Cell Prolif|肖金刚团队揭示LncRNA-AK137033介导糖尿病性骨质疏松症中脂肪干细胞成骨潜能的作用机制

2022年8月3日 0条评论 312次阅读 0人点赞

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糖尿病（DM）是一种以高血糖为特征的全身性代谢疾病，葡萄糖代谢紊乱引起骨骼和关节系统的严重并发症，即糖尿病性骨质疏松症（DOP），对骨骼系统产生严重的负面影响。对于DOP患者的骨缺损，目前的治疗方法并不理想。研究显示，基于脂肪干细胞（ASCs）的骨组织工程有望成为DOP患者骨缺损的新疗法。2021年12月24日，西南医科大学附属口腔医院口腔种植科肖金刚团队在Cell Proliferation上发表了题为“LncRNA-AK137033 inhibits the osteogenic potential of adipose-derived stem cells in diabetic osteoporosis by regulating Wnt signaling pathway via DNA methylation”的研究论文。在本研究中，团队旨在从表观遗传学角度探讨DOP-ASCs成骨潜能的分子机制，为DOP骨缺损患者的骨修复提供可能的治疗靶点。结果证明，LncRNA-AK137033可通过调节sFrp2启动子区的DNA甲基化，来调节Wnt信号通路，从而抑制DOP-ASCs成骨潜力，为寻找治疗DOP患者骨缺损的新靶点提供重要参考。

团队前期研究表明，与对照ASCs（CON-ASCs）相比，DOP-ASCs的成骨潜力较低与Wnt信号通路有关。Wnt信号通路是一组通过Wnt配体与细胞膜受体结合而激活的多功能信号转导通路，参与细胞的各种生理和病理过程，包括各种骨组织疾病，如骨质疏松症和干细胞相关的骨再生。干细胞中Wnt信号通路的激活提高了它们的骨分化能力，而Wnt信号通路的抑制会减少骨形成。

哺乳动物DNA甲基化是指DNA的CpG二核苷酸中胞嘧啶上第五个碳原子的甲基化，由四种DNA甲基转移酶即DNMT1、DNMT2、DNMT3a和DNMT3b催化。一般认为DNA的高甲基化与抑制基因表达有关，而DNA去甲基化则具有相反的作用。由于许多基因的启动子区域包含高密度CpG二核苷酸聚集区域，即CpG岛，因此DNA甲基化在哺乳动物细胞生物学中起着重要作用。最近研究表明，DNA甲基化可通过调节特定基因的表达来影响干细胞的多向分化，从而导致包括骨质疏松症和骨关节炎在内的各种骨骼疾病。

随着对LncRNAs研究的逐步深入，许多研究报道了LncRNAs在表观遗传、转录和转录后水平调控基因表达，对间充质干细胞的细胞命运和发生发展具有潜在调控作用。研究证实，LncRNAs可通过调节其启动子区域DNA甲基化水平影响特定基因的表达。在本研究中，团队对CON-ASC和DOP-ASC进行了mRNA/LncRNA表达谱和MeDIP测序。结果表明，与LncRNA-AK137033相关的Wnt信号分子sFrp2启动子区DNA甲基化水平存在显著差异。随后，对sFrp2和LncRNA-AK137033进行了体内外功能研究，从表观遗传学的角度探讨了调控DOP-ASCs骨分化的分子机制。结果发现，与CON-ASCs相比，DOP-ASCs中AK137033表达、sFrp2启动子区DNA甲基化水平、Wnt信号通路标志物和成骨分化潜能均降低；机制上，AK137033通过调控sFrp2启动子区DNA甲基化水平来介导Wnt信号通路和成骨能力，其在DOP-ASC中的过表达挽救了DOP引起的一系列变化。

《Cell Prolif|肖金刚团队揭示LncRNA-AK137033介导糖尿病性骨质疏松症中脂肪干细胞成骨潜能的作用机制》

图本文部分实验结果，成功构建糖尿病骨质疏松小鼠模型。

期刊及DOI号

Cell Prolif. 2021 Dec 24.

doi: 10.1111/cpr.13174.

题目

LncRNA-AK137033 inhibits the osteogenic potential of adipose-derived stem cells in diabetic osteoporosis by regulating Wnt signaling pathway via DNA methylation

摘要

目的：Bone tissue engineering based on adipose-derived stem cells (ASCs) is expected to become a new treatment for diabetic osteoporosis (DOP) patients with bone defects. However, compared with control ASCs (CON-ASCs), osteogenic potential of DOP-ASCs is decreased, which increased the difficulty of bone reconstruction in DOP patients. Moreover, the cause of the poor osteogenesis of ASCs in a hyperglycemic microenvironment has not been elucidated. Therefore, this study explored the molecular mechanism of the decline in the osteogenic potential of DOP-ASCs from the perspective of epigenetics to provide a possible therapeutic target for bone repair in DOP patients with bone defects.

材料和方法：An animal model of DOP was established in mice. CON-ASCs and DOP-ASCs were isolated from CON and DOP mice, respectively. AK137033 small interfering RNA (SiRNA) and an AK137033 overexpression plasmid were used to regulate the expression of AK137033 in CON-ASCs and DOP-ASCs in vitro. Lentiviruses that carried shRNA-AK137033 or AK137033 cDNA were used to knockdown or overexpress AK137033, respectively, in CON-ASCs and DOP-ASCs in vivo. Hematoxylin and eosin (H&E), Masson’s, alizarin red, and alkaline phosphatase (ALP) staining, micro-computed tomography (Micro-CT), flow cytometry, qPCR, western blotting, immunofluorescence, and bisulfite-specific PCR (BSP) were used to analyze the functional changes of ASCs.

结果：The DOP mouse model was established successfully. Compared with CON-ASCs, AK137033 expression, the DNA methylation level of the sFrp2 promoter region, Wnt signaling pathway markers, and the osteogenic differentiation potential were decreased in DOP-ASCs. In vitro experiments showed that AK137033 silencing inhibited the Wnt signaling pathway and osteogenic ability of CON-ASCs by reducing the DNA methylation level in the sFrp2 promoter region. Additionally, overexpression of AK137033 in DOP-ASCs rescued these changes caused by DOP. Moreover, the same results were obtained in vivo.

结论：LncRNA-AK137033 inhibits the osteogenic potential of DOP-ASCs by regulating the Wnt signaling pathway via modulating the DNA methylation level in the sFrp2 promoter region. This study provides an important reference to find new targets for the treatment of bone defects in DOP patients.