Downregulation of DNA methyltransferase-3a ameliorates the osteogenic differentiation ability of adipose-derived stem cells in diabetic osteoporosis via Wnt/β-catenin signaling pathway.
Downregulation of DNA methyltransferase-3a ameliorates the osteogenic differentiation ability of adipose-derived stem cells in diabetic osteoporosis via Wnt/β-catenin signaling pathway.
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DNA 甲基转移酶-3a 的下调通过 Wnt/β-catenin 信号通路改善糖尿病骨质疏松症中脂肪干细胞的成骨分化能力
DOI：
10.1186/s13287-022-03088-4
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发表时间：
2022-08-04
影响因子：
7.5
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中科院分区：
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--
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糖尿病相关性骨质疏松症（Diabetes-related osteoporosis，DOP）是由高糖环境引起的骨细胞和成骨细胞相关间充质干细胞代谢紊乱引起的慢性疾病。DOP使骨缺损修复和再生过程变得非常困难。脂肪源性干细胞作为骨组织工程技术的种子细胞，为DOP患者的骨再生提供了一种有希望的治疗途径。在DOP模型中，ASCs的成骨能力低于对照ASCs。DNA甲基化作为一种表观遗传调控机制，可能参与多种基因的DNA甲基化，从而参与多种细胞的生物学行为。新的证据表明，DNA甲基化水平的增加与Wnt/β-catenin信号通路的激活有关。本研究旨在探讨糖尿病环境对ASCs成骨分化能力的影响，并通过Wnt/β-catenin信号通路探讨DNA甲基化在DOP-ASCs成骨分化中的作用，以提高DOP诱导的ASCs的成骨分化能力。DOP-ASC和对照ASC分别从DOP C57 BL/6和对照小鼠中分离。通过茜素红-S、油红-O和阿尔新蓝染色证实DOP-ASC的多能性。采用实时聚合酶链反应（RT-PCR）、免疫荧光和蛋白质印迹法分析成骨分化、DNA甲基化和Wnt/β-catenin信号转导标志物的变化。茜素红-S染色也用于确认成骨能力的变化。使用DNMT小干扰RNA（siRNA）、shRNA-Dnmt 3a和LVRNA-Dnmt 3a来评估Dnmt 3a在对照ASC和DOP-ASC的成骨分化中的作用。使用显微计算机断层扫描、苏木精和伊红染色以及Masson染色来分析用慢病毒下调DOP小鼠体内Dnmt 3a时成骨能力的变化。DOP-ASCs的增殖能力低于对照ASCs。DOP-ASCs的成骨分化能力下降，Wnt/β-catenin信号通路活性降低，Dnmt 3a水平升高。当siRNA和shRNA下调Dnmt 3a表达时，成骨相关因子Runt相关转录因子2和骨桥蛋白以及Wnt/β-catenin信号通路的活性增加，从而挽救了DOP-ASCs不良的成骨能力。当LVRNA-Dnmt 3a上调Dnmt 3a时，成骨能力受到抑制。在体内获得了相同的结果。Dnmt 3a沉默可以缓解DOP对ASC的负面影响，并为糖尿病骨质疏松症患者的骨组织再生提供了一种可能的方法。
Diabetes-related osteoporosis (DOP) is a chronic disease caused by the high glucose environment that induces a metabolic disorder of osteocytes and osteoblast-associated mesenchymal stem cells. The processes of bone defect repair and regeneration become extremely difficult with DOP. Adipose-derived stem cells (ASCs), as seed cells in bone tissue engineering technology, provide a promising therapeutic approach for bone regeneration in DOP patients. The osteogenic ability of ASCs is lower in a DOP model than that of control ASCs. DNA methylation, as a mechanism of epigenetic regulation, may be involved in DNA methylation of various genes, thereby participating in biological behaviors of various cells. Emerging evidence suggests that increased DNA methylation levels are associated with activation of Wnt/β-catenin signaling pathway. The purpose of this study was to investigate the influence of the diabetic environment on the osteogenic potential of ASCs, to explore the role of DNA methylation on osteogenic differentiation of DOP-ASCs via Wnt/β-catenin signaling pathway, and to improve the osteogenic differentiation ability of ASCs with DOP. DOP-ASCs and control ASCs were isolated from DOP C57BL/6 and control mice, respectively. The multipotency of DOP-ASCs was confirmed by Alizarin Red-S, Oil Red-O, and Alcian blue staining. Real-time polymerase chain reaction (RT-PCR), immunofluorescence, and western blotting were used to analyze changes in markers of osteogenic differentiation, DNA methylation, and Wnt/β-catenin signaling. Alizarin Red-S staining was also used to confirm changes in the osteogenic ability. DNMT small interfering RNA (siRNA), shRNA-Dnmt3a, and LVRNA-Dnmt3a were used to assess the role of Dnmt3a in osteogenic differentiation of control ASCs and DOP-ASCs. Micro-computed tomography, hematoxylin and eosin staining, and Masson staining were used to analyze changes in the osteogenic capability while downregulating Dnmt3a with lentivirus in DOP mice in vivo. The proliferative ability of DOP-ASCs was lower than that of control ASCs. DOP-ASCs showed a decrease in osteogenic differentiation capacity, lower Wnt/β-catenin signaling pathway activity, and a higher level of Dnmt3a than control ASCs. When Dnmt3a was downregulated by siRNA and shRNA, osteogenic-related factors Runt-related transcription factor 2 and osteopontin, and activity of Wnt/β-catenin signaling pathway were increased, which rescued the poor osteogenic potential of DOP-ASCs. When Dnmt3a was upregulated by LVRNA-Dnmt3a, the osteogenic ability was inhibited. The same results were obtained in vivo. Dnmt3a silencing rescues the negative effects of DOP on ASCs and provides a possible approach for bone tissue regeneration in patients with diabetic osteoporosis.
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影响因子：
39.3
作者：
Liu J;Xiao Q;Xiao J;Niu C;Li Y;Zhang X;Zhou Z;Shu G;Yin G
通讯作者：
Yin G
影响因子：
5.7
作者：
Seman NA;Mohamud WN;Östenson CG;Brismar K;Gu HF
通讯作者：
Gu HF
影响因子：
6.7
作者：
Dutra Oliveira, Peterson Antonio;Soares Dutra Oliveira, Alcione Maria;Cury, Patricia Ramos
通讯作者：
Cury, Patricia Ramos
影响因子：
6.2
作者：
Heilmeier, Ursula;Hackl, Matthias;Link, Thomas M.
通讯作者：
Link, Thomas M.
影响因子：
39.3
作者：
Ma W;Zhan Y;Zhang Y;Mao C;Xie X;Lin Y
通讯作者：
Lin Y