TNF-a对BMP-2诱导下小鼠BMSCs成骨分化miRNA表达的影响
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摘要
目的探讨肿瘤坏死因子α(TNF-α)对骨形态发生蛋白-2(BMP-2)诱导下小鼠骨髓间充质干细胞(BMSCs)成骨分化的微小核糖核酸(micro ribonucleicacid,miRNA)表达谱的影响。方法给予小鼠BMSCs以下分组刺激:1阴性对照组(ctrl);2阳性对照组(pos):BMP-2(200ng/ml)刺激48h;3实验组(48h):BMP-2(200ng/ml)+TNF-α(10ng/ml)刺激48h;4实验组(72h):BMP-2(200ng/ml)+TNF-α(10ng/ml)刺激72h,48h和72h后分别提取RNA,应用miRNA芯片检测获得miRNA表达谱,筛选部分差异表达的miRNA进行荧光实时定量PCR(RT-PCR)验证。结果 miRNA表达谱分析结果表明,与阳性对照组相比,48h双因子刺激下检测到表达上调超过1.5倍的miRNA有44个,72h刺激下检测到22个miRNA,72h与48h相比,检测到24个表达上调超过1.5倍的miRNA,RT-PCR验证与芯片结果基本相符合。结论 miRNA参与调控TNF-α模拟炎症状态下BMP-2诱导BMSCs的成骨分化过程,且相关miRNA在TNF-α作用下表达上调,可能参与调控TNF-α的抑制成骨作用。由此可进一步解释炎性因子对成骨细胞分化的抑制机制,为发现新的药物靶点提供理论依据。
Objective In this study, we aimed to investigate the micro ribonucleicacid( miRNA) expression profile in the tumor necrosis factor(TNF)-α-inhibited osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs) introduced by BMP-2. Methods The BMSCs was cultured with/without TNF-α and bone morphogenetic protein(BMP)-2 induction: 1 negative control group(ctrl); 2 positive control(pos):stimulation with BMP-2(200ng/ml)for 48 h; 3 experimental group(48h):stimulation with BMP-2(200ng/ml) and TNF-α(10ng/ml)for 48 h; 4 experimental group(72h):stimulation with BMP-2(200ng/ml)and TNF-α(10ng/ml)for 72 h. Then the cells were harvested at the indicated times(48h, 72h). miRNA microar-ray technology was used to detect the expression profile of miRNA, and the expression of miRNA was verified by real time quantification-polymerase chain reaction(RT-PCR). Results Microarray analysis found that the expression profile of miRNA changed dramatically. 44 miRNA were upregulated significantly more than 1.5-fold in 48 h BMP-2/TNF-α group and 22 miRNA in 72 h BMP-2/TNF-α group, compared with positive control group(P<0.01). Compared with 48 h, 24 of up-regulated more than 1.5 times miRNA were detected in 72 h BMP-2/TNF-α group, which were consistent with the results of miRNA microarray. Conclusion miRNA were involved in the BMP-2-induced osteogenic differentiation process with TNF-α to mimic a state of inflammation, and significantly upregulated miRNA may be involved in the suppression of osteogenesis by TNF-α. These findings can further explain the inhibitory mechanism of inflammatory cytokines on osteoblast differentiation and has important clinical significance.
Objective In this study, we aimed to investigate the micro ribonucleicacid( miRNA) expression profile in the tumor necrosis factor(TNF)-α-inhibited osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs) introduced by BMP-2. Methods The BMSCs was cultured with/without TNF-α and bone morphogenetic protein(BMP)-2 induction: 1 negative control group(ctrl); 2 positive control(pos):stimulation with BMP-2(200ng/ml)for 48 h; 3 experimental group(48h):stimulation with BMP-2(200ng/ml) and TNF-α(10ng/ml)for 48 h; 4 experimental group(72h):stimulation with BMP-2(200ng/ml)and TNF-α(10ng/ml)for 72 h. Then the cells were harvested at the indicated times(48h, 72h). miRNA microar-ray technology was used to detect the expression profile of miRNA, and the expression of miRNA was verified by real time quantification-polymerase chain reaction(RT-PCR). Results Microarray analysis found that the expression profile of miRNA changed dramatically. 44 miRNA were upregulated significantly more than 1.5-fold in 48 h BMP-2/TNF-α group and 22 miRNA in 72 h BMP-2/TNF-α group, compared with positive control group(P<0.01). Compared with 48 h, 24 of up-regulated more than 1.5 times miRNA were detected in 72 h BMP-2/TNF-α group, which were consistent with the results of miRNA microarray. Conclusion miRNA were involved in the BMP-2-induced osteogenic differentiation process with TNF-α to mimic a state of inflammation, and significantly upregulated miRNA may be involved in the suppression of osteogenesis by TNF-α. These findings can further explain the inhibitory mechanism of inflammatory cytokines on osteoblast differentiation and has important clinical significance.
