MAGE-D1对大鼠牙胚来源外胚间充质干细胞增殖迁移能力的影响
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摘要
目的研究黑色素瘤相关抗原D1(Melanoma associated antigens,MAGE-D1)对大鼠牙胚来源的外胚间充质干细胞(Ectomensenchymal stem cells,EMSCs)增殖及迁移能力的影响。方法取SD大鼠孕19. 5 d胎鼠牙胚组织,组织块法培养牙胚来源外胚间充质干细胞,流式细胞仪进行外胚间充质干细胞表面分子鉴定CD14、CD29、CD44、CD45、CD90、CD105、CD146、CD166和p75NTR,MAGE-D1 siRNA转染入外胚间充质干细胞,实验组分为:对照组(Control)和MAGE-D1 siRNA组(MAGEsiRNA-EMSCs),CCK8法和划痕法分别检测两组细胞增殖能力与迁移能力,Real Time-PCR及Western blot检测迁移及WNT通路相关基因MAGE-D1、β-catenin、E-cadherin的mRNA及蛋白表达量。结果组织块法成功培养出牙胚来源的外胚间充质干细胞,经流式细胞仪检测细胞阳性表达CD14(92. 16%)、CD29(94. 53%)、CD44(99. 51%)、CD90(95. 18%)、CD105(34. 22%)、CD146(92. 39%)、CD166(98. 15%)和p75NTR(88. 56%),阴性表达CD45(0. 5%);CCK8法检测MAGE-D1 siRNA组EMSCs增殖能力受到抑制;划痕试验法检测MAGE-D1 siRNA组细胞48 h迁移量高于对照组细胞;Real Time-PCR检测Mage-D1、β-catenin和E-cadherin表达量,MAGE-D1 siRNA组EMSCs MAGE-D1和E-cadherin表达较对照组低,而β-catenin表达较高,Western blot检测MAGE-D1、β-catenin和E-cadherin表达水平趋势与Real Time-PCR结果相一致。结论抑制EMSCs中MAGE-D1的表达上调β-catenin,下调E-cadherin,能够提高细胞的迁移能力,但是其导致增殖能力降低,MAGE-D1可能作为EMSCs迁移增殖能力的关键因子。
Objective To study the effects of MAGE-D1 on the proliferation and migration of ectomesenchymal stem cells originated from rat teeth germ. Methods Ectomesenchymal stem cells( EMSCs) were cultures by tissue mass technique. The mesenchymal membrane surface molecular CD14,CD29,CD44,CD45,CD90,CD105,CD146,CD166 and p75 NTR of ectomesenchymal stem cells were detected. This study was divided into non-treatment EMSCs group( Control) and MAGE-D1 siRNA group( MAGE siRNA-EMSCs). Cell proliferation was detected by CCK8 assay. The migration ability of EMSCs was analyzed by scratch wound healing assay. Real-time PCR and western blot assay was applied to detect the mRNA and protein level of migration and WNT related genes MAGE-D1,β-catenin and E-cadherin. Results EMSCs were successfully cultured by tissue mass technique. The cell membrane molecular expression were CD14( 92. 16%),CD29( 94. 53%),CD44(99. 51%),CD90( 95. 18%),CD105( 34. 22%),CD146( 92. 39%),CD166( 98. 15%),p75 NTR(88. 56%)and CD45(0. 5%)in teeth germ originated EMSCs. The proliferation ability in MAGE siRNA-EMSCs group was lower than that in control group. A higher wound-healing rate was shown in MAGE siRNA-EMSCs group compared to control group. Lower expressions of MAGE-D1 and E-cadherin in MAGE siRNA-EMSCs group and the higher expression of β-catenin were indicated. Conclusion The inhibition of MAGE-D1 expression leads to upregulating β-catenin and downregulating E-cadherin and further promoting the migration rate of EMSCs,but EMSCs proliferation ability is decreased,which suggesting that MAGE-D1 might be the key factor of proliferation and migration in EMSCs.
