	0.095	0.097

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