釉基质蛋白诱导MC3T3-E1细胞成骨分化过程中微小核糖核酸的表达
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摘要
目的探讨微小核糖核酸(micro ribonucleicacid,mi RNA)是否参与到釉基质蛋白(Enamel Matrix Derivative,EMD)诱导的小鼠前成骨细胞系MC3T3-E1细胞成骨分化的过程,并对发生显著变化的mi RNA进行分析。方法将MC3T3-E1用含EMD(刺激组)/不含EMD(对照组)的培养液培养0天、7天和14天,检测碱性磷酸酶(alkaline phosphatase,ALP)活性,实时聚合酶链式反应(RT-PCR)技术分析成骨分化标记物ALP、骨涎蛋白(bone sialoprotein,BSP)和骨钙素(osteocalcin,OC)的信使RNA(m RNA)水平的表达变化。利用基因芯片技术分析mi RNA的相对表达。结果 0天、7天、14天的ALP染色结果显示随着培养时间的增加,两组ALP活性均逐渐增强,EMD刺激组ALP活性增强较对照组更为明显。RT-PCR结果表明,与对照组相比,ALP、BSP、OC的m RNA表达量均显著增加,ALP与OC均于14天时表达量达到峰值,BSP则于7天时处于峰值表达,EMD刺激组MC3T3-E1成骨活性更强;各期11个mi RNA表达上调,28个mi RNA表达下调,其中mi R-335-5p,mi R-503已被证实可参与促进骨形成,而mi R-30家族(mi R-30a,-30b,-30c和-30d)则被证实参与抑制成骨。结论 mi RNA参与EMD诱导的MC3T3-E1细胞的成骨分化过程,这一发现可以为了解EMD促进成骨分化的机理及临床应用提供指导。
Objective In this study, we aimed to identify whether micro ribonucleicacid(micro RNA, mi RNA) participated in the process of osteogenic differentiation induced by Enamel Matrix Derivative( EMD) and to anlayze the significant change of mi RNAs in response to EMD. Methods The mouse pre-osteoblast cell line MC3T3-E1 was cultured with / without(the EMD stimulating /negative control group)EMD induction(0, 7 and 14 days), and osteogenic differentiation was detected by alkaline phosphatase(ALP) staining and real-time quantitative polymerase chain reaction( RT-PCR) analysis. Taq Man Micro RNA Arrays(0, 7 and 14 days) was used to analyse the relative expression of mi RNAs. Results The results of the ALP staining indicated that EMD could enhance osteogenic differentiation in MC3T3-E1 cells, the results of EMD group were more significant than the negative group. The results of RT-PCR showed that when compared to the negative group, the expression of ALP, bone sialoprotein(BSP) and osteocalcin(OC) increased significantly. In addition, both ALP and OC reached their peak levels at 14 days, while BSP reached its peak at 7 days. These findings indicate that EMD enhanced osteogenic differentiation in MC3T3-E1 cells. At 0, 7 and 14 days, 11 mi RNAs were upregulated and 28 mi RNAs were downregulated significantly during the process of osteogenic differentiation induced by EMD. And mi R-335-5p, mi R-503 have been confirmed to promote bone formation, while mi R-30 family have been confirmed to be negative regulators of osteogenic differentiation. Conclusion mi RNAs were involved in EMD induced MC3T3-E1 osteogenic differentiation. These findings could be used to understand the mechanism of the regulatory process of osteogenic differentiation induced by EMD and guide clinical application.
Objective In this study, we aimed to identify whether micro ribonucleicacid(micro RNA, mi RNA) participated in the process of osteogenic differentiation induced by Enamel Matrix Derivative( EMD) and to anlayze the significant change of mi RNAs in response to EMD. Methods The mouse pre-osteoblast cell line MC3T3-E1 was cultured with / without(the EMD stimulating /negative control group)EMD induction(0, 7 and 14 days), and osteogenic differentiation was detected by alkaline phosphatase(ALP) staining and real-time quantitative polymerase chain reaction( RT-PCR) analysis. Taq Man Micro RNA Arrays(0, 7 and 14 days) was used to analyse the relative expression of mi RNAs. Results The results of the ALP staining indicated that EMD could enhance osteogenic differentiation in MC3T3-E1 cells, the results of EMD group were more significant than the negative group. The results of RT-PCR showed that when compared to the negative group, the expression of ALP, bone sialoprotein(BSP) and osteocalcin(OC) increased significantly. In addition, both ALP and OC reached their peak levels at 14 days, while BSP reached its peak at 7 days. These findings indicate that EMD enhanced osteogenic differentiation in MC3T3-E1 cells. At 0, 7 and 14 days, 11 mi RNAs were upregulated and 28 mi RNAs were downregulated significantly during the process of osteogenic differentiation induced by EMD. And mi R-335-5p, mi R-503 have been confirmed to promote bone formation, while mi R-30 family have been confirmed to be negative regulators of osteogenic differentiation. Conclusion mi RNAs were involved in EMD induced MC3T3-E1 osteogenic differentiation. These findings could be used to understand the mechanism of the regulatory process of osteogenic differentiation induced by EMD and guide clinical application.
