MicroRNA-125b对人牙周膜干细胞成骨分化的影响

doi:10.19439/j.sjos.2018.01.003

上海口腔医学 ›› 2018, Vol. 27 ›› Issue (1): 11-17.doi: 10.19439/j.sjos.2018.01.003

MicroRNA-125b对人牙周膜干细胞成骨分化的影响

杜莉, 曹伟靖, 田莹, 王原明

延安大学附属医院口腔科,陕西延安 716000

收稿日期:2017-04-25 修回日期:2017-08-21 出版日期:2018-02-25 发布日期:2018-03-05
通讯作者: 王原明,E-mail：yuanmingkq@163.com
作者简介:杜莉（1974-）,女,学士,副主任医师,E-mail：272855787@qq.com

MicroRNA-125b regulates osteogenic differentiation of human periodontal ligament stem cells

DU Li, CAO Wei-jing, TIAN Ying, WANG Yuan-ming

Department of Stomatology, Yanan University Affiliated Hospital. Yanan 716000, Shaanxi Province, China

Received:2017-04-25 Revised:2017-08-21 Online:2018-02-25 Published:2018-03-05

摘要/Abstract

摘要： 目的: 探讨microRNA-125b（miR-125b）对人牙周膜干细胞（periodontal ligament stem cell, PDLSCs）成骨分化的影响。方法: 利用组织块法分离培养PDLSCs,流式细胞术检测细胞表面标志分子。PDLSCs经miR-125b模拟物或miR-125b抑制剂转染后,采用MTT法和LDH试剂盒分别检测细胞活性和细胞毒性,碱性磷酸酶（alkaline phosphatase,ALP）活性和细胞内钙离子水平均以相应试剂盒检测,Western 印迹法和RT-qPCR检测成骨相关基因水平。双荧光素酶基因报告实验检测miR-125b对细胞间隙连接蛋白43（connexin43,Cx43）的靶向调控作用。采用SPSS17.0软件包对数据进行统计学分析。结果: 体外分离培养的PDLSCs呈现间充质干细胞特性。转染miR-125b mimic后,PDLSCs的活性下降,毒性上升,ALP活性、钙离子水平以及成骨相关分子水平均显著下降;相反,anti-miR125b提高了PDLSCs的细胞活性、ALP活性、钙离子水平以及成骨相关分子的表达。双荧光素酶基因报告实验结果表明,miR-125b能够靶向降低Cx43基因水平。而在Cx43过表达的PDLSCs中,miR-125b模拟物对PDLSCs成骨分化的作用被反转。结论: miR-125b通过靶向下调Cx43而抑制PDLSCs的成骨分化。

关键词: 牙周膜干细胞, microRNA-125b, 细胞间隙连接蛋白43

Abstract: PURPOSE: To investigate the effect of microRNA-125b (miR-125b) on osteogenic differentiation of periodontal ligament stem cell (PDLSCs). METHODS: The surface factor of isolated PDLSCs was detected by flow cytometry. After transfected with miR-125b or anti-miR125b in PDLSCs, MTT and LDH were used to detect cell viability and cytotoxicity; ALP activity and calcium level were detected by ALP assay kit and calcium colorimetric assay kit. Western blot and RT-qPCR were used to determine the expression of osteogenesis-related genes. The interaction between miR-125b and connexin 43 (Cx43) was detected by dual luciferase gene reporter assay. The data were analyzed with SPSS 17.0 software. RESULTS: The cultured PDLSCs showed the characteristics of mesenchymal stem cells. After transfected with miR-125b in PDLSCs, the cell viability was decreased, cytotoxicity was increased; ALP activity, calcium level and the expression of osteogenesis-related genes were significantly decreased. On the contrary, cell viability, ALP activity, calcium level and the expression of osteogenesis-related genes in PDLSCs were increased after anti-miR-125b transfection. Dual luciferase gene reporter assay showed that miR-125b could target Cx43. In addition, the effect of miR-125b on osteogenic differentiation of PDLSCs was reversed after Cx43 overexpression. CONCLUSIONS: miR-125b may inhibit osteogenic differentiation of PDLSCs by targeting Cx43.

Key words: Periodontal ligament stem cell, MicroRNA-125b, Connexin43

中图分类号:

R781.4

杜莉, 曹伟靖, 田莹, 王原明. MicroRNA-125b对人牙周膜干细胞成骨分化的影响[J]. 上海口腔医学, 2018, 27(1): 11-17.

DU Li, CAO Wei-jing, TIAN Ying, WANG Yuan-ming. MicroRNA-125b regulates osteogenic differentiation of human periodontal ligament stem cells[J]. Shanghai Journal of Stomatology, 2018, 27(1): 11-17.

