LncRNA通过DNA甲基化调控Wnt信号通路在II型糖尿病性骨质疏松症脂肪干细胞骨向分化中的作用及机制

Autologous adipose-derived stem cells (ASCs) have a good prospect applicated in the treatment of fractures and bone defects associated with diabetic osteoporosis (DOP). Our previous study found that the osteogenic differentiation of ASCs was weakened, the degree of DNA methylation was increased, and the Wnt/β-catenin signaling pathway was inhibited under glycation end products (AGEs) treatment which simulated the microenvironment of diabetes. Moreover, we constructed an II-DOP mouse model, and isolated II-DOP-ASCs from adipose tissues of II-DOP mice. The changes in II-DOP-ASCs were consistent with the effects of AGEs on ASCs, suggesting that DNA methylation modulating Wnt signaling pathway might participate in the process of inhibiting bone differentiation of ASCs in DOP microenvironment. We completed the lncRNA/mRNA gene chip and DNA methylation sequencing, and screened 5 pairs of Wnt signaling molecules with differentially expressed DNA methylation levels and their associated differential lncRNAs. Therefore, our study intends to perform functional studies on five pairs of differential lncRNA-mRNAs, to clarify the molecular mechanism of lncRNA regulating differential Wnt signaling pathway through DNA methylation in osteogenic differentiation of DOP-ASCs, and to promote bone regeneration in the DOP microenvironment by the targeted intervention of key genes.

自体脂肪干细胞（ASCs）用于治疗糖尿病性骨质疏松症（DOP）伴发的骨折和骨缺损具有良好的应用前景。本课题组前期研究发现，糖基化终末产物（AGEs）处理ASCs模拟糖尿病微环境，ASCs骨向分化能力减弱，DNA甲基化程度增强，Wnt/β-catenin信号通路被抑制；构建Ⅱ-DOP小鼠模型，分离培养的Ⅱ-DOP-ASCs变化与AGEs作用于ASCs一致，提示DNA甲基化调控Wnt信号通路参与DOP微环境下ASCs骨向分化被抑制的过程；完成了LncRNA/mRNA基因芯片和DNA甲基化测序，筛选出了5对DNA甲基化水平改变的差异Wnt信号分子和与之相关联的差异LncRNA。因此，本课题拟对5对差异LncRNA-mRNA进行功能学研究，明确LncRNA通过DNA甲基化调控Wnt信号通路在Ⅱ-DOP-ASCs骨向分化中的作用和调控机理；靶向干预关键基因促进DOP微环境下骨修复再生。

糖尿病性骨质疏松症（DOP）患者除血糖异常升高以外，还具有骨量减少、骨强度降低易发生骨折、骨缺损不易愈合等特征。然而，针对DOP伴骨折或骨缺损的患者，目前应用的治疗手段不甚理想。随着组织工程的快速发展，基于脂肪干细胞（ASCs）的骨组织工程被认为是最有前景的骨缺损修复方法之一，但既往研究表明DOP-ASCs成骨能力下降，限制了其在DOP患者骨折或骨缺损的应用。本课题组前期研究发现：相比于对照组ASCs（CON-ASCs），AGEs模拟糖尿病微环境下ASCs成骨能力下降，DNA甲基化水平增加，Wnt/β-catenin信号通路被抑制，但其具体分子机制尚需进一步研究。本课题通过构建DOP小鼠动物模型，分离培养CON-ASCs和DOP-ASCs，进行LncRNA/mRNA基因芯片和DNA甲基化测序，筛选出CON-ASCs和DOP-ASCs中DNA甲基化水平改变的差异Wnt分子（JKAMP，sFrp2），靶向干预目标基因检测Wnt信号通路和成骨能力表达变化，阐明了JKAMP和sFrp2的DNA甲基化改变可通过Wnt信号通路调控DOP-ASCs成骨能力。基于此，课题组进一步深入研究发现sFrp2相关LncRNA-AK137033可通过影响sFrp2启动子区域DNA甲基化水平，调控Wnt信号通路从而改变DOP-ASCs体内外成骨能力。本课题首次从表观转录水平阐明DOP微环境下LncRNA通过DNA甲基化调控Wnt信号通路改变DOP-ASCs成骨能力的分子机制。通过体内外实验证明了从表观转录水平靶向干预Wnt信号通路从而恢复DOP-ASCs骨向分化潜能的可行性，对以自体DOP-ASCs为基础的骨组织工程治疗DOP患者骨折或骨缺损具有重要意义。