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

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

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。

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.