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miR-148a-3p靶向SMURF2调节牙髓干细胞和口腔上皮细胞共培养体系成骨分化及牙釉质发育的作用机制

徐倩 崔玉梅 马思明 林云红 熊依菁 宋子珺 李旭东

徐倩, 崔玉梅, 马思明, 林云红, 熊依菁, 宋子珺, 李旭东. miR-148a-3p靶向SMURF2调节牙髓干细胞和口腔上皮细胞共培养体系成骨分化及牙釉质发育的作用机制[J]. 昆明医科大学学报, 2023, 44(11): 16-21. doi: 10.12259/j.issn.2095-610X.S20231103
引用本文: 徐倩, 崔玉梅, 马思明, 林云红, 熊依菁, 宋子珺, 李旭东. miR-148a-3p靶向SMURF2调节牙髓干细胞和口腔上皮细胞共培养体系成骨分化及牙釉质发育的作用机制[J]. 昆明医科大学学报, 2023, 44(11): 16-21. doi: 10.12259/j.issn.2095-610X.S20231103
Qian XU, Yumei CUI, Siming MA, Yunhong LIN, Yijing XIONG, Zijun SONG, Xudong LI. miR-148a-3p Targeting SMURF2 in Regulating Osteogenic Differentiation and Enamel Development during In Vitro Tooth Organogenesis[J]. Journal of Kunming Medical University, 2023, 44(11): 16-21. doi: 10.12259/j.issn.2095-610X.S20231103
Citation: Qian XU, Yumei CUI, Siming MA, Yunhong LIN, Yijing XIONG, Zijun SONG, Xudong LI. miR-148a-3p Targeting SMURF2 in Regulating Osteogenic Differentiation and Enamel Development during In Vitro Tooth Organogenesis[J]. Journal of Kunming Medical University, 2023, 44(11): 16-21. doi: 10.12259/j.issn.2095-610X.S20231103

miR-148a-3p靶向SMURF2调节牙髓干细胞和口腔上皮细胞共培养体系成骨分化及牙釉质发育的作用机制

doi: 10.12259/j.issn.2095-610X.S20231103
基金项目: 云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目(202001AY070001-249);云南省医学学科后备人才培养项目(H-2019019)
详细信息
    作者简介:

    徐倩(1984~),女,云南昆明人,博士,副教授,主要从事口腔种植修复与牙周研究工作

    崔玉梅与徐倩对本文有同等贡献

    通讯作者:

    李旭东,E-mail:kmulxd@163.com

  • 中图分类号: R78

miR-148a-3p Targeting SMURF2 in Regulating Osteogenic Differentiation and Enamel Development during In Vitro Tooth Organogenesis

  • 摘要:   目的  探讨miR-148a-3p在体外牙齿发生中成骨分化和牙釉质发育中的作用,揭示其分子机制。  方法  获取人牙髓干细胞和口腔上皮细胞。将人牙髓干细胞转染miR-148a-3p mimics、miR-148a-3p抑制剂或阴性对照。通过在Matrigel基质凝胶上接种人牙髓干细胞并覆盖口腔上皮细胞,建立三维共培养系统。采用MTT实验评估miR-148a-3p对共培养细胞增殖和蛋白表达的影响,采用Western blotting实验检测成骨细胞分化(RUNX2)和牙釉质发育标志物(E-cadherin)蛋白表达水平,采用荧光素酶报告实验验证SMURF2(SMAD 特异性 E3泛素蛋白连接酶2)与miR-148a-3p的相互作用。  结果  在人牙髓干细胞中使用抑制剂下调miR-148a-3p后,3D共培养中的细胞活力降低(P < 0.05)。使用模拟物上调miR-148a-3p后,共培养中成骨标志物RUNX2和牙釉质发育标志物E-cadherin的表达增加 (P < 0.05)。TargetScan软件预测miR-148a-3p在SMURF2的3'-UTR中有结合位点。荧光素酶报告实验表明miR-148a-3p mimics抑制野生型载体的荧光素酶活性(P < 0.05),同时 Western blot 结果显示miR-148a-3p mimics下调SMURF2的表达(P < 0.05)。  结论  miR-148a-3p可能通过靶向SMURF2,调控RUNX2和E-cadherin的表达,参与了人牙髓干细胞成骨分化和上皮细胞牙釉质发育。为牙齿修复和治疗提供了潜在的治疗靶点。
  • 图  1  各组细胞MTT活力检测

    与Con比较,*P < 0.05。

    Figure  1.  MTT Detection of cell viability

    图  2  Western blotting法检测miR-148a-3p对RUNX2和N-cadherin蛋白表达水平的影响

    A:RT-PCR检测miR-148a-3p表达水平;B:Western blotting法检测RUNX2和N-cadherin蛋白表达水平;C:灰度值分析RUNX2和N-cadherin蛋白相对表达量。与Con比较,*P < 0.05。

    Figure  2.  The effects of miR-148a-3p on the expression levels of RUNX2 and N-cadherin proteins were detected by Western blotting

    图  3  miR-148a-3p通过结合SMURF2 3′ -UTR调节SMURF2表达水平

    A:miR-148a-3p与SMURF2 3′ -UTR的互补位点;B:双荧光素酶报告实验;C:Western blotting法检测SMURF2蛋白表达水平;D:灰度值分析SMURF2蛋白相对表达量。与NC mimics比较,*P < 0.05;与Con比较,#P < 0.05。

    Figure  3.  miR-148a-3p regulates SMURF2 expression by binding to SMURF2 3’ -UTR

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出版历程
  • 收稿日期:  2023-09-06
  • 网络出版日期:  2023-11-13
  • 刊出日期:  2023-11-30

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