干扰foxM1基因表达可增强骨髓间充质干细胞的成骨分化能力
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摘要
背景:foxM1基因被认为参与了干细胞分化命运的调控,但其对间充质干细胞成骨分化的影响尚未见报道,关于foxM1基因的研究也主要集中于肿瘤领域。目的:探索干扰foxM1基因表达水平对骨髓间充质干细胞成骨分化能力的影响。方法:全骨髓贴壁法培养SD大鼠(中山大学实验动物中心提供)骨髓间充质干细胞,构建含嘌呤霉素抗性基因的foxM1 shRNA重组慢病毒载体并转染大鼠骨髓间充质干细胞,经嘌呤霉素筛选建立foxM1稳定敲低细胞株,另设置空病毒载体组以及未转染组。使用骨髓间充质干细胞成骨诱导分化培养基诱导培养并行茜素红染色检测其成骨分化能力,使用定量RT-PCR检测成骨相关基因的表达水平,同时提取胞核蛋白,Western blot检测β-catenin蛋白表达水平。结果与结论:(1)与空病毒载体组以及未转染组比较,foxM1敲低的大鼠骨髓间充质干细胞经成骨诱导分化后形成的骨结节数量明显增多,成骨相关基因col1a1和runx2表达水平均显著升高,但胞核β-catenin蛋白水平无明显改变;(2)干扰foxM1基因表达可增强骨髓间充质干细胞的成骨能力。
BACKGROUND:FoxM1 is thought to be involved in the regulation of stem cell differentiation fate, but its effect on osteogenic differentiation of mesenchymal stem cells has not been reported. The research on foxM1 gene is mainly focused on the tumor field.OBJECTIVE:To investigate the effect of shRNA-mediated knockdown of foxM1 on the osteogenic differentiation ability of bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells from Sprague-Dawley rats were isolated and cultured by the whole bone marrow adherence method. Recombinant lentivirus carrying foxM1-specific shRNA and puromycin-resistance gene was constructed and transfected into the rat bone marrow mesenchymal stem cells, and foxM1-knockdown bone marrow mesenchymal stem cells were acquired after puromycin screening. Alizarin red staining was performed to investigate the osteogenic differentiation ability of bone marrow mesenchymal stem cells cultured in osteogenic differentiation medium. Expression levels of several osteogenesis-related genes were examined using quantitative PCR. The nuclear expression of β-catenin was detected using western blot.RESULTS AND CONCLUSION:Knockdown of foxM1 in bone marrow mesenchymal stem cells enhanced the capability to form mineralized nodules and significantly increased the m RNA expressions of col1a1 and runx2, while no significant difference was found in the nuclear protein expressions of β-catenin. These results suggest that knockdown of foxM1 can promote the osteogenic differentiation of bone marrow mesenchymal stem cells.
BACKGROUND:FoxM1 is thought to be involved in the regulation of stem cell differentiation fate, but its effect on osteogenic differentiation of mesenchymal stem cells has not been reported. The research on foxM1 gene is mainly focused on the tumor field.OBJECTIVE:To investigate the effect of shRNA-mediated knockdown of foxM1 on the osteogenic differentiation ability of bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells from Sprague-Dawley rats were isolated and cultured by the whole bone marrow adherence method. Recombinant lentivirus carrying foxM1-specific shRNA and puromycin-resistance gene was constructed and transfected into the rat bone marrow mesenchymal stem cells, and foxM1-knockdown bone marrow mesenchymal stem cells were acquired after puromycin screening. Alizarin red staining was performed to investigate the osteogenic differentiation ability of bone marrow mesenchymal stem cells cultured in osteogenic differentiation medium. Expression levels of several osteogenesis-related genes were examined using quantitative PCR. The nuclear expression of β-catenin was detected using western blot.RESULTS AND CONCLUSION:Knockdown of foxM1 in bone marrow mesenchymal stem cells enhanced the capability to form mineralized nodules and significantly increased the m RNA expressions of col1a1 and runx2, while no significant difference was found in the nuclear protein expressions of β-catenin. These results suggest that knockdown of foxM1 can promote the osteogenic differentiation of bone marrow mesenchymal stem cells.
引文
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[17]Gartel AL.FOXM1 in Cancer:Interactions and Vulnerabilities.Cancer Res.2017;77(12):3135-3139.
[18]Hou Y,Li W,Sheng Y,et al.The transcription factor Foxm1 is essential for the quiescence and maintenance of hematopoietic stem cells.Nat Immunol.2015;16(8):810-818.
[19]Sun X,Luo LH,Feng L,et al.Down-regulation of lncRNA MEG3promotes endothelial differentiation of bone marrow derived mesenchymal stem cells in repairing erectile dysfunction.Life Sci.2018;208:246-252.
[20]Xu J,Huang Z,Lin L,et al.miRNA-130b is required for the ERK/FOXM1 pathway activation-mediated protective effects of isosorbide dinitrate against mesenchymal stem cell senescence induced by high glucose.Int J Mol Med.2015;35(1):59-71.
