miR-199a-3p 通过靶向 CABLES-1 调控流体剪切力介导的成骨细胞增殖
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国家自然科学基金项目(81874017,81960403,82060405),兰州市科技计划项目(2021-RC-102),兰州大学第二医院“萃英科技创新” 计划(CY2017-ZD02, CY2021-MS-A07)


Mir-199a-3p Mediates Fluid Shear Stress-Induced Osteoblast Proliferation by Targeting CABLES-1
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    摘要:

    目的 探讨 miR-199a-3p 在流体剪切力(fluid shear stress, FSS)诱导成骨细胞增殖中的作用及其可能的分子机制。 方法 对成骨细胞 MC3T3-E1 加载 1. 2 Pa FSS,时间分别为 0、15、30、45、60、75、90 min。 使用 miR-199a-3p模拟物或 miR-199a-3p 抑制物转染 MC3T3-E1 细胞。 使用将过表达的 miR-199a-3p 以及其阴性对照分别转染MC3T3-E1 细胞,并以 1. 2 Pa FSS 处理 45 min。 将 pcDNA NC、pcDNA-CABLES-1、si RNA NC、si RNA CABLES-1 转染至 MC3T3-E1 细胞中。 分别共转染 pc DNA-CABLES-1 与 miR-199a-3p mimic 以及 si RNA-CABLES-1 与 miR-199a-3pinhibitor。 CCK-8 实验检测细胞活性;RT-qPCR 检测 CABLES-1、 miR-199a-3p、CDK 6、Cyclin D1、PCNA 表达水平;荧光素酶报告实验检测 CABLES-1 和 miR-199a-3p 的靶向关系。 免疫荧光检测 CABLES-1 蛋白表达。 Western blot 检测 CABLES-1、CDK 6、PCNA、Cyclin D1 的蛋白表达。 结果 FSS 作用下 MC3T3-E1 细胞中的 miR-199a-3p 出现显著下调。 过表达的 miR-199a-3p 抑制成骨细胞增殖,下调 miR-199a-3p 表达促进成骨细胞增殖。 miR-199a-3p 可以逆转 FSS 诱导的成骨细胞增殖。 双荧光素酶实验表明,miR-199a-3p 靶向作用于 CABLES-1,过表达的 miR-199a-3p 可抑制 CBALES-1 蛋白表达。 CABLES-1 能够促进成骨细胞增殖。 miR-199a-3p 通过 CABLES-1 抑制 FSS 诱导的成骨细胞增殖。 结论 FSS 诱导的成骨细胞增殖通过下调 miR-199a-3p 并通过靶向作用于 CABLES-1 实现。 研究结果为 FSS 诱导成骨细胞增殖机制的研究提供新方向,也为未来机械刺激在骨关节疾病治疗中的临床应用研究提供新思路。

    Abstract:

    Objective To explore the role of miR-199a-3p in osteoblast proliferation induced by fluid shear stress (FSS) and the potential molecular mechanism. Methods Osteoblast MC3T3-E1 was treated with 1. 2 Pa FSS with time gradients of 0, 15, 30, 45, 60, 75 and 90 min, respectively. MC3T3-E1 cells were transfected with miR-199a-3p mimic or miR-199a-3p inhibitor. MC3T3-E1 cells were transfected with miR-199a-3p mimic and itsnegative control and then treated with 1. 2 Pa FSS for 45 min. The pc DNA NC, pc DNA-CABLES -1, si RNA NC and si RNA CABLES-1 were transfected into MC3T3-E1 cells. The pc DNA-CABLES-1 and mir-199a-3p mimic and SI NA-cables-1 and miR-199a-3p inhibitor were co-transfected, respectively. Cell activity was detected by CCK-8 assay. Real-time quantitative PCR (RT-qPCR) was used to detect expression levels of CABLES-1, miR-199a-3p, CDK 6, Cyclin D1 and PCNA. Luciferase reporting assay was used to detect targeting relationship between CABLES-1 and miR-199a-3p. Immunofluorescence was used to detect protein expression of CABLES-1.Western blot was used to detect protein expression of CABLES-1, CDK 6, PCNA and Cyclin D1. Results Mir- 199a-3p in MC3T3-E1 cells was significantly down-regulated by FSS. Over-expressed miR-199a-3p inhibitedosteoblast proliferation, and down-regulated miR-199a-3p expression promoted osteoblast proliferation. miR-199a- 3p could reverse the FSS-induced proliferation in osteoblasts. Dual luciferase assay showed that miR-199a-3p targeted to CABLES-1 and over-expressed miR-199a-3p inhibited expression of CBALES-1 protein. CABLES-1 could promote proliferation of osteoblasts. miR-199a-3p inhibited osteoblast proliferation induced by FSS through CABLES-1. Conclusions FSS-induced osteoblast proliferation can be realized by down-regulated miR-199a-3p expression via targeting CABLES-1. The findings in this study provide new direction for researches on mechanism of FSS-induced osteoblast proliferation, as well as new ideas for future research on clinical application of mechanical loading in the treatment of bone and joint diseases.

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王力夫,张 坤,移 穷,刘众成,刘雪宁,耿 彬,夏亚一. miR-199a-3p 通过靶向 CABLES-1 调控流体剪切力介导的成骨细胞增殖[J].医用生物力学,2023,38(2):268-275

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  • 收稿日期:2022-04-04
  • 最后修改日期:2022-04-13
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  • 在线发布日期: 2023-04-25
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