Down-regulation of Noggin and miR-138 coordinately promote osteogenesis of mesenchymal stem cells
Down-regulation of Noggin and miR-138 coordinately promote osteogenesis of mesenchymal stem cells
复制标题
Noggin 和 miR-138 的下调协同促进间充质干细胞的成骨。
DOI：
10.1007/s10735-017-9740-5
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发表时间：
2017-12-01
影响因子：
3.2
通讯作者：
Wang, Li-Jun
中科院分区：
文献类型：
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作者：
Sun, Xing-Kun;Zhou, Jin;Wang, Li-Jun
骨髓间充质干细胞（Mesenchymal stem cells，MSCs）在适当条件下可分化为骨细胞。近年来，出于安全性的考虑，微核苷酸逐渐被用于调节细胞中的基因表达。我们目前的研究旨在研究Noggin-siRNA和antimiR-138的共同递送是否增强了MSC的成骨作用。使用鼠MSC系C3 H/10 T1/2细胞，通过定量实时聚合酶链反应（qRT-PCR）评估Noggin-siRNA和antimiR-138向MSC中的递送效率。流式细胞仪检测细胞表型，MTT法检测细胞增殖能力。通过碱性磷酸酶（ALP）染色、qRT-PCR和western blot分析来测试MSC的成骨能力。我们的研究结果表明，Noggin-siRNA和/或antimiR-138可显著沉默MSCs中Noggin和miR-138的表达，而MSCs的表型和增殖能力不受影响。下调Noggin和miR-138协同促进MSCs的成骨分化。ALP阳性细胞达83.57 ± 10.18%。与单独转染相比，两种寡核苷酸联合转染的成骨相关基因Alp、Col-1、Bmp 2、Ocn和Runx 2的表达最高。RUNX 2蛋白表达水平、pSMAD 1/5/SMAD 1/5和pERK 1/2/ERK 1/2比值均显著升高。Smad、Erk信号通路的激活可能是促进成骨过程的潜在机制。综上所述，我们的研究为骨髓间充质干细胞在骨骼缺损的临床康复应用提供了一种安全的策略。
Mesenchymal stem cells (MSCs) can differentiate to osteocytes under suitable conditions. In recent years, micro-nucleotides have been progressively used to modulate gene expression in cells due to the consideration of safety. Our present study aimed to investigate whether co-delivery of Noggin-siRNA and antimiR-138 enhances the osteogenic effect of MSCs. Using a murine MSC line, C3H/10T1/2 cells, the delivery efficiency of Noggin-siRNA and antimiR-138 into MSCs was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Cell phenotype and proliferation capacity was assessed by flow cytometry and MTT assay respectively. The osteogenesis of MSCs was tested by Alkaline Phosphatase (ALP) staining, qRT-PCR, and western blot analyses. Our results demonstrated that the expression of Noggin and miR-138 were significantly silenced in MSCs by Noggin-siRNA and/or antimiR-138 delivery, while the phenotype and proliferation capacity of MSCs were not affected. Down-regulation of Noggin and miR-138 cooperatively promoted osteogenic differentiation of MSCs. The ALP positive cells reached about 83.57 +/- 10.18%. Compared with single delivery, the expression of osteogenic related genes, such as Alp, Col-1, Bmp2, Ocn and Runx2, were the highest in cells with co-delivery of the two oligonucleotides. Moreover, the protein level of RUNX2, and the ratios of pSMAD1/5/SMAD1/5 and pERK1/2/ERK1/2 were significantly increased. The activation of Smad, Erk signaling may constitute the underlying mechanism of the enhanced osteogenesis process. Taken together, our study provides a safe strategy for the clinical rehabilitation application of MSCs in skeletal deficiency.