抑制Hmga2促进小鼠脂肪间充质干细胞成骨分化并加速骨缺损修复

doi:10.12122/j.issn.1673-4254.2024.07.02

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (7): 1227-1235.doi: 10.12122/j.issn.1673-4254.2024.07.02

抑制Hmga2促进小鼠脂肪间充质干细胞成骨分化并加速骨缺损修复

柯志勇¹(), 黄子城¹(), 何若琳¹, 张倩¹, 陈思旭¹, 崔忠凯¹(), 丁晶²()

^1.南方医科大学基础医学院细胞生物学教研室，广东广州 510515
^2.上海交通大学医学院附属新华医院儿骨科，上海 200092

收稿日期:2024-05-31 出版日期:2024-07-20 发布日期:2024-07-25
通讯作者: 崔忠凯,丁晶 E-mail:kezhy@smu.edu.cn;huangzicheng2@qq.com;zhongkaicui@smu.edu.cn;doctor2049@sina.com
作者简介:柯志勇，硕士，高级实验师，E-mail: kezhy@smu.edu.cn
黄子城，在读硕士研究生，E-mail: huangzicheng2@qq.com
第一联系人：柯志勇、黄子城共同为第一作者
基金资助:
国家自然科学基金(32270792);上海交通大学医工交叉“多学科交叉项目培育（转化）”(YQ2021QN48)

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

Zhiyong KE¹(), Zicheng HUANG¹(), Ruolin HE¹, Qian ZHANG¹, Sixu CHEN¹, Zhong-Kai CUI¹(), Jing DING²()

^1.Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515 China
^2.Department of Pediatric Orthopedics, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200092, China

Received:2024-05-31 Online:2024-07-20 Published:2024-07-25
Contact: Zhong-Kai CUI, Jing DING E-mail:kezhy@smu.edu.cn;huangzicheng2@qq.com;zhongkaicui@smu.edu.cn;doctor2049@sina.com
Supported by:
National Natural Science Foundation of China(32270792)

摘要/Abstract

摘要：

目的探讨高迁移率族蛋白A2（HMGA2）在脂肪间充质干细胞（ADSCs）成骨分化进程中的作用及其在骨缺损修复中的应用。方法通过GEO数据库和Rstudio软件，挖掘出在ADSCs“成脂-成骨”分化平衡中的关键节点因子HMGA2，并通过在线蛋白互作网络分析工具String和绘图软件Cytoscape，绘制HMGA2在成骨分化中的互作关系网络，预测其下游作用靶点。设计Hmga2 siRNA并转染小鼠原代脂肪间充质干细胞（mADSCs），诱导其体外成骨分化，在不同时间点（Day 3，Day 7，Day 14）收集样本，通过碱性磷酸酶染色和茜素红染色评估成骨分化能力，并通过RT-qPCR和Western blotting检测成骨特异性标志物Runt相关转录因子2（RUNX2）、骨桥蛋白（OPN）和骨钙素（OCN）的表达。将敲低 Hmga2 的mADSCs移植至小鼠不可自愈合颅骨缺损处，术后6周通过μCT扫描、骨组织学染色检测成骨标志物，评价骨缺损修复效果。结果 GEO数据库分析结果显示HMGA2在ADSCs成脂分化进程中表达上调。蛋白互作网络分析提示在ADSCs成骨分化中，HMGA2的潜在作用靶点包括SMAD7、CDH1、CDH2、SNAI1、SMAD9、IGF2BP3、ALDH1A1。抑制Hmga2后，mADSCs中成骨分化相关标志物RUNX2、OPN和OCN的表达显著上调，且碱性磷酸酶的表达和钙结节的形成增加（P<0.05）。在小鼠颅骨缺损模型中，敲低Hmga2促进了骨缺损部位的新骨形成（P<0.05）。结论 HMGA2是调控ADSCs成骨分化的重要因子，抑制Hmga2能显著促进ADSCs成骨分化，并加速体内骨缺损的修复。

关键词: 脂肪间充质干细胞, 高迁移率族蛋白A2, 成骨分化, 骨缺损修复

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

柯志勇, 黄子城, 何若琳, 张倩, 陈思旭, 崔忠凯, 丁晶. 抑制Hmga2促进小鼠脂肪间充质干细胞成骨分化并加速骨缺损修复[J]. 南方医科大学学报, 2024, 44(7): 1227-1235.

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.

图/表 9

表1 RT-qPCR 所用引物序列

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

图1 hADSCs成脂分化进程中的差异基因表达情况

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.

图2 抑制Hmga2在体外促进ADSCs成骨分化

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.

图3 抑制Hmga2对ADSCs成骨成脂分化标志的影响

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.

图4 HMGA2影响ADSCs成骨分化的作用机制图

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

图5 术后6周小鼠颅骨μCT三维重建图及骨计量学分析

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.

图6 术后6周小鼠颅骨缺损骨再生的组织学HE和Masson染色

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).

图7 HMGA2的蛋白互作网络分析

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

表2 HMGA2互作蛋白评分

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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抑制Hmga2促进小鼠脂肪间充质干细胞成骨分化并加速骨缺损修复

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

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