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

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

昆明医科大学附属口腔医院口腔修复科，云南昆明　650106

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
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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

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

Dept. of Oral Restorations，Affiliated Stomatological Hospital of Kunming Medical University，Kunming Yunnan 650106，China

摘要

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。
- miR-148a-3p /
- 牙源性间充质干细胞 /
- 成骨分化 /
- 牙釉质发育 /
- SMURF2
Abstract: Objective To explore the role of miR-148a-3p in osteogenic differentiation and enamel development during in vitro tooth organogenesis and uncover its underlying molecular mechanism. Methods Human dental pulp stem cells and oral epithelial cells were obtained. Human dental pulp stem cells were transfected with miR-148a-3p mimics, miR-148a-3p inhibitors, or a negative control. A three-dimensional co-culture system was established by seeding human dental pulp stem cells onto the Matrigel matrix and overlaying them with oral epithelial cells. The impact of miR-148a-3p on cell proliferation and protein expression was assessed using MTT assay. The expression levels of osteogenic marker RUNX2 and enamel development marker E-cadherin were determined through Western blotting. The interaction between SMURF2（SMAD-specific E3 ubiquitin-protein ligase 2） and miR-148a-3p was validated via a luciferase reporter assay. Results Downregulation of miR-148a-3p using its inhibitor in human dental pulp stem cells led to reduced cell viability in the 3D co-culture system（P < 0.05）. Conversely, upregulation of miR-148a-3p using its mimic increased the expression of osteogenic marker RUNX2 and enamel development marker E-cadherin in the co-culture setting（P < 0.05）. TargetScan software predicted a binding site for miR-148a-3p within the 3’ -UTR of SMURF2. Luciferase report Experiments showed that miR-148a-3p mimics significantly inhibited the luciferase activity of wild-type（P < 0.05）, while Western blot results showed that miR-148a-3p mimics significantly down-regulated the expression of SMURF2（P < 0.05）. Conclusion The research findings suggest that miR-148a-3p potentially regulates the expression of RUNX2 and E-cadherin by targeting SMURF2, thereby participating in osteogenic differentiation of human dental pulp stem cells and enamel development in oral epithelial cells during in vitro tooth organogenesis. This study provides promising therapeutic targets for dental repair and treatment.
- MiR-148a-3p /
- Dental pulp stem cells /
- Osteogenic differentiation /
- Enamel development /
- SMURF2

HTML全文

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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