miR-203靶向RUNX2对牙周膜干细胞成骨分化能力的调控作用
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摘要
目的:体外研究miR-203对人牙周膜干细胞成骨分化能力的影响及调控机制。方法:分离培养人牙周膜干细胞,检测牙周膜干细胞矿化诱导前后miR-203和RUNX2的表达;分别转染miR-203沉默及过表达模拟物;qPCR检测miR-203和RUNX2 mRNA水平的表达;Western blot检测RUNX2蛋白水平的表达,茜素红染色观察细胞成骨分化能力的变化;生物信息学分析miR-203的靶基因,双荧光素酶报告实验验证;共转染miR-203 inhibitor和RUNX2 siRNA,分析共转染后对RUNX2表达及细胞成骨分化能力的影响。结果:成骨诱导液诱导后牙周膜干细胞中miR-203的表达水平显著下调,而RUNX2的表达水平明显升高;miR-203过表达可明显抑制h PDLSCs的成骨分化能力和RUNX2的表达,抑制miR-203的表达,则结果相反;通过生物信息学分析miR-203的潜在靶基因含有RUNX2,双荧光素酶报告实验验证RUNX2为miR-203的靶基因;进一步实验结果表明沉默RUNX2可逆转miR-203 inhibitor对牙周膜干细胞成骨分化的促进作用。结论:miR-203可靶向调控RUNX2对人牙周膜干细胞成骨分化能力产生影响,提示miR-203在h PDLSCs组织工程损伤修复过程中发挥着重要作用。
Objective: To investigate regulation of miR-203 on the osteogenic differentiation capacity of human periodontal ligamentstem cell(hPDLSCs) in vitro. Methods: The expression of miR-203 and RUNX2 were detected in h PDLSCs under conduction of bone induction for two weeks. Periodontal ligament stem cells were isolated from human healthy periodontal ligament tissue and infected with miR-203. Then the osteogenic differentiation capacity were evaluated alizarin red assay. The expression of miR-203 and RUNX2 were detected by q-RCR and Western blot. The potential target genes of miR-203 were analyzed by bioinformatics and verified by dual luciferase report. Results: The expression of miR-203 were significant down-regulated but RUNX2 were up-regulated under the conduction of bone induction. The osteogenic differentiation capacity of h PDLSCs were inhibited after overexpression of miR-203. RUNX2 was the potential target gene of miR-203, which verified by bioinformatics analysis and dual luciferase report. In addition, si-RUNX2 was able to reverse the induced effect of miR-203 inhibitor on the osteogenic differentiation of hPDLSCs. Conclusion: miR-203 inhibit the osteogenic differentiation ability of hPDLSCs via inhibiting the expression of RUNX2, it was suggested that miR-203 plays an important role in hPDLSCs involved in injury repair.
Objective: To investigate regulation of miR-203 on the osteogenic differentiation capacity of human periodontal ligamentstem cell(hPDLSCs) in vitro. Methods: The expression of miR-203 and RUNX2 were detected in h PDLSCs under conduction of bone induction for two weeks. Periodontal ligament stem cells were isolated from human healthy periodontal ligament tissue and infected with miR-203. Then the osteogenic differentiation capacity were evaluated alizarin red assay. The expression of miR-203 and RUNX2 were detected by q-RCR and Western blot. The potential target genes of miR-203 were analyzed by bioinformatics and verified by dual luciferase report. Results: The expression of miR-203 were significant down-regulated but RUNX2 were up-regulated under the conduction of bone induction. The osteogenic differentiation capacity of h PDLSCs were inhibited after overexpression of miR-203. RUNX2 was the potential target gene of miR-203, which verified by bioinformatics analysis and dual luciferase report. In addition, si-RUNX2 was able to reverse the induced effect of miR-203 inhibitor on the osteogenic differentiation of hPDLSCs. Conclusion: miR-203 inhibit the osteogenic differentiation ability of hPDLSCs via inhibiting the expression of RUNX2, it was suggested that miR-203 plays an important role in hPDLSCs involved in injury repair.
