中国汉族先天性脊柱侧凸患者与正常人群血清miRNA的对比研究
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摘要
研究背景
先天性脊柱侧凸(Congenital scoliosis,CS)是常见的脊柱畸形之一。根据影像学表现可分为三种类型：分节障碍型,形成障碍型以及同时存在以上两种畸形类型的混合型；每一种亚型的影像学表现以及自然史显著不同。先天性脊柱侧凸发病于胚胎时期“体节”形成的过程中,目前认为遗传因素以及环境因素均在其发病中发挥作用。先天性脊柱侧凸病因尚不明确,目前的研究结果支持CS是一种多基因遗传疾病。但具体的发病机制尚不清楚。
近年来,miRNA在基因转录后的调节作用使其成为多个学科的研究热点。多项研究表明miRNA在骨的形成与代谢中发挥重要调控作用。已经有研究表明miRNA可以对鸟类的体节形成过程进行调控。人血清中存在稳定的miRNA,且在正常人群中表达一致,已有研究表明血清中的miRNA在机体处于疾病状态时会发生特异性改变；异常表达的特异miRNA与疾病导致的异常代谢机制有关,通过对其的研究有助于更深入的了解疾病的发病机制、疾病的早期诊断以及预后等。
研究表明先天性脊柱侧凸患者骨密度较正常人低,提示其可能存在相应的代谢异常。由此推测参与该异常调控的miRNA会在血清中有所体现。迄今为止,未见有先天性脊柱侧凸患者与正常人群血清miRNA对比的相关研究。
目的
1.利用miRNA微阵列芯片技术筛选CS患者与正常对照间血清差异miRNA。
2.利用生物信息学技术对差异表达的miRNA进行分析，对其靶基因进行预测，并对miRNA可能起作用的细胞间信号通路进行预测。
3.结合目前已有CS病因学研究成果,选取可能的目标miRNA,并在后续研究中进行验证,以期寻找与之相关的异常的调控机制,了解其在CS发病中的作用。为CS的预防、早期诊断以及治疗提供新的线索。
研究方法
1.本研究收集了3例分节障碍型、3例形成障碍型先天性脊柱侧凸患者以及3例年龄、性别匹配的正常对照人群的血清。提取血清中总RNA,经质控合格后,利用miRNA微阵列芯片技术对两种亚型CS患者与正常对照之间差异表达的miRNA进行筛选。筛选差异表达的miRNA的标准为：上调的Foldchange值>2,P值<0.05；下调的Foldchange值<0.5,P值<0.05。
2.通过查询miRbase数据库对差异的miRNA进行序列分析；查询miRbase以及Targetscan数据库进行靶基因预测；通过http://diana.cslab.ece.ntua.gr/pathways/预测差异miRNA与细胞间信号传导通路的关系；在Pumbmed以及NCBI数据库查询靶基因位点注释信息。结合目前已有CS病因学研究成果,选取可能的目标miRNA。
研究结果
1.标本经质控确认合格后,根据标准进行miRNA筛选。分节障碍型先天性脊柱侧凸患者同正常对照相比上调的miRNA有54个,下调的有42个；形成障碍型先天性脊柱侧凸患者同正常对照组相比上调的miRNA有19个,下调的有28个。
2.通过对每个miRNA靶基因的预测以及相关细胞间信号传导通路的查询,结合目前已知的CS病因学研究资料筛选出可能的目标miRNA。其中分节障碍组上调选取11个,分别为：hsa-miR-10a-3p、hsa-miR-10a-5p、hsa-miR-3915、hsa-miR-548j、hsa-miR-378g、hsa-miR-3173-5p、hsa-miR-604、hsa-miR-3913-3p、hsa-miR-4312、hsa-miR-520a-5p以及hsa-miR-378c;下调选取9个,分别为hsa-miR-4726-5p、hsa-miR-4472、hsa-miR-181c-5p、hsa-miR-1293、hsa-miR-181d、hsa-miR-138-5p、hsa-miR-144-3p、hsa-miR-30e-3p和hsa-miR-181b-5p。形成障碍组上调选取5个,分别为：hsa-miR-574-5p、hsa-miR-4796-5p、hsa-miR-548a-5p、hsa-miR-604以及hsa-miR-3913-3p;下调选取5个,分别为hsa-miR-4262、hsa-miR-10b-5p、hsa-miR-30e-3p、 hsa-miR-4483以及hsa-miR-3148。选取的miRNA的靶基因均为目前已知CS的候选基因,或表达产物为可能参与发病相关细胞间信号通路组成部分的基因；选取目标miRNA相关的细胞间信号传导通路为"Notch通路”、"Wnt通路”以及"Dorso-ventral axis formation通路”,其中Notch通路已经被证实在CS的发病中发挥重要调控作用；而另两者为胚胎早期的体节发育过程中重要的调控通路。
3. hsa-miR-548a-5p (hsa-miR-548j), hsa-miR-604以及hsa-miR-3913-3p在分节障碍组以及形成障碍组均表现为上调,涉及的与CS发病相关的靶基因有：LMX1A、DVL3以及Notch2。其相关细胞间信号传导通路为Notch通路以及Wnt通路。hsa-miR-30e-3p在两组均表现为下调,其涉及的与CS发病相关的靶基因为Notch1以及JAG2,分别编码Notch通路的受体与配体。
结论
1.本研究首次对CS患者进行血清miRNA微阵列芯片研究,发现CS患者的血清miRNA与正常人存在差异,并成功构建了CS患者与正常对照组患者血清差异miRNA资料库。
2.利用生物信息学技术对差异表达的miRNA进行分析,结合现有先天性脊柱侧凸病因学研究成果,分别在分节障碍型CS患者和形成障碍型CS患者的血清中筛选出可能的目标miRNA:其中两组存在共同的目标miRNA:3种表现为上调,1种表现为下调。目标miRNA的靶基因及这些miRNA参与调控的细胞间信号传导通路均与CS发病以及胚胎早期体节发育相关。而两组存在相同的差异miRNA提示两种亚型可能存在共同的异常代谢以及相关调控机制,需要在后续研究中进一步探讨。
Background
Congenital scoliosis is a lateral curvature of the spine caused by vertebral anomalies that result in an imbalance of the longitudinal growth of the spine. The congenital scoliosis could be divided into three types. Type I is failure of formation. Type Ⅱ is failure of segmentation. Type III is a combination of both of these conditions. Congenital anomalies occur in the phase of somitogenesis during embryogenesis. Disruptions in somitogenesis have been shown to result in vertebral malformations. Vertebral defects can arise from the disruption of genes involved in development, drugs, enviromental insults during gestation, or a combination of these factors. Many evidences suggest that CS may be multigenetic disease.