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[22]Qian J,Luo Y,Gu X,et al.Twist1 promotes gastric cancer cell proliferation through up-regulation of FoxM1.PLoS One.2013;8(10):e77625.
[23]Chen Q,Shou P,Zhang L,et al.An osteopontin-integrin interaction plays a critical role in directing adipogenesis and osteogenesis by mesenchymal stem cells.Stem Cells.2014;32(2):327-337.
[24]Xie Y,Li Y,Kong Y.OPN induces FoxM1 expression and localization through ERK 1/2,AKT,and p38 signaling pathway in HEC-1A cells.Int J Mol Sci.2014;15(12):23345-23358.
[25]Zhang N,Wei P,Gong A,et al.FoxM1 promotesβ-catenin nuclear localization and controls Wnt target-gene expression and glioma tumorigenesis.Cancer Cell.2011;20(4):427-442.
[26]陈钦桂,郑海崇,何婉媚,等.小鼠骨髓间充质干细胞的分离培养及其诱导分化为肺泡上皮细胞[J].医学研究生学报,2017,30(12):1283-1288.
[27]Kelleher FC,O'Sullivan H.FOXM1 in sarcoma:role in cell cycle,pluripotency genes and stem cell pathways.Oncotarget.2016;7(27):42792-42804.
[28]Lee Y,Kim KH,Kim DG,et al.FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2.PLoS One.2015;10(10):e0137703.
[29]Yuan B,Liu Y,Yu X,et al.FOXM1 contributes to taxane resistance by regulating UHRF1-controlled cancer cell stemness.Cell Death Dis.2018;9(5):562.
[30]Kwok CT,Leung MH,Qin J,et al.The Forkhead box transcription factor FOXM1 is required for the maintenance of cell proliferation and protection against oxidative stress in human embryonic stem cells.Stem Cell Res.2016;16(3):651-661.
[31]Besharat ZM,Abballe L,Cicconardi F,et al.Foxm1 controls a pro-stemness microRNA network in neural stem cells.Sci Rep.2018;8(1):3523.
[32]Zhang F,Luo K,Rong Z,et al.Periostin Upregulates Wnt/β-Catenin Signaling to Promote the Osteogenesis of CTLA4-Modified Human Bone Marrow-Mesenchymal Stem Cells.Sci Rep.2017;7:41634.
[33]Yuan Z,Li Q,Luo S,et al.PPARγand Wnt Signaling in Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells.Curr Stem Cell Res Ther.2016;11(3):216-225.
[34]Chen Y,Li Y,Xue J,et al.Wnt-induced deubiquitination FoxM1ensures nucleusβ-catenin transactivation.EMBO J.2016;35(6):668-684.
[35]Thiagarajan L,Abu-Awwad HAM,Dixon JE.Osteogenic Programming of Human Mesenchymal Stem Cells with Highly Efficient Intracellular Delivery of RUNX2.Stem Cells Transl Med.2017;6(12):2146-2159.
[2]Gupta A,Thussbas C,Koch M,et al.Management of glenoid bone defects with reverse shoulder arthroplasty-surgical technique and clinical outcomes.J Shoulder Elbow Surg.2018;27(5):853-862.
[3]Wang W,Yeung KWK.Bone grafts and biomaterials substitutes for bone defect repair:A review.Bioact Mater.2017;2(4):224-247.
[4]Croes M,Kruyt MC,Loozen L,et al.Local induction of inflammation affects bone formation.Eur Cell Mater.2017;33:211-226.
[5]Kobolak J,Dinnyes A,Memic A,et al.Mesenchymal stem cells:Identification,phenotypic characterization,biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche.Methods.2016;99:62-68.
[6]Tam WL,Luyten FP,Roberts SJ.From skeletal development to the creation of pluripotent stem cell-derived bone-forming progenitors.Philos Trans R Soc Lond B Biol Sci.2018;373(1750):20170218.
[7]Kon E,Filardo G,Roffi A,et al.Bone regeneration with mesenchymal stem cells.Clin Cases Miner Bone Metab.2012;9(1):24-27.
[8]van der Stok J,Koolen MK,Jahr H,et al.Chondrogenically differentiated mesenchymal stromal cell pellets stimulate endochondral bone regeneration in critical-sized bone defects.Eur Cell Mater.2014;27:137-148.
[9]Nauth A,Schemitsch EH.Stem cells for the repair and regeneration of bone.Indian J Orthop.2012;46(1):19-21.
[10]Cordeiro-Spinetti E,de Mello W,Trindade LS,et al.Human bone marrow mesenchymal progenitors:perspectives on an optimized in vitro manipulation.Front Cell Dev Biol.2014;2:7.
[11]Samsonraj RM,Rai B,Sathiyanathan P,et al.Establishing criteria for human mesenchymal stem cell potency.Stem Cells.2015;33(6):1878-1891.
[12]James AW.Review of Signaling Pathways Governing MSCOsteogenic and Adipogenic Differentiation.Scientifica(Cairo).2013;2013:684736.