引文
[1]Seo B M,Miura M,Gronthos S,et al.Investigation of multipotent postnatal stem cells from human periodontal ligament[J].Lancet,2004,364(9429):149-155
[2]Liu Y,Zh eng Y,Ding G,et al.Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine[J].Stem Cells,2008,26(4):1065-1073
[3]Tang Y,Wu X,Lei W,et al.TGF-beta1-induced migration of bone mesench ymal stem cells couples bone resorption with formation[J].Nat Med,2009,15(7):757-765
[4]Wada N,Menicanin D,Shi S,et al.Immunomodulatory properties of human periodontal ligament stem cells[J].J Cell Ph ysiol,2009,219(3):667-676
[5]Yoo Y J,Oh J H,Lee W,et al.Regenerative Characteristics of Apical Papilla-derived Cells from Immature Teeth with Pulpal and Periapical Path osis[J].J Endod,2016,42(11):1626-1632
[6]贺慧霞,刘洪臣,李华,et al.犬牙周膜干细胞用于附着龈组织工程化再生的实验研究[J].口腔颌面修复学杂志,2013,14(4):193-198
[7]Kidwai F,Edwards J,Zou L,et al.Fibrinogen Induces RUNX2 Activity and Osteogenic Development from Human Pluripotent Stem Cells[J].Stem Cells,2016,34(8):2079-2089
[8]Xu J,Li Z,Hou Y,et al.Potential mechanisms underlying the Runx 2 induced osteogenesis of bone marrow mesenchymalstem cells[J].Am J Transl Res,2015,7(12):2527-2535
[9]Deng B,Wang B,Fang J,et al.MiRNA-203 suppresses cell proliferation,migration and invasion in colorectal cancer via targeting of EIF5A2[J].Sci Rep,2016,6:28301
[10]Pillai R S.MicroRNA function:multiple mechanisms for a tiny RNA?[J].Rna,2005,11(12):1753-1761
[11]Yin Q,Wang J,Fu Q,et al.CircRUNX2 through hasmiR-203 regulates RUNX2 to prevent osteoporosis[J].J Cell Mol Med,2018,22(12):6112-6121
[12]Tu B,Liu S,Yu B,et al.miR-203 inh ibits the traumatich eterotopicossification by targeting Runx 2[J].Cell Death Dis,2016,7(10):e2436
[13]Bueno C,Ramirez C,Rodriguez-Lozano F J,et al.Human adult periodontal ligament-derived cells integrate and differentiate after implantation into the adult mammalian brain[J].Cell Transplant,2013,22(11):2017-2028
[14]Bruderer M,Rich ards R G,Alini M,et al.Role and regulation of RUNX2 in osteogenesis[J].Eur Cell Mater,2014,28:269-286
[15]Komori T.Roles of Runx 2 in Skeletal Development[J].Adv Exp Med Biol,2017,962:83-93
[16]Li B,Zhang Y,Wang Q,et al.Periodontal ligament stem cells modulate root resorption of human primary teeth via Runx 2 regulating RANKL/OPG system[J].Stem Cells Dev,2014,23(20):2524-2534
[2]Liu Y,Zh eng Y,Ding G,et al.Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine[J].Stem Cells,2008,26(4):1065-1073
[3]Tang Y,Wu X,Lei W,et al.TGF-beta1-induced migration of bone mesench ymal stem cells couples bone resorption with formation[J].Nat Med,2009,15(7):757-765
[4]Wada N,Menicanin D,Shi S,et al.Immunomodulatory properties of human periodontal ligament stem cells[J].J Cell Ph ysiol,2009,219(3):667-676
[5]Yoo Y J,Oh J H,Lee W,et al.Regenerative Characteristics of Apical Papilla-derived Cells from Immature Teeth with Pulpal and Periapical Path osis[J].J Endod,2016,42(11):1626-1632
[6]贺慧霞,刘洪臣,李华,et al.犬牙周膜干细胞用于附着龈组织工程化再生的实验研究[J].口腔颌面修复学杂志,2013,14(4):193-198
[7]Kidwai F,Edwards J,Zou L,et al.Fibrinogen Induces RUNX2 Activity and Osteogenic Development from Human Pluripotent Stem Cells[J].Stem Cells,2016,34(8):2079-2089
[8]Xu J,Li Z,Hou Y,et al.Potential mechanisms underlying the Runx 2 induced osteogenesis of bone marrow mesenchymalstem cells[J].Am J Transl Res,2015,7(12):2527-2535
[9]Deng B,Wang B,Fang J,et al.MiRNA-203 suppresses cell proliferation,migration and invasion in colorectal cancer via targeting of EIF5A2[J].Sci Rep,2016,6:28301
[10]Pillai R S.MicroRNA function:multiple mechanisms for a tiny RNA?[J].Rna,2005,11(12):1753-1761
[11]Yin Q,Wang J,Fu Q,et al.CircRUNX2 through hasmiR-203 regulates RUNX2 to prevent osteoporosis[J].J Cell Mol Med,2018,22(12):6112-6121
[12]Tu B,Liu S,Yu B,et al.miR-203 inh ibits the traumatich eterotopicossification by targeting Runx 2[J].Cell Death Dis,2016,7(10):e2436
[13]Bueno C,Ramirez C,Rodriguez-Lozano F J,et al.Human adult periodontal ligament-derived cells integrate and differentiate after implantation into the adult mammalian brain[J].Cell Transplant,2013,22(11):2017-2028
[14]Bruderer M,Rich ards R G,Alini M,et al.Role and regulation of RUNX2 in osteogenesis[J].Eur Cell Mater,2014,28:269-286
[15]Komori T.Roles of Runx 2 in Skeletal Development[J].Adv Exp Med Biol,2017,962:83-93
[16]Li B,Zhang Y,Wang Q,et al.Periodontal ligament stem cells modulate root resorption of human primary teeth via Runx 2 regulating RANKL/OPG system[J].Stem Cells Dev,2014,23(20):2524-2534