引文
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2 Shahriari S,Houshmand B,Razavian H,et al.Effect of the combination of enamel matrix derivatives and deproteinized bovine bone materials on bone formation in rabbits'calvarial defects.Dent Res J(Isfahan),2012,9:422~426.
3 Kawana F,Sawae Y,Sahara T,et al.Porcine enamel matrix derivative enhances trabecular bone regeneration during wound healing of injured rat femur.Anat Rec,2001,264:438~446.
4 Lee RC,Feinbaum RL,Ambros V.The C.Elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75(5):843~854.
5 Li H,Xie H,Liu W,et al.A novel micro RNA targeting HDAC5regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans.J Clin Invest,2009,119:3666~3677.
6 Li Z,Hassan MQ,Volinia S,et al.A micro RNA signature for a BMP2-induced osteoblast Lineage commitment program.PNAS,2008,105:13906~13911.
7 Li Z,Hassan MQ,Jafferji M,et al.Biological Functions of mi R-29 b Contribute to Positive Regulation of Osteoblast Differentiation.J Biol Chem,2009;284:15676~15684.
8 Itoh T,Nozawa Y,Akao Y.Micro RNA-141 and-200a Are Involved in Bone Morphogenetic Protein-2-induced Mouse Pre-osteoblast Differentiation by Targeting Distal-less Homeobox5.J Biol Chem,2009,284:19272~19279.
9 Moe D,Salling E,Kirkeby S.Extraction of enamel proteins.Scand J Dent Res,1984,92:503~507.
10 Shu R,Li C,Liu Z.The extraction and N-terminal amino acid sequence analysis of porcine enamel matrix proteins.J.Comprehensive Stomatol,1999,15(2):67~68.
11 Li Z,Hassan MQ,Jafferji M,et al.Biological Functions of mi R-29 b Contribute to Positive Regulation of Osteoblast Differentiation.J Biol Chem,2009;284:15676~15684.
12 Bosshardt DD.Biological mediators and periodontal regeneration:a review of enamel matrix proteins at the cellular and molecular levels.J Clin Periodontol,2008;35:87~105.
13 Kawase T,Okuda K,Momose M,et al.Enamel matrix derivative(EMDOGAIN)rapidly stimulates phosphorylation of the MAP kinase family and nuclear accumulation of smad2 in both oral epithelial and fibroblastic human cells.J Periodontal Res,2001,36 :367~376.
14 Lee AZ,Jiang J,He J,et al.Stimulation of cytokines in osteoblasts cultured on enamel matrix derivative.Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2008,106:133~138.
15 Kawase T,Okuda K,Yoshie H,Burns DM.Anti-TGF-beta antibody blocks enamel matrix derivative-induced upregulation of p21WAF1/cip1 and prevents its inhibition of human oral epithelial cell proliferation.J Periodont Res,2002,37:255~262.
16 束蓉,刘正,葛琳华.猪釉基质蛋白对MC3T3-E1成骨细胞增殖分化的影响,华西口腔医学杂志2000年18卷第4期,226~228.
17 Miron RJ,Caluseru OM,Guillemette V,et al.Influence of enamel matrix derivative on cells at different maturation stages of differentiation.PLo S One 2013;8(8):e71008.
18 Guida L,Annunziata M,Carinci F,Di Feo A,Passaro I,Oliva A(2007)In vitro biologic response of human bone marrow stromal cells to enamel matrix derivative.J.Periodontol.78,2190~2196.
19 van den Dolder J,Vloon AP,Jansen JA(2006)The effect of Emdogain on the growth and differentiation of rat bone marrow cells.J.Periodontal.Res.41,471~476.
20 Takayama T,Suzuki N,Narukawa M,et al.Ito K(2005)Enamel matrix derivative stimulates core binding factor alpha1/Runtrelated transcription factor-2 expression via activation of Smad1 in C2C12 cells.J.Periodontol.76,244~249.
21 Wang J,Zhao H,Tang D,et al.Overexpressions of Micro RNA-9and Micro RNA-200c in Human Breast Cancers Are Associated with Lymph Node Metastasis.Cancer Biother Radiopharm2013;28:283~288.
22 Zhang J,Tu Q,Bonewald LF,et al.Effects of mi R-335-5p in modulating osteogenic differentiation by specifically downregulating Wnt antagonist DKK1.J Bone Miner Res 2011;26:1953~1963.
23 Chen C,Cheng P,Xie H,et al.Mi R-503 regulates osteoclastogenesis via targeting RANK.J Bone Miner Res 2013;doi:10.1002/jbmr.2032.
24 Liao L,Yang X,Su X,et al.Redundant mi R-3077-5p and mi R-705mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow.Cell Death Dis 2013;4:e600.
