Department of Oral and Maxillofacial Surgery, Affiliated Stomatological Hospital of Nanjing Medical University, Hanzhong Road No.136, Nanjing, 210029, Jiangsu Province, People's Republic of China.
Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, 210029, Jiangsu Province, People's Republic of China.
Stem Cell Res Ther. 2020 Jul 25;11(1):318. doi: 10.1186/s13287-020-01842-0.
BACKGROUND: MicroRNAs (miRNAs) play a role in regulating osteogenic differentiation (OD) of mesenchymal stem cells by inhibiting mRNAs translation under cyclic strain. miR-503-3p was downregulated in OD of human adipose-derived stem cells (hASCs) in vivo under cyclic strain in our previous study, while it might target the Wnt/β-catenin (W-β) pathway. In this study, we explored miR-503-3p's role in OD of hASCs under cyclic strain. METHODS: OD of hASCs was induced by cyclic strain. Bioinformatic and dual luciferase analyses were used to confirm the relationship between Wnt2/Wnt7b and miR-503-3p. Immunofluorescence was used to detect the effect of miR-503-3p on Wnt2/Wnt7b and β-catenin in hASCs transfected with miR-503-3p mimic and inhibitor. Mimic, inhibitor, and small interfering RNA (siRNA) transfected in hASCs to against Wnt2 and Wnt7b. Quantitative real-time PCR (RT-PCR) and western blot were used to examine the OD and W-β pathway at the mRNA and protein levels, respectively. Immunofluorescence was performed to locate β-catenin. ALP activity and calcium were detected by colorimetric assay. RESULTS: Results of immunophenotypes by flow cytometry and multi-lineage potential confirmed that the cultured cells were hASCs. Results of luciferase reporter assay indicated that miR-503-3p could regulate the expression levels of Wnt2 and Wnt7b by targeting their respective 3'-untranslated region (UTR). Under cyclic strain, gain- or loss-function of miR-503-3p studies by mimic and inhibitor revealed that decreasing expression of miR-503-3p could significantly bring about promotion of OD of hASCs, whereas increased expression of miR-503-3p inhibited OD. Furthermore, miR-503-3p high-expression reduced the activity of the W-β pathway, as indicated by lowering expression of Wnt2 and Wnt7b, inactive β-catenin in miR-503-3p-treated hASCs. By contrast, miR-503-3p inhibition activated the W-β pathway. CONCLUSIONS: Collectively, our findings indicate that miR-503-3p is a negative factor in regulating W-β pathway by Wnt2 and Wnt7b, which inhibit the OD of hASCs under cyclic strain.
背景：微小 RNA（miRNA）通过在循环应变下抑制 mRNA 翻译，在间充质干细胞的成骨分化（OD）中发挥作用。在我们之前的研究中，在体内循环应变下，人脂肪来源干细胞（hASC）的 OD 中 miR-503-3p 下调，而其可能靶向 Wnt/β-连环蛋白（W-β）途径。在这项研究中，我们探讨了 miR-503-3p 在循环应变下对 hASC 的 OD 的作用。
方法：通过循环应变诱导 hASC 的 OD。生物信息学和双荧光素酶分析用于确认 Wnt2/Wnt7b 与 miR-503-3p 之间的关系。免疫荧光用于检测 miR-503-3p 对转染 miR-503-3p 模拟物和抑制剂的 hASC 中 Wnt2/Wnt7b 和 β-连环蛋白的影响。转染 hASC 以针对 Wnt2 和 Wnt7b 的模拟物、抑制剂和小干扰 RNA（siRNA）。实时定量 PCR（RT-PCR）和 Western blot 分别用于检测 OD 和 W-β 通路的 mRNA 和蛋白水平。免疫荧光用于定位 β-连环蛋白。通过比色法检测碱性磷酸酶（ALP）活性和钙。
结果：流式细胞术免疫表型和多谱系潜能的结果证实培养的细胞是人 ASC。荧光素酶报告实验结果表明，miR-503-3p 可以通过靶向它们各自的 3'-非翻译区（UTR）来调节 Wnt2 和 Wnt7b 的表达水平。在循环应变下，通过模拟物和抑制剂进行 miR-503-3p 的增益或失能研究表明，miR-503-3p 表达的降低可显著促进 hASC 的 OD，而 miR-503-3p 的表达增加则抑制 OD。此外，miR-503-3p 的高表达降低了 W-β 通路的活性，表现为 miR-503-3p 处理的 hASC 中 Wnt2 和 Wnt7b 的表达降低，β-连环蛋白失活。相反，miR-503-3p 抑制激活了 W-β 通路。
结论：总之，我们的研究结果表明，miR-503-3p 通过 Wnt2 和 Wnt7b 是调节 W-β 通路的负性因子，抑制循环应变下 hASC 的 OD。
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