人牙髓干细胞增殖分化过程中的miR-431
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摘要
背景:利用现代细胞分子生物学和组织工程技术,将牙髓干细胞应用于修复和重建牙组织具有积极的临床价值。目的:探索miR-431在牙髓干细胞牙向分化中的作用以及miR-431对牙髓干细胞体外增殖能力的影响。方法:酶消化法分离培养牙髓干细胞,转染miR-431模拟剂或抑制剂促进或抑制miR-431表达,诱导牙髓干细胞成牙分化,通过碱性磷酸酶活性检测试剂盒检测碱性磷酸酶活性,Westernblot检测成牙分化标志物牙本质涎磷蛋白、骨钙素、骨唾液蛋白的表达,qRT-PCR检测miR-431表达,MTT法检测细胞增殖率,克隆形成实验检测细胞克隆形成能力。结果与结论:①牙髓干细胞成牙分化过程中,碱性磷酸酶活性上升,牙本质涎磷蛋白、骨钙素、骨唾液蛋白表达上升,miR-431表达下调;②过表达miR-431后,牙髓干细胞成牙分化受到抑制,而抑制miR-431表达后,牙髓干细胞成牙分化得到促进,表明miR-431对牙髓干细胞成牙分化有负调控作用;③miR-431对牙髓干细胞的增殖和克隆形成能力有抑制作用。
BACKGROUND: Dental pulp stem cells have great clinical potential in dental repair and reconstruction through modern cellular and molecular biology and tissue engineering approaches.OBJECTIVE: To explore the effect of microRNA-431(miR-431) on the odontogenetic differentiation and in vitro proliferation of dental pulp stem cells.METHODS: Dental pulp stem cells were isolated by enzyme digestion and induced to odontogenetic differentiation. Alkaline phosphatase activity was measured by Alkaline Phosphatase Assay Kit. Expression of odontogenetic differentiation markers, dentin sialophosphoprotein,osteocalcin and bone sialoprotein, was detected by western blot. Expression of miR-431 was detected by qRT-PCR. Cell proliferation and colony formation ability of dental pulp stem cells were detected by MTT and colony formation assay, respectively.RESULTS AND CONCLUSION: Alkaline phosphatase activity and the expression of dentin sialophosphoprotein, osteocalcin and bone sialoprotein were increased but the expression of miR-431 reduced during the odontogenetic differentiation. Odontogenetic differentiation was inhibited by miR-431 overexpression, while miR-431-knockdown promoted the odontogenetic differentiation of dental pulp stem cells. These results indicate that miR-431 negatively regulates the odontogenetic differentiation of dental pulp stem cells, and it also inhibits the proliferation and colony formation ability of dental pulp stem cells.
BACKGROUND: Dental pulp stem cells have great clinical potential in dental repair and reconstruction through modern cellular and molecular biology and tissue engineering approaches.OBJECTIVE: To explore the effect of microRNA-431(miR-431) on the odontogenetic differentiation and in vitro proliferation of dental pulp stem cells.METHODS: Dental pulp stem cells were isolated by enzyme digestion and induced to odontogenetic differentiation. Alkaline phosphatase activity was measured by Alkaline Phosphatase Assay Kit. Expression of odontogenetic differentiation markers, dentin sialophosphoprotein,osteocalcin and bone sialoprotein, was detected by western blot. Expression of miR-431 was detected by qRT-PCR. Cell proliferation and colony formation ability of dental pulp stem cells were detected by MTT and colony formation assay, respectively.RESULTS AND CONCLUSION: Alkaline phosphatase activity and the expression of dentin sialophosphoprotein, osteocalcin and bone sialoprotein were increased but the expression of miR-431 reduced during the odontogenetic differentiation. Odontogenetic differentiation was inhibited by miR-431 overexpression, while miR-431-knockdown promoted the odontogenetic differentiation of dental pulp stem cells. These results indicate that miR-431 negatively regulates the odontogenetic differentiation of dental pulp stem cells, and it also inhibits the proliferation and colony formation ability of dental pulp stem cells.
引文
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[8]La Noce M, Paino F, Spina A, et al. Dental pulp stem cells:state of the art and suggestions for a true translation of research into therapy. J Dent. 2014;42(7):761-768.