[13]潘京华,黄浩,查振刚.间充质干细胞向成骨细胞分化中的Wnt信号通路[J].中国组织工程研究,2013,17(40):7144-7149.
[14]陈小静,高艳虹.Wnt信号通路调控间充质干细胞成骨分化的研究进展[J].上海交通大学学报(医学版),2013,33(1):99-103.
[15]Lampert FM,Kütscher C,Stark GB,et al.Overexpression of Hif-1αin Mesenchymal Stem Cells Affects Cell-Autonomous Angiogenic and Osteogenic Parameters.J Cell Biochem.2016;117(3):760-768.
[16]Baker N,Sohn J,Tuan RS.Promotion of human mesenchymal stem cell osteogenesis by PI3-kinase/Akt signaling,and the influence of caveolin-1/cholesterol homeostasis.Stem Cell Res Ther.2015;6:238.
[17]Gartel AL.FOXM1 in Cancer:Interactions and Vulnerabilities.Cancer Res.2017;77(12):3135-3139.
[18]Hou Y,Li W,Sheng Y,et al.The transcription factor Foxm1 is essential for the quiescence and maintenance of hematopoietic stem cells.Nat Immunol.2015;16(8):810-818.
[19]Sun X,Luo LH,Feng L,et al.Down-regulation of lncRNA MEG3promotes endothelial differentiation of bone marrow derived mesenchymal stem cells in repairing erectile dysfunction.Life Sci.2018;208:246-252.
[20]Xu J,Huang Z,Lin L,et al.miRNA-130b is required for the ERK/FOXM1 pathway activation-mediated protective effects of isosorbide dinitrate against mesenchymal stem cell senescence induced by high glucose.Int J Mol Med.2015;35(1):59-71.
[21]Kronenberg HM.Twist genes regulate Runx2 and bone formation.Dev Cell.2004;6(3):317-318.
[22]Qian J,Luo Y,Gu X,et al.Twist1 promotes gastric cancer cell proliferation through up-regulation of FoxM1.PLoS One.2013;8(10):e77625.
[23]Chen Q,Shou P,Zhang L,et al.An osteopontin-integrin interaction plays a critical role in directing adipogenesis and osteogenesis by mesenchymal stem cells.Stem Cells.2014;32(2):327-337.
[24]Xie Y,Li Y,Kong Y.OPN induces FoxM1 expression and localization through ERK 1/2,AKT,and p38 signaling pathway in HEC-1A cells.Int J Mol Sci.2014;15(12):23345-23358.
[25]Zhang N,Wei P,Gong A,et al.FoxM1 promotesβ-catenin nuclear localization and controls Wnt target-gene expression and glioma tumorigenesis.Cancer Cell.2011;20(4):427-442.
[26]陈钦桂,郑海崇,何婉媚,等.小鼠骨髓间充质干细胞的分离培养及其诱导分化为肺泡上皮细胞[J].医学研究生学报,2017,30(12):1283-1288.
[27]Kelleher FC,O'Sullivan H.FOXM1 in sarcoma:role in cell cycle,pluripotency genes and stem cell pathways.Oncotarget.2016;7(27):42792-42804.
[28]Lee Y,Kim KH,Kim DG,et al.FoxM1 Promotes Stemness and Radio-Resistance of Glioblastoma by Regulating the Master Stem Cell Regulator Sox2.PLoS One.2015;10(10):e0137703.
[29]Yuan B,Liu Y,Yu X,et al.FOXM1 contributes to taxane resistance by regulating UHRF1-controlled cancer cell stemness.Cell Death Dis.2018;9(5):562.
[30]Kwok CT,Leung MH,Qin J,et al.The Forkhead box transcription factor FOXM1 is required for the maintenance of cell proliferation and protection against oxidative stress in human embryonic stem cells.Stem Cell Res.2016;16(3):651-661.
[31]Besharat ZM,Abballe L,Cicconardi F,et al.Foxm1 controls a pro-stemness microRNA network in neural stem cells.Sci Rep.2018;8(1):3523.
[32]Zhang F,Luo K,Rong Z,et al.Periostin Upregulates Wnt/β-Catenin Signaling to Promote the Osteogenesis of CTLA4-Modified Human Bone Marrow-Mesenchymal Stem Cells.Sci Rep.2017;7:41634.
[33]Yuan Z,Li Q,Luo S,et al.PPARγand Wnt Signaling in Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells.Curr Stem Cell Res Ther.2016;11(3):216-225.
[34]Chen Y,Li Y,Xue J,et al.Wnt-induced deubiquitination FoxM1ensures nucleusβ-catenin transactivation.EMBO J.2016;35(6):668-684.
[35]Thiagarajan L,Abu-Awwad HAM,Dixon JE.Osteogenic Programming of Human Mesenchymal Stem Cells with Highly Efficient Intracellular Delivery of RUNX2.Stem Cells Transl Med.2017;6(12):2146-2159.
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