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山东大学学报 (医学版) ›› 2020, Vol. 58 ›› Issue (3): 87-93.doi: 10.6040/j.issn.1671-7554.0.2019.1367

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miR-222-5p在人根尖乳头干细胞成骨/成牙本质向分化中的作用

宋孟晓1,王燕2,3,4,刘进忠1   

  1. 1.郑州大学第一附属医院口腔科, 河南 郑州 450052;2.山东大学口腔医学院·口腔医院特诊科, 山东 济南 250012;3.山东省口腔组织再生重点实验室, 山东 济南 250012;4.山东省口腔生物材料与组织再生工程实验室, 山东 济南 250012
  • 出版日期:2020-03-10 发布日期:2022-09-27
  • 通讯作者: 刘进忠. E-mail:liujz@zzu.edu.cn

MiR-222-5p promotes osteo/odontogenic differentiation of stem cells from human apical papilla

SONG Mengxiao1, WANG Yan2,3,4, LIU Jinzhong1   

  1. 1. Department of Stomatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China;
    2. Department of VIP Center, School and Hospital of Stomatology, Shandong University, Jinan 250012, Shandong, China;
    3. Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan 250012, Shandong, China;
    4. Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, Jinan 250012, Shandong, China
  • Online:2020-03-10 Published:2022-09-27

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摘要： 目的 探讨miR-222-5p在人根尖乳头干细胞(SCAP)成骨/成牙本质向分化中的作用。 方法 选取芯片研究中差异表达的miRNAs 19个,采用qPCR验证SCAP成骨/成牙本质分化中以上miRNAs的表达;将19个miRNAs转染SCAP,成骨/成牙本质向诱导分化,qPCR检测DSPP、Runx2的表达,挑选表达量高的miR-222-5p组,Western blotting检测DSPP、Runx2蛋白水平的表达。生物信息学靶基因预测miR-222-5p的潜在靶基因,构建靶基因野生型/突变型双荧光素酶报告基因载体,与miR-222-5p共转染HEK 293T,双荧光素酶报告基因实验检测荧光素酶活性,证明miR-222-5p和预测的靶基因之间的关系。Western blotting验证 miR-222-5p转染SCAP后靶基因的表达。进一步研究抑制内源性miR-222-5p和干扰靶基因对SCAP成骨/成牙本质向分化的影响。 结果 19个miRNAs表达与芯片结果一致,其中miR-222-5p表达量高;转染miR-222-5p组DSPP、Runx2的mRNA及蛋白表达明显上调;生物信息学及双荧光素酶报告基因实验确认人分泌型卷曲相关蛋白4(SFRP4)是miR-222-5p的直接靶基因,而且过表达miR-222-5p抑制SFRP4表达;干扰SFRP4可以有效地逆转miR-222-5p inhibitor对DSPP表达的抑制作用。 结论 miR-222-5p通过靶向调节SFRP4促进SCAP成骨/成牙本质向分化。

关键词: miR-222-5p, 根尖乳头干细胞, 成骨/成牙本质向分化, SFRP4, Wnt信号通路

Abstract: Objective To investigate the role of miR-222-5p in osteo/odontogenic differentiation of human stem cells from apical papilla(SCAP)and the related mechanism. Methods The expression of 19 miRNAs in osteo/odontogenic differentiation of SCAP was verified with qPCR. The mRNA expressions of DSPP and Runx2 were measured after SCAP was transfected with 19miRNAs to induce osteo/odontogenic differentiation. The protein expressions of DSPP and Runx2 in the high-expression miR-222-5p group were determined with Western blotting. The potential target gene of miR-222-5p was predicted with bioinformatics analysis. The wild-type(WT)/mutant-type(Mut)reporter vector was constructed to co-transfect HEK293T cells with miR-222-5p. The luciferase activities were evaluated using the dual-luciferase reporter assay system to prove the relationship between miR-222-5p and the target gene. Then the expression of the target gene was tested with Western blotting after overexpression of miR-222-5p. The effects of miR-222-5p and target gene on osteo/odontogenic differentiation of SCAP were further explored. Results The expression of 19 miRNAs detected by qPCR was consistent with that detected by microarray in which miR-222-5p was highly expressed. The mRNA and protein expressions of DSPP and Runx2 were upregulated in miR-222-5p transfected group. Bioinformatics analysis and dual luciferase reporter assay identified SFRP4 as the direct target gene of miR-222-5p, and overexpression of miR-222-5p could inhibit SFRP4 expression. SFRP4 knockdown could effectively reverse the inhibitory effect of miR-222-5p inhibitor on the mRNA expression of DSPP. Conclusion miR-222-5p targetsd SFRP4 and promoted osteo/odontogenic differentiation of SCAP.

Key words: miR-222-5p, Stem cells from apical papilla, Osteo/odontogenic differentiation, SFRP4, Wnt signaling pathway

中图分类号: 

  • R781.3
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