Objective To study the effects of MAGE-D1 on the proliferation and migration of ectomesenchymal stem cells originated from rat teeth germ. Methods Ectomesenchymal stem cells( EMSCs) were cultures by tissue mass technique. The mesenchymal membrane surface molecular CD14,CD29,CD44,CD45,CD90,CD105,CD146,CD166 and p75 NTR of ectomesenchymal stem cells were detected. This study was divided into non-treatment EMSCs group( Control) and MAGE-D1 siRNA group( MAGE siRNA-EMSCs). Cell proliferation was detected by CCK8 assay. The migration ability of EMSCs was analyzed by scratch wound healing assay. Real-time PCR and western blot assay was applied to detect the mRNA and protein level of migration and WNT related genes MAGE-D1,β-catenin and E-cadherin. Results EMSCs were successfully cultured by tissue mass technique. The cell membrane molecular expression were CD14( 92. 16%),CD29( 94. 53%),CD44(99. 51%),CD90( 95. 18%),CD105( 34. 22%),CD146( 92. 39%),CD166( 98. 15%),p75 NTR(88. 56%)and CD45(0. 5%)in teeth germ originated EMSCs. The proliferation ability in MAGE siRNA-EMSCs group was lower than that in control group. A higher wound-healing rate was shown in MAGE siRNA-EMSCs group compared to control group. Lower expressions of MAGE-D1 and E-cadherin in MAGE siRNA-EMSCs group and the higher expression of β-catenin were indicated. Conclusion The inhibition of MAGE-D1 expression leads to upregulating β-catenin and downregulating E-cadherin and further promoting the migration rate of EMSCs,but EMSCs proliferation ability is decreased,which suggesting that MAGE-D1 might be the key factor of proliferation and migration in EMSCs.
引文
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[9]Xue B,Wen C,Shi Y,etal. Human NRAGE disrupts Ecadherin/beta-catenin regulated homotypic cell-cell adhesion[J]. Biochemical&Biophysical Research Communications,2005,336(1):247-251.
[10]Shen W G,Xue Q Y,Zhu J,et al. Inhibition of adenovirus-mediated human MAGE-D1 on angiogenesis in vitro and in vivo[J]. Molecular&Cellular Biochemistry,2007,300(1-2):89-99.
[11]Cao S,Zhang F,Zhao P,et al. The effects of annexin a2expression on the proliferation,migration and invasion of human breast cancer cells[J]. Chinese Journal of Clinical Oncology,2011,38(6):304-307.
[12]Chu C S,Xue B,Tu C,et al. NRAGE suppresses metastasis of melanoma and pancreatic cancer in vitro and in vivo[J]. Cancer Letters,2007,250(2):268-275.
[13]Lai S S,Xue B,Yang Y,et al. Ror2-Src signaling in metastasis of mouse melanoma cells is inhibited by NRAGE[J]. Cancer Genet,2012,205(11):552-562.
[14]Chang X,Xue X,Zhang Y,et al. The role of NRAGE subcellular location and epithelial-mesenchymal transition on radiation resistance of esophageal carcinoma cell[J].Journal of Cancer Research&Therapeutics,2018,14(1):46.
[15]Yang K,Wang Y,Ju Y,et al. p75 neurotrophin receptor regulates differential mineralization of rat ectomesenchymal stem cells[J]. Cell Proliferation,2016,50(1):1-6.
[16]Abe S,Hamada K,Miura M,et al. Neural crest stem cell property of apical pulp cells derived from human developing tooth[J]. Cell Biology International,2013,36(10):927-936.
[17]Karbalaie K,Tanhaei S,Rabiei F,et al. Stem cells from human exfoliated deciduous tooth exhibit stromal-derived inducing activity and leadto generation of neural crest cells from human embryonic stem cells[J]. Cell Journal,2015,17(1):37-48.
[18]Wen X,Liu L,Deng M,et al. Characterization of p75(+)ectomesenchymal stem cells from rat embryonic facial process tissue[J]. Biochemical&Biophysical Research Communications,2012,427(1):5-10.
[19]Wen X,Liu L,Deng M,et al. In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75(+)stem cells with dental follicle cell conditioned medium[J]. Experimental Cell Research,2015,337(1):76-86.
[20]Jordan B,Dinev D V,Troppmair J,et al. Neurotrophin receptor-interacting mage homologue is an inducible inhibitor of apoptosis protein-interacting protein that augments cell death[J]. Journal of Biological Chemistry,2001,276(43):39985.