引文
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20 Li H,Li T,Wang S,et al.miR-17-5p and miR-106a are involved in the balance between osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells.Stem Cell Res,2013,10 (3):313~324.
21 W Liu,Y Liu,T Guo,et al.TCF3,a novel positive regulator of osteogenesis,plays a crucial role in miR-17 modulating the diverse effect of canonical Wnt signaling in different microenvironments.Cell Death Dis,2013,14(4):e539.
2 Duarte PM,de Mendona AC,Máximo MB,et al.Differential cytokine expressions affect the severity of peri-implant disease.Clin Oral Implants Res,2009,20:514~520.
3 Lachmann S,Kimmerle-Müller E,Axmann D,et al.Associations between peri-implant crevicular fluid volume,concentrations of crevicular inflammatory mediators,and composite IL-1A-889 and IL-1B+3954 genotype.A cross-sectional study on implant recall patients with and without clinical signs of peri-implantitis.Clin Oral Implants Res,2007,18:212~223.
4 Venza I,Visalli M,Cucinotta M,et al.Proinflammatory gene expression at chronic periodontitis and peri-implantitis sites in patients with or without type 2 diabetes.J Periodontol,2010,81:99 ~108.
5 Beklen A,Ainola M,Hukkanen M,et al.MMPs,IL-and TNF are regulated by IL-17 in periodontitis.J Dent Res,2007,86:347~351.
6 Liu W,Konermann A,Guo T,et al.Canonical Wnt signaling differently modulates osteogenic differentiation of mesenchymal stem cells derived from bone marrow and from periodontal ligament under inflammatory conditions.Biochimica et Biophysica Acta,2014,1840(3):1125~1134.
7 Stefan Renvert,Ioannis Polyzois,Noel Claffey.Surgical therapy for the control of peri-implantitis.Clin.Oral Implants Res,2012,23 (6):84~94.
8 Kim VN.MicroRNA biogenesis:coordinated cropping and dicing.Nat Rev Mol Cell Biol,2005,6(5):376~385.
9 Wu T,Zhou H,Hong Y,et al.miR-30 family members negatively regulate osteoblast differentiation.J Biol Chem,2012,287 (10):7503~7511.
10 Itoh T,Nozawa Y,Akao Y.MicroRNA-141 and-200a are involved in bone morphogenetic protein-2-induced mouse pre-osteoblast differentiation by targeting distal-less homeobox 5.Biochemical and Biophysical Research Communications,2012,418:587~591.
11 Li Z,Hassan MQ,Volinia S,et al.A microRNA signature for a BMP2-induced osteoblast lineage commitment program.Proc Natl Acad Sci U S A,2008,105(37):13906~13911.
12 Kagiya T,Nakamura S.Expression profiling of microRNAs in RAW264.7 cells treated with a combination of tumor necrosis factor alpha and RANKL during osteoclast differentiation.J Periodontal Res,2013,48(3):373~385.
13 Wu T,Xie M,Wang X,et al.miR-155 modulates TNF-a-inhibited osteogenic differentiation by targeting SOCS1 expression.Bone,2012,51:498~505.
14 Murata K,Furu M,Yoshitomi H,et al.Comprehensive microRNA analysis identifies miR-24 and miR-125a-5p as plasma biomarkers for rheumatoid arthritis.PLoS One,2013,8(7):e69118.
15 Seeliger C,Karpinski K,Haug A,et al.Five Freely Circulating miRNAs and Bone Tissue miRNAs are Associated with Osteoporotic Fractures.J Bone Miner Res.2014;doi:10.1002/jbmr.2175.
16 Perri R,Nares S,Zhang S,et al.MicroRNA modulation in obesity and periodontitis.J Dent Res,2012,91(1):33~38.
17 Xie YF,Shu R,Jiang SY,et al.Comparison of microRNA profiles of human periodontal diseased and healthy gingival tissues.Int J Oral Sci,2011,3(3):125-134.
18 Lee YH,Na HS,Jeong SY,et al.Comparison of inflammatory microRNA expression in healthy and periodontitis tissues.Biocell,2011,35(2):43~49.
19 Yang N,Wang G,Hu C,et al.Tumor necrosis factor a supresses the mesenchymal stem cell osteogenesis promoter miR-21 in estrogendeficiency-induced osteoporosis.J Bone Miner Res,2013,28(3):559 ~573.
20 Li H,Li T,Wang S,et al.miR-17-5p and miR-106a are involved in the balance between osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells.Stem Cell Res,2013,10 (3):313~324.
21 W Liu,Y Liu,T Guo,et al.TCF3,a novel positive regulator of osteogenesis,plays a crucial role in miR-17 modulating the diverse effect of canonical Wnt signaling in different microenvironments.Cell Death Dis,2013,14(4):e539.