[9]Alge DL, Zhou D, Adams LL, et al. Donor-matched comparison of dental pulp stem cells and bone marrow-derived mesenchymal stem cells in a rat model. J Tissue Eng Regen Med. 2010;4(1):73-81.
[10]Kim S, Shin SJ, Song Y, et al. In Vivo Experiments with Dental Pulp Stem Cells for Pulp-Dentin Complex Regeneration.Mediators Inflamm. 2015;2015:409347.
[11]Lee SM, Zhang Q, Le AD. Dental stem cells:sources and potential applications. Current Oral Health Reports. 2014;1(1):34-42.
[12]Bansal R, Jain A. Current overview on dental stem cells applications in regenerative dentistry. J Nat Sci Biol Med.2015;6(1):29-34.
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[14]Stroynowska-Czerwinska A, Fiszer A, Krzyzosiak WJ. The panorama of miRNA-mediated mechanisms in mammalian cells. Cell Mol Life Sci. 2014;71(12):2253-2270.
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[16]Huang TC, Pinto SM, Pandey A. Proteomics for understanding miRNA biology. Proteomics. 2013;13(3-4):558-567.
[17]Shenoy A, Blelloch RH. Regulation of microRNA function in somatic stem cell proliferation and differentiation. Nat Rev Mol Cell Biol. 2014;15(9):565-576.
[18]Miyoshi N, Ishii H, Nagano H, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs.Cell Stem Cell. 2011;8(6):633-638.
[19]Baglìo SR, Devescovi V, Granchi D, et al. MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31. Gene. 2013;527(1):321-331.
[20]Hamam D, Ali D, Kassem M, et al. microRNAs as regulators of adipogenic differentiation of mesenchymal stem cells. Stem Cells Dev. 2015;24(4):417-425.
[21]Zhang W, Walboomers XF, Van Kuppevelt TH, et al. In vivo evaluation of human dental pulp stem cells differentiated towards multiple lineages. J Tissue Eng Regen Med. 2008;2(2-3):117-125.
[22]Hilkens P, Gervois P, Fanton Y, et al. Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells. Cell Tissue Res. 2013;353(1):65-78.
[23]Gay I, Cavender A, Peto D, et al. Differentiation of human dental stem cells reveals a role for microRNA-218. J Periodontal Res. 2014;49(1):110-120.
[24]Gong Q, Wang R, Jiang H, et al. Alteration of microRNA expression of human dental pulp cells during odontogenic differentiation. J Endod. 2012;38(10):1348-1354.
[25]Liu W, Gong Q, Ling J, et al. Role of miR-424 on angiogenic potential in human dental pulp cells. J Endod. 2014;40(1):76-82.
[26]Hara ES, Ono M, Eguchi T, et al. miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells. PLoS One. 2013;8(12):e83545.
[27]Wheeler G, Ntounia-Fousara S, Granda B, et al. Identification of new central nervous system specific mouse microRNAs.FEBS Lett. 2006;580(9):2195-2200.
[28]Wu D, Murashov AK. MicroRNA-431 regulates axon regeneration in mature sensory neurons by targeting the Wnt antagonist Kremen1. Front Mol Neurosci. 2013;6:35.
[29]Liu R, Ma X, Xu L, et al. Differential microRNA expression in peripheral blood mononuclear cells from Graves'disease patients.J Clin Endocrinol Metab. 2012;97(6):E968-972.
[30]Pan L, Ren F, Rong M, et al. Correlation between down-expression of miR-431 and clinicopathological significance in HCC tissues. Clin Transl Oncol. 2015;17(7):557-563.
[31]Sun K, Zeng T, Huang D, et al. MicroRNA-431 inhibits migration and invasion of hepatocellular carcinoma cells by targeting the ZEB1-mediated epithelial-mensenchymal transition. FEBS Open Bio. 2015;5:900-907.
[32]Sun K, Zeng T, Huang D, et al. MicroRNA-431 inhibits migration and invasion of hepatocellular carcinoma cells by targeting the ZEB1-mediated epithelial-mensenchymal transition. Genes Dev. 2015;29(15):1605-1617.