[21]Sasaki A,Masuda Y,Iwai K,et al. A RING finger protein Praja1 regulates Dlx5-dependent transcription through its ubiquitin ligase activity for the Dlx/Msx-interacting MAGE/Necdin family protein,Dlxin-1[J]. Journal of Biological Chemistry,2002,277(25):22541.
[22]Chen Y,Jin L,Xue B,et al. NRAGE inducesβ-catenin/Arm O-Glc NAcylation and negatively regulates Wnt signaling[J]. Biochemical&Biophysical Research Communications,2017,487(2):433-437.
[23]Wen C J,Xue B,Qin W X,et al. h NRAGE,a human neurotrophin receptor interacting MAGE homologue,regulates p53 transcriptional activity and inhibits cell proliferation[J]. Febs Letters,2004,564(1):171-176.
[24]Kuwajima T,Taniura H,Nishimura I,et al. Necdin interacts with the Msx2 homeodomain protein via MAGE-D1to promote myogenic differentiation of C2C12 cells[J].Journal of Biological Chemistry,2004,279(39):40484.
[25]Salehi A H,Roux P P,Kubu C J,et al. NRAGE,A novel MAGE protein,interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis[J]. Neuron,2000,27(2):279-288.
[26]Salehi A H,Xanthoudakis S,Barker P A. NRAGE,a p75neurotrophin receptor-interacting protein, induces caspase activation and cell death through a JNK-dependent mitochondrial pathway[J]. Journal of Biological Chemistry,2002,277(50):48043-48050.
[27]Cowin P,Rowlands T M,Hatsell S J. Cadherins and catenins in breast cancer[J]. Current Opinion in Cell Biology,2005,17(5):499-508.
[28]Shang Y C,Wang S H,Xiong F,et al. Wnt3a signaling promotes proliferation,myogenic differentiation,and migration of rat bone marrow mesenchymal stem cells[J].Acta Pharmacologica Sinica,2010,28(11):1761-1774.
[29]Matsuzawa M,Sheu T J,Lee Y J,et al. Putative signaling action of amelogenin utilizes the Wnt/beta-catenin pathway[J]. Journal of Periodontal Research,2010,44(3):289-296.
[30]薛斌. NRAGE抑制细胞生长、粘附和迁移分子机理的研究[D].南京:南京师范大学,2006.
[2]Mantesso A,Sharpe P. Dental stem cells for tooth regeneration and repair[J]. Expert Opinion on Biological Therapy,2009,9:1143-1154.
[3]Barker P A,Salehi A. The MAGE proteins:emerging roles in cell cycle progression,apoptosis,and neurogenetic disease[J]. J Neurosci Res,2002,67:705-712.
[4]Sasaki A,Hinck L,Watanabe K. Rum MAGE-D the members:structure and function of a new adaptor family of MAGE-D proteins[J]. Journal of Receptor&Signal Transduction Research,2005,25(3):181-198.
[5]Tsai J R,Chong I W,Chen Y H,et al. Differential expression profile of MAGE family in non-small-cell lung cancer[J]. Lung Cancer,2007,56(2):185-192.
[6]Kirkin A F,Dzhandzhugazyan K N,Zeuthen J. Cancer/testis antigens:structural and immunobiologicalproperties[J]. Cancer Investigation,2002,20(2):222-236.
[7]Stone B,Schummer M,Paley P J,et al. MAGE-F1,a novel ubiquitously expressed member of the MAGE superfamily[J]. Gene,2001,267(2):173-182.
[8]Chomez P,Backer O D,Bertrand M,et al. An Overview of the MAGE Gene Family with the Identification of All Human Members of the Family[J]. Cancer Research,2001,61(14):5544-5551.
[9]Xue B,Wen C,Shi Y,etal. Human NRAGE disrupts Ecadherin/beta-catenin regulated homotypic cell-cell adhesion[J]. Biochemical&Biophysical Research Communications,2005,336(1):247-251.
[10]Shen W G,Xue Q Y,Zhu J,et al. Inhibition of adenovirus-mediated human MAGE-D1 on angiogenesis in vitro and in vivo[J]. Molecular&Cellular Biochemistry,2007,300(1-2):89-99.
[11]Cao S,Zhang F,Zhao P,et al. The effects of annexin a2expression on the proliferation,migration and invasion of human breast cancer cells[J]. Chinese Journal of Clinical Oncology,2011,38(6):304-307.