25 Wu T,Zhou H,Hong Y,et al.Mi R-30 family members negatively regulate osteoblast differentiation.J Biol Chem 2012;287:7503~7511.
2 Shahriari S,Houshmand B,Razavian H,et al.Effect of the combination of enamel matrix derivatives and deproteinized bovine bone materials on bone formation in rabbits'calvarial defects.Dent Res J(Isfahan),2012,9:422~426.
3 Kawana F,Sawae Y,Sahara T,et al.Porcine enamel matrix derivative enhances trabecular bone regeneration during wound healing of injured rat femur.Anat Rec,2001,264:438~446.
4 Lee RC,Feinbaum RL,Ambros V.The C.Elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75(5):843~854.
5 Li H,Xie H,Liu W,et al.A novel micro RNA targeting HDAC5regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans.J Clin Invest,2009,119:3666~3677.
6 Li Z,Hassan MQ,Volinia S,et al.A micro RNA signature for a BMP2-induced osteoblast Lineage commitment program.PNAS,2008,105:13906~13911.
7 Li Z,Hassan MQ,Jafferji M,et al.Biological Functions of mi R-29 b Contribute to Positive Regulation of Osteoblast Differentiation.J Biol Chem,2009;284:15676~15684.
8 Itoh T,Nozawa Y,Akao Y.Micro RNA-141 and-200a Are Involved in Bone Morphogenetic Protein-2-induced Mouse Pre-osteoblast Differentiation by Targeting Distal-less Homeobox5.J Biol Chem,2009,284:19272~19279.
9 Moe D,Salling E,Kirkeby S.Extraction of enamel proteins.Scand J Dent Res,1984,92:503~507.
10 Shu R,Li C,Liu Z.The extraction and N-terminal amino acid sequence analysis of porcine enamel matrix proteins.J.Comprehensive Stomatol,1999,15(2):67~68.
11 Li Z,Hassan MQ,Jafferji M,et al.Biological Functions of mi R-29 b Contribute to Positive Regulation of Osteoblast Differentiation.J Biol Chem,2009;284:15676~15684.
12 Bosshardt DD.Biological mediators and periodontal regeneration:a review of enamel matrix proteins at the cellular and molecular levels.J Clin Periodontol,2008;35:87~105.
13 Kawase T,Okuda K,Momose M,et al.Enamel matrix derivative(EMDOGAIN)rapidly stimulates phosphorylation of the MAP kinase family and nuclear accumulation of smad2 in both oral epithelial and fibroblastic human cells.J Periodontal Res,2001,36 :367~376.
14 Lee AZ,Jiang J,He J,et al.Stimulation of cytokines in osteoblasts cultured on enamel matrix derivative.Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2008,106:133~138.
15 Kawase T,Okuda K,Yoshie H,Burns DM.Anti-TGF-beta antibody blocks enamel matrix derivative-induced upregulation of p21WAF1/cip1 and prevents its inhibition of human oral epithelial cell proliferation.J Periodont Res,2002,37:255~262.
16 束蓉,刘正,葛琳华.猪釉基质蛋白对MC3T3-E1成骨细胞增殖分化的影响,华西口腔医学杂志2000年18卷第4期,226~228.
17 Miron RJ,Caluseru OM,Guillemette V,et al.Influence of enamel matrix derivative on cells at different maturation stages of differentiation.PLo S One 2013;8(8):e71008.
18 Guida L,Annunziata M,Carinci F,Di Feo A,Passaro I,Oliva A(2007)In vitro biologic response of human bone marrow stromal cells to enamel matrix derivative.J.Periodontol.78,2190~2196.
19 van den Dolder J,Vloon AP,Jansen JA(2006)The effect of Emdogain on the growth and differentiation of rat bone marrow cells.J.Periodontal.Res.41,471~476.
20 Takayama T,Suzuki N,Narukawa M,et al.Ito K(2005)Enamel matrix derivative stimulates core binding factor alpha1/Runtrelated transcription factor-2 expression via activation of Smad1 in C2C12 cells.J.Periodontol.76,244~249.
21 Wang J,Zhao H,Tang D,et al.Overexpressions of Micro RNA-9and Micro RNA-200c in Human Breast Cancers Are Associated with Lymph Node Metastasis.Cancer Biother Radiopharm2013;28:283~288.
22 Zhang J,Tu Q,Bonewald LF,et al.Effects of mi R-335-5p in modulating osteogenic differentiation by specifically downregulating Wnt antagonist DKK1.J Bone Miner Res 2011;26:1953~1963.
23 Chen C,Cheng P,Xie H,et al.Mi R-503 regulates osteoclastogenesis via targeting RANK.J Bone Miner Res 2013;doi:10.1002/jbmr.2032.
24 Liao L,Yang X,Su X,et al.Redundant mi R-3077-5p and mi R-705mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow.Cell Death Dis 2013;4:e600.
25 Wu T,Zhou H,Hong Y,et al.Mi R-30 family members negatively regulate osteoblast differentiation.J Biol Chem 2012;287:7503~7511.