Department of Oral Implantology, The Affiliated Stomatology Hospital of Southwest Medical University, Luzhou, China.
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
Cell Prolif. 2022 Jan;55(1):e13174. doi: 10.1111/cpr.13174. Epub 2021 Dec 24.
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.
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.
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.
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.
基于脂肪来源干细胞（ASCs）的骨组织工程有望成为治疗糖尿病骨质疏松症（DOP）伴骨缺损患者的新方法。然而，与对照 ASCs（CON-ASCs）相比，DOP-ASCs 的成骨潜力降低，这增加了 DOP 患者骨重建的难度。此外，高血糖微环境中 ASCs 成骨能力下降的原因尚不清楚。因此，本研究从表观遗传学角度探讨了 DOP-ASCs 成骨能力下降的分子机制，为 DOP 伴骨缺损患者的骨修复提供了可能的治疗靶点。
建立 DOP 小鼠模型。分别从 CON 和 DOP 小鼠中分离 CON-ASCs 和 DOP-ASCs。体外使用 AK137033 小干扰 RNA（siRNA）和 AK137033 过表达质粒调节 CON-ASCs 和 DOP-ASCs 中 AK137033 的表达。体内使用携带 shRNA-AK137033 或 AK137033 cDNA 的慢病毒分别在 CON-ASCs 和 DOP-ASCs 中敲低或过表达 AK137033。苏木精和伊红（H&E）、马松氏、茜素红和碱性磷酸酶（ALP）染色、微计算机断层扫描（Micro-CT）、流式细胞术、qPCR、western blot、免疫荧光和亚硫酸氢盐特异性 PCR（BSP）用于分析 ASCs 的功能变化。
成功建立了 DOP 小鼠模型。与 CON-ASCs 相比，DOP-ASCs 中 AK137033 表达、sFrp2 启动子区域的 DNA 甲基化水平、Wnt 信号通路标志物和成骨分化潜能降低。体外实验表明，AK137033 沉默通过降低 sFrp2 启动子区域的 DNA 甲基化水平抑制 CON-ASCs 的 Wnt 信号通路和成骨能力。此外，在 DOP-ASCs 中转染 AK137033 过表达可挽救由 DOP 引起的这些变化。此外，体内实验也得到了相同的结果。
长链非编码 RNA-AK137033 通过调节 sFrp2 启动子区域的 DNA 甲基化水平来调节 Wnt 信号通路，从而抑制 DOP-ASCs 的成骨潜能。本研究为寻找治疗 DOP 患者骨缺损的新靶点提供了重要参考。
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