张应力通过下调miR-148a-3p而促进hASCs成骨分化的机制研究

Oral and maxillofacial bone defects can cause facial functional disturbance and dysmorphism, which seriously affects the physiological and psychological health of patients. Nowadays, bone flap transplantation is usually applied to repair these defects in clinic, but it causes problems such as damage of donor site and so on at the same time. Bone tissue engineering provides new methods to repair these defects, and human adipose derived stem cells (hASCs) as seed cells is the key point. Tensile stress can promote the osteogenic differentiation of hASCs in vitro, but the specific mechanism of how it works is still not very clear. Our previous studies have confirmed that Wnt/β-catenin signaling pathway is activated under tensile stress.Our primary data also showed that miR-148a-3p was markedly downregulated in hASCs under tensile stress treatment. Bioinformatic analysis revealed that Wnt10b and TCF-4, the components of Wnt/β-catenin signaling pathway, were the potential targets of miR-148a-3p. And results of western blot also demonstrated that both Wnt10b and TCF-4 were regulated by miR-148a-3p. Based on our findings and information from the literatures, we postulate that activation of Wnt/β-catenin signaling pathway caused by the downregulation of miR-148a-3p are involve in the promoting osteogenic differentiation of hASCs under the tensile stress treatment. We will test this hypothesis using molecular biological methods, and provide a theoretical and experimental basis for the application of mechanical factors in bone tissue engineering.

口颌面骨缺损常导致面部功能障碍和容貌畸形，严重影响患者的身心健康。现临床常用骨组织瓣移植进行修复，但同时也造成了供区的损伤等问题。骨组织工程为修复此类缺损提供了新方法，人脂肪干细胞（hASCs）作为种子细胞是其研究重点。张应力在体外可促进hASCs成骨分化，但其机制尚不清楚。我们既往的研究表明张应力条件下Wnt/β-catenin信号通路被激活。而预实验结果显示张应力显著下调miR-148a-3p的表达；生物信息学分析表明Wnt/β-catenin通路的Wnt10b和TCF-4是miR-148a-3p的靶基因；初步证明Wnt10b和TCF-4的表达确实被miR-148a-3p调控。于是我们推测：张应力通过下调miR-148a-3p而激活Wnt/β-catenin通路，进而促进hASCs成骨分化。本项目拟通过分子生物学手段验证此假说，从而为促进力学因素在组织工程化骨组织构建中的应用提供基础。

微小RNA（miRNA）通过抑制循环应变下的mRNA翻译来调节间充质干细胞的成骨分化。在我们先前的研究中，miR-148a-3p在人脂肪干细胞（hASCs）的成骨分化下在张应力诱导中被下调，而它可能靶向Wnt /β-catenin（W-β）途径。在这项研究中，我们探讨了miR-148a-3p在张应力下在hASCs 成骨分化中的作用。通过流式细胞术免疫表型结果和向分化潜能证实，培养的细胞是hASCs。荧光素酶报告基因分析的结果表明，miR-148a-3p可以通过靶向结合Wnt10b和TCF-4的3'-非翻译区（UTR）来调节其表达水平。在张应力下，利用miR-148a-3p mimic或inhibitor研究表明，减少miR-503-3p的表达可显着促进hASC的成骨分化，而增加miR-148a-3p的表达抑制成骨分化。此外，miR-148a-3p的高表达降低了W-β信号传导途径的活性，如降低miR-148a-3p处理的hASC中Wnt10b和TCF-4的表达升高。相反，miR-148a-3p抑制激活了W-β信号通路。总体而言，我们的发现表明，miR-148a-3p是Wnt10b和TCF-4调控W-β途径的负因子，抑制了hASCs在循环应变下的成骨分化。