参考文献

[1] Hajishengallis G.Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response[J]. Trends Immunol, 2014, 35(1): 3-11.
[2] Moshaverinia A, Xu X, Chen C, et al.Application of stem cells derived from the periodontal ligament or gingival tissue sources for tendon tissue regeneration[J]. Biomaterials, 2014, 35(9): 2642-2650.
[3] Chen FM, Gao LN, Tian BM, et al.Treatment of periodontal intrabony defects using autologous periodontal ligament stem cells: a randomized clinical trial[J]. Stem Cell Res Ther, 2016, 7: 33.
[4] Seo BM, Miura M, Gronthos S, et al.Investigation of multipotent postnatal stem cells from human periodontal ligament[J]. Lancet, 2004, 364(9429): 149-155.
[5] 吕红兵, 卢兆杰, 郑碧琼, 等. 人牙周膜干细胞与牙周膜非干细胞差异表达microRNA靶基因及基因功能分析[J]. 中国实用口腔科杂志, 2014, 7(7): 403-406.
[6] Zhou Q, Zhao ZN, Cheng JT, et al.Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs[J]. Biochem Biophys Res Commun, 2011, 404(1): 127-132.
[7] 吕红兵. 应用基因芯片分析牙周膜干细胞向成骨细胞分化过程中miRNA表达谱的差异[J]. 中国医学工程, 2014, 22(5): 3-4.
[8] Kumar PS, Saxena R, Patil S, et al.Clinical investigation of periodontal ligament distraction osteogenesis for rapid orthodontic canine retraction[J]. Aust Orthod J, 2009, 25(2): 147-152.
[9] Genna F, Paganelli C, Salgarello S, et al.Mechanical response of bone under short-term loading of a dental implant with an internal layer simulating the nonlinear behaviour of the periodontal ligament[J]. Comput Methods Biomech Biomed Engin, 2003, 6(5-6): 305-318.
[10] Oliveto S, Mancino M, Manfrini N, et al.Role of microRNAs in translation regulation and cancer[J]. World J Biol Chem, 2017, 8(1): 45-56.
[11] Wang H, Gou X, Jiang T, et al.The effects of microRNAs on glucocorticoid responsiveness[J]. J Cancer Res Clin Oncol, 2017, 143(6): 1005-1011.
[12] Zhang Q, Zhang K, Zhang C, et al.MicroRNAs as big regulators of neural stem/progenitor cell proliferation, differentiation and migration: a potential treatment for stroke[J]. Curr Pharm Des, 2017, 23(15): 2252-2257.
[13] 陆细红, 邓敏, 贺洪辉, 等. miR-125b通过靶向抑制Smad4调控骨髓间充质干细胞成骨分化[J]. 中南大学学报(医学版), 2013, 38(4): 341-346.
[14] Mizuno Y, Yagi K, Tokuzawa Y, et al.miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation[J]. Biochem Biophys Res Commun, 2008, 368(2): 267-272.
[15] Xu J, Li Z, Hou Y, et al.Potential mechanisms underlying the Runx2 induced osteogenesis of bone marrow mesenchymal stem cells[J]. Am J Transl Res, 2015, 7(12): 2527-2535.
[16] Ching HS, Luddin N, Rahman IA, et al.Expression of odontogenic and osteogenic markers in dpscs and shed: a review[J]. Curr Stem Cell Res Ther, 2017, 12(1): 71-79.
[17] Shrivats AR, Mcdermott MC, Klimak M, et al.Nanogel-mediated rnai against runx2 and osx inhibits osteogenic differentiation in constitutively active bmpr1a osteoblasts[J]. ACS Biomater Sci Eng, 2015, 1(11): 1139-1150.
[18] Jin H, Choung HW, Lim KT, et al.Recombinant human plasminogen activator inhibitor-1 promotes cementogenic differentiation of human periodontal ligament stem cells[J]. Tissue Eng Part A, 2015, 21(23-24): 2817-2828.
[19] Chen D, Liu Y, Yang H, et al.Connexin 43 promotes ossification of the posterior longitudinal ligament through activation of the ERK1/2 and p38 MAPK pathways[J]. Cell Tissue Res, 2016, 363(3): 765-773.
[20] João SM, Arana-Chavez VE. Expression of connexin 43 and ZO-1 in differentiating ameloblasts and odontoblasts from rat molar tooth germs[J]. Histochem Cell Biol, 2003, 119(1): 21-26.
[21] 徐智云, 刘鑫, 黄兴, 等. 细胞间隙连接蛋白43基因在斑马鱼牙齿早期发育中的表达[J]. 华西口腔医学杂志, 2015, 33(4): 347-351.
[22] Yang H, Shi L, Shi G, et al.Connexin 43 affects osteogenic differentiation of the posterior longitudinal ligament cells via regulation of ERK activity by stabilizing Runx2 in ossification[J]. Cell Physiol Biochem, 2016, 38(1): 237-247.

MicroRNA-125b对人牙周膜干细胞成骨分化的影响

MicroRNA-125b regulates osteogenic differentiation of human periodontal ligament stem cells

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