Diabetic osteoporosis (DOP) is a systemic metabolic bone disease caused by diabetes mellitus (DM). Adipose-derived stem cells (ASCs) play an important role in bone regeneration. Our previous study confirmed that ASCs from DOP mice (DOP-ASCs) have a lower osteogenesis potential compared with control ASCs (CON-ASCs). However, the cause of this poor osteogenesis has not been elucidated. Therefore, this study investigated the underlying mechanism of the decline in the osteogenic potential of DOP-ASCs from the perspective of epigenetics and explored methods to enhance their osteogenic capacity. The expression level of JNK1-associated membrane protein (JKAMP) and degree of DNA methylation in CON-ASCs and DOP-ASCs were measured by mRNA expression profiling and MeDIP sequencing, respectively. JKAMP small interfering RNA (siRNA) and a Jkamp overexpression plasmid were used to assess the role of JKAMP in osteogenic differentiation of CON-ASCs and DOP-ASCs. Immunofluorescence, qPCR, and western blotting were used to measure changes in expression of Wnt signaling pathway-related genes and osteogenesis-related molecules after osteogenesis induction. Alizarin red and ALP staining was used to confirm the osteogenic potential of stem cells. Bisulfite-specific PCR (BSP) was used to detect JKAMP methylation degree. Expression of JKAMP and osteogenesis-related molecules (RUNX2 and OPN) in DOP-ASCs was decreased significantly in comparison with CON-ASCs. JKAMP silencing inhibited the Wnt signaling pathway and reduced the osteogenic ability of CON-ASCs. Overexpression of JKAMP in DOP-ASCs rescued the impaired osteogenic capacity caused by DOP. Moreover, JKAMP in DOP-ASCs contained intragenic DNA hypermethylated regions related to the downregulation of JKAMP expression. Intragenic DNA methylation inhibits the osteogenic ability of DOP-ASCs by suppressing expression of JKAMP and the Wnt signaling pathway. This study shows an epigenetic explanation for the reduced osteogenic ability of DOP-ASCs and provides a potential therapeutic target to prevent and treat osteoporosis.

中文翻译：

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JKAMP 通过基因内 DNA 甲基化调节 Wnt 信号通路抑制脂肪干细胞在糖尿病性骨质疏松症中的成骨能力

糖尿病性骨质疏松症（DOP）是由糖尿病（DM）引起的全身代谢性骨病。脂肪干细胞 (ASCs) 在骨再生中发挥重要作用。我们之前的研究证实，与对照 ASCs (CON-ASCs) 相比，来自 DOP 小鼠的 ASCs (DOP-ASCs) 具有较低的成骨潜力。然而，这种不良成骨的原因尚未阐明。因此，本研究从表观遗传学的角度研究了DOP-ASCs成骨能力下降的潜在机制，并探索了增强其成骨能力的方法。分别通过 mRNA 表达谱和 MeDIP 测序测量 CON-ASC 和 DOP-ASC 中 JNK1 相关膜蛋白 (JKAMP) 的表达水平和 DNA 甲基化程度。JKAMP 小干扰 RNA (siRNA) 和 Jkamp 过表达质粒用于评估 JKAMP 在 CON-ASCs 和 DOP-ASCs 成骨分化中的作用。免疫荧光、qPCR 和蛋白质印迹用于测量成骨诱导后 Wnt 信号通路相关基因和成骨相关分子的表达变化。茜素红和 ALP 染色用于确认干细胞的成骨潜力。亚硫酸氢盐特异性PCR（BSP）用于检测JKAMP甲基化程度。与 CON-ASCs 相比，DOP-ASCs 中 JKAMP 和成骨相关分子（RUNX2 和 OPN）的表达显着降低。JKAMP 沉默抑制 Wnt 信号通路并降低 CON-ASCs 的成骨能力。DOP-ASCs 中 JKAMP 的过表达挽救了由 DOP 引起的成骨能力受损。此外，DOP-ASCs 中的 JKAMP 含有与 JKAMP 表达下调相关的基因内 DNA 高甲基化区域。基因内 DNA 甲基化通过抑制 JKAMP 和 Wnt 信号通路的表达来抑制 DOP-ASCs 的成骨能力。该研究显示了 DOP-ASCs 成骨能力降低的表观遗传学解释，并为预防和治疗骨质疏松症提供了潜在的治疗靶点。