[12]Chu C S,Xue B,Tu C,et al. NRAGE suppresses metastasis of melanoma and pancreatic cancer in vitro and in vivo[J]. Cancer Letters,2007,250(2):268-275.
[13]Lai S S,Xue B,Yang Y,et al. Ror2-Src signaling in metastasis of mouse melanoma cells is inhibited by NRAGE[J]. Cancer Genet,2012,205(11):552-562.
[14]Chang X,Xue X,Zhang Y,et al. The role of NRAGE subcellular location and epithelial-mesenchymal transition on radiation resistance of esophageal carcinoma cell[J].Journal of Cancer Research&Therapeutics,2018,14(1):46.
[15]Yang K,Wang Y,Ju Y,et al. p75 neurotrophin receptor regulates differential mineralization of rat ectomesenchymal stem cells[J]. Cell Proliferation,2016,50(1):1-6.
[16]Abe S,Hamada K,Miura M,et al. Neural crest stem cell property of apical pulp cells derived from human developing tooth[J]. Cell Biology International,2013,36(10):927-936.
[17]Karbalaie K,Tanhaei S,Rabiei F,et al. Stem cells from human exfoliated deciduous tooth exhibit stromal-derived inducing activity and leadto generation of neural crest cells from human embryonic stem cells[J]. Cell Journal,2015,17(1):37-48.
[18]Wen X,Liu L,Deng M,et al. Characterization of p75(+)ectomesenchymal stem cells from rat embryonic facial process tissue[J]. Biochemical&Biophysical Research Communications,2012,427(1):5-10.
[19]Wen X,Liu L,Deng M,et al. In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75(+)stem cells with dental follicle cell conditioned medium[J]. Experimental Cell Research,2015,337(1):76-86.
[20]Jordan B,Dinev D V,Troppmair J,et al. Neurotrophin receptor-interacting mage homologue is an inducible inhibitor of apoptosis protein-interacting protein that augments cell death[J]. Journal of Biological Chemistry,2001,276(43):39985.
[21]Sasaki A,Masuda Y,Iwai K,et al. A RING finger protein Praja1 regulates Dlx5-dependent transcription through its ubiquitin ligase activity for the Dlx/Msx-interacting MAGE/Necdin family protein,Dlxin-1[J]. Journal of Biological Chemistry,2002,277(25):22541.
[22]Chen Y,Jin L,Xue B,et al. NRAGE inducesβ-catenin/Arm O-Glc NAcylation and negatively regulates Wnt signaling[J]. Biochemical&Biophysical Research Communications,2017,487(2):433-437.
[23]Wen C J,Xue B,Qin W X,et al. h NRAGE,a human neurotrophin receptor interacting MAGE homologue,regulates p53 transcriptional activity and inhibits cell proliferation[J]. Febs Letters,2004,564(1):171-176.
[24]Kuwajima T,Taniura H,Nishimura I,et al. Necdin interacts with the Msx2 homeodomain protein via MAGE-D1to promote myogenic differentiation of C2C12 cells[J].Journal of Biological Chemistry,2004,279(39):40484.
[25]Salehi A H,Roux P P,Kubu C J,et al. NRAGE,A novel MAGE protein,interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis[J]. Neuron,2000,27(2):279-288.
[26]Salehi A H,Xanthoudakis S,Barker P A. NRAGE,a p75neurotrophin receptor-interacting protein, induces caspase activation and cell death through a JNK-dependent mitochondrial pathway[J]. Journal of Biological Chemistry,2002,277(50):48043-48050.
[27]Cowin P,Rowlands T M,Hatsell S J. Cadherins and catenins in breast cancer[J]. Current Opinion in Cell Biology,2005,17(5):499-508.
[28]Shang Y C,Wang S H,Xiong F,et al. Wnt3a signaling promotes proliferation,myogenic differentiation,and migration of rat bone marrow mesenchymal stem cells[J].Acta Pharmacologica Sinica,2010,28(11):1761-1774.
[29]Matsuzawa M,Sheu T J,Lee Y J,et al. Putative signaling action of amelogenin utilizes the Wnt/beta-catenin pathway[J]. Journal of Periodontal Research,2010,44(3):289-296.
[30]薛斌. NRAGE抑制细胞生长、粘附和迁移分子机理的研究[D].南京:南京师范大学,2006.