MicroRNAs (miRNAs) are classified as regulatory RNAs, and have been proved to play important roles in the development, proliferation and differentiation of various types of cells. It has been reported that miRNA played an important role in the bone development and formation. Recent studies have indicated that miRNA also circulates in serum and plasma. Levels of miRNAs in serum are stable, and consistent among individuals of the same species. For instance, disease-specific serum miRNA has been found in cancer, diabetes, and other diseases.
It has been found that both BMD (bone mineral desity) and trabecular bone microsturcture in CS patients were lower than those in normal person, suggesting abnormal bone metabolism in these patients. Until now there has been no study on miRNA in serum of patients with congenital scoliosis.
Objectives:
1. To detect differential expressed microRNAs in serum between CS and control.
2. To analyze the differential expressed microRNAs with bioinformatics analysis.
3. To find key microRNA for further research through comparative analysis combined with bioinformatic prediction.
Methods
1. Serum was collected from3patients with formation failure,3patients with segmentation failure and3age-and gender-matched normal persons. Then total RNA was isolated and tested with Exiqon miRNA Arrays. The slides were scanned and followed by data extraction. After statistical analysis we got the list of differential expressed microRNAs.
2. Target gene and pathway prediction was made.
3. The key miRNA was selected after comparative analysis combined with bioinformatic prediction.
Results
1. A list of differential expressed microRNAs was made by setting the hreshold of up/down regulated microRNAs as Foldchange>2and P value<0.05.54up-regulated miRNAs and42down-regulated miRNAs were detected in the segmentation failure group VS control group.19up-regulated miRNAs and28down-regulated miRNAs were detected in formation failure group VS control group.
2. The key miRNAs were selected. For the segmental failure group,11up-regulated:hsa-miR-10a-3p, hsa-miR-10a-5p, hsa-miR-3915, hsa-miR-548j, hsa-miR-378g, hsa-miR-3173-5p, hsa-miR-604, hsa-miR-3913-3p, hsa-miR-4312, hsa-miR-520a-5p and hsa-miR-378c;9down-regulated:hsa-miR-4726-5p, hsa-miR-4472, hsa-miR-181c-5p, hsa-miR-1293, hsa-miR-181d, hsa-miR-138-5p, hsa-miR-144-3p, hsa-miR-30e-3p and hsa-miR-181b-5p. For the formation failure group,5up-regulated:hsa-miR-574-5p, hsa-miR-4796-5p, hsa-miR-548a-5p, hsa-miR-604and hsa-miR-3913-3p;5down-regulated: hsa-miR-4262, hsa-miR-10b-5p, hsa-miR-30e-3p, hsa-miR-4483and hsa-miR-3148.
Conclusions
1. This is the first time to study comparative serum miRNA of CS. And differential regulated miRNAs in serum were detected between patients and normal persons.
2. The key miRNAs of the group of segmentation failure and formation failure were selected for the following research.
先天性脊柱侧凸(Congenital scoliosis,CS)是常见的脊柱畸形之一。根据影像学表现可分为三种类型：分节障碍型,形成障碍型以及同时存在以上两种畸形类型的混合型；每一种亚型的影像学表现以及自然史显著不同。先天性脊柱侧凸发病于胚胎时期“体节”形成的过程中,目前认为遗传因素以及环境因素均在其发病中发挥作用。先天性脊柱侧凸病因尚不明确,目前的研究结果支持CS是一种多基因遗传疾病。但具体的发病机制尚不清楚。
近年来,miRNA在基因转录后的调节作用使其成为多个学科的研究热点。多项研究表明miRNA在骨的形成与代谢中发挥重要调控作用。已经有研究表明miRNA可以对鸟类的体节形成过程进行调控。人血清中存在稳定的miRNA,且在正常人群中表达一致,已有研究表明血清中的miRNA在机体处于疾病状态时会发生特异性改变；异常表达的特异miRNA与疾病导致的异常代谢机制有关,通过对其的研究有助于更深入的了解疾病的发病机制、疾病的早期诊断以及预后等。
研究表明先天性脊柱侧凸患者骨密度较正常人低,提示其可能存在相应的代谢异常。由此推测参与该异常调控的miRNA会在血清中有所体现。迄今为止,未见有先天性脊柱侧凸患者与正常人群血清miRNA对比的相关研究。
目的
1.利用miRNA微阵列芯片技术筛选CS患者与正常对照间血清差异miRNA。
2.