[33]Salmon B, Bardet C, Khaddam M, et al. MEPE-derived ASARM peptide inhibits odontogenic differentiation of dental pulp stem cells and impairs mineralization in tooth models of X-linked hypophosphatemia. PLoS One. 2013;8(2):e56749.
[34]Tsukamoto Y, Fukutani S, Shin-Ike T, et al. Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts.Arch Oral Biol. 1992;37(12):1045-1055.
[35]Tsukamoto Y, Fukutani S, Shin-Ike T, et al. Loss of bone sialoprotein leads to impaired endochondral bone development and mineralization. Bone. 2015;71:145-154.
[36]Yang L, Cheng P, Chen C, et al. miR-93/Sp7 function loop mediates osteoblast mineralization. J Bone Miner Res. 2012;27(7):1598-1606.
[37]Guo S, Lim D, Dong Z, et al. Dentin sialophosphoprotein:a regulatory protein for dental pulp stem cell identity and fate.Stem Cells Dev. 2014;23(23):2883-2894.
[38]Sreenath T, Thyagarajan T, Hall B, et al. Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III. J Biol Chem. 2003;278(27):24874-24880.
[39]Liu P, Zhao Y, Yan Y, et al. Construction of extracellular microenvironment to improve surface endothelialization of NiTi alloy substrate. Mater Sci Eng C Mater Biol Appl. 2015;55:1-7.
[40]何飞,谭颖徽,张纲.人牙髓干细胞的体外培养和鉴定[J].华西口腔医学杂志, 2005, 23(1):75-78.
[2]Tatullo M, Marrelli M, Shakesheff KM, et al. Dental pulp stem cells:function, isolation and applications in regenerative medicine. J Tissue Eng Regen Med. 2015;9(11):1205-1216.
[3]Shi S, Bartold PM, Miura M, et al. The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod Craniofac Res. 2005;8(3):191-199.
[4]Liu H, Gronthos S, Shi S. Dental pulp stem cells. Methods Enzymol. 2006;419:99-113.
[5]杜丽娟,苗勇,胡志奇.成体细胞转化为多能干细胞诱导方法的研究进展[J].中华实验外科杂志, 2014, 31(11):2630-2631.
[6]Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells(DPSCs)in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97(25):13625-13630.
[7]Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002;81(8):531-535.
[8]La Noce M, Paino F, Spina A, et al. Dental pulp stem cells:state of the art and suggestions for a true translation of research into therapy. J Dent. 2014;42(7):761-768.
[9]Alge DL, Zhou D, Adams LL, et al. Donor-matched comparison of dental pulp stem cells and bone marrow-derived mesenchymal stem cells in a rat model. J Tissue Eng Regen Med. 2010;4(1):73-81.
[10]Kim S, Shin SJ, Song Y, et al. In Vivo Experiments with Dental Pulp Stem Cells for Pulp-Dentin Complex Regeneration.Mediators Inflamm. 2015;2015:409347.
[11]Lee SM, Zhang Q, Le AD. Dental stem cells:sources and potential applications. Current Oral Health Reports. 2014;1(1):34-42.
[12]Bansal R, Jain A. Current overview on dental stem cells applications in regenerative dentistry. J Nat Sci Biol Med.2015;6(1):29-34.
[13]Modino SA, Sharpe PT. Tissue engineering of teeth using adult stem cells. Arch Oral Biol. 2005;50(2):255-258.
[14]Stroynowska-Czerwinska A, Fiszer A, Krzyzosiak WJ. The panorama of miRNA-mediated mechanisms in mammalian cells. Cell Mol Life Sci. 2014;71(12):2253-2270.
[15]Friedman RC, Farh KK, Burge CB, et al. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res.2009;19(1):92-105.
[16]Huang TC, Pinto SM, Pandey A. Proteomics for understanding miRNA biology. Proteomics. 2013;13(3-4):558-567.
[17]Shenoy A, Blelloch RH. Regulation of microRNA function in somatic stem cell proliferation and differentiation. Nat Rev Mol Cell Biol. 2014;15(9):565-576.
[18]Miyoshi N, Ishii H, Nagano H, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs.Cell Stem Cell. 2011;8(6):633-638.
[19]Baglìo SR, Devescovi V, Granchi D, et al. MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31. Gene. 2013;527(1):321-331.
[20]Hamam D, Ali D, Kassem M, et al. microRNAs as regulators of adipogenic differentiation of mesenchymal stem cells. Stem Cells Dev. 2015;24(4):417-425.
[21]Zhang W, Walboomers XF, Van Kuppevelt TH, et al. In vivo evaluation of human dental pulp stem cells differentiated towards multiple lineages. J Tissue Eng Regen Med. 2008;2(2-3):117-125.
[22]Hilkens P, Gervois P, Fanton Y, et al. Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells. Cell Tissue Res. 2013;353(1):65-78.
[23]Gay I, Cavender A, Peto D, et al. Differentiation of human dental stem cells reveals a role for microRNA-218. J Periodontal Res. 2014;49(1):110-120.
[24]Gong Q, Wang R, Jiang H, et al. Alteration of microRNA expression of human dental pulp cells during odontogenic differentiation. J Endod. 2012;38(10):1348-1354.
[25]Liu W, Gong Q, Ling J, et al. Role of miR-424 on angiogenic potential in human dental pulp cells. J Endod. 2014;40(1):76-82.
[26]Hara ES, Ono M, Eguchi T, et al. miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells. PLoS One. 2013;8(12):e83545.
[27]Wheeler G, Ntounia-Fousara S, Granda B, et al. Identification of new central nervous system specific mouse microRNAs.FEBS Lett. 2006;580(9):2195-2200.
[28]Wu D, Murashov AK. MicroRNA-431 regulates axon regeneration in mature sensory neurons by targeting the Wnt antagonist Kremen1. Front Mol Neurosci. 2013;6:35.
[29]Liu R, Ma X, Xu L, et al. Differential microRNA expression in peripheral blood mononuclear cells from Graves'disease patients.J Clin Endocrinol Metab. 2012;97(6):E968-972.
[30]Pan L, Ren F, Rong M, et al. Correlation between down-expression of miR-431 and clinicopathological significance in HCC tissues. Clin Transl Oncol. 2015;17(7):557-563.
[31]Sun K, Zeng T, Huang D, et al. MicroRNA-431 inhibits migration and invasion of hepatocellular carcinoma cells by targeting the ZEB1-mediated epithelial-mensenchymal transition. FEBS Open Bio. 2015;5:900-907.
[32]Sun K, Zeng T, Huang D, et al. MicroRNA-431 inhibits migration and invasion of hepatocellular carcinoma cells by targeting the ZEB1-mediated epithelial-mensenchymal transition. Genes Dev. 2015;29(15):1605-1617.
[33]Salmon B, Bardet C, Khaddam M, et al. MEPE-derived ASARM peptide inhibits odontogenic differentiation of dental pulp stem cells and impairs mineralization in tooth models of X-linked hypophosphatemia. PLoS One. 2013;8(2):e56749.
[34]Tsukamoto Y, Fukutani S, Shin-Ike T, et al. Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts.Arch Oral Biol. 1992;37(12):1045-1055.
[35]Tsukamoto Y, Fukutani S, Shin-Ike T, et al. Loss of bone sialoprotein leads to impaired endochondral bone development and mineralization. Bone. 2015;71:145-154.
[36]Yang L, Cheng P, Chen C, et al. miR-93/Sp7 function loop mediates osteoblast mineralization. J Bone Miner Res. 2012;27(7):1598-1606.
[37]Guo S, Lim D, Dong Z, et al. Dentin sialophosphoprotein:a regulatory protein for dental pulp stem cell identity and fate.Stem Cells Dev. 2014;23(23):2883-2894.
[38]Sreenath T, Thyagarajan T, Hall B, et al. Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III. J Biol Chem. 2003;278(27):24874-24880.
[39]Liu P, Zhao Y, Yan Y, et al. Construction of extracellular microenvironment to improve surface endothelialization of NiTi alloy substrate. Mater Sci Eng C Mater Biol Appl. 2015;55:1-7.
[40]何飞,谭颖徽,张纲.人牙髓干细胞的体外培养和鉴定[J].华西口腔医学杂志, 2005, 23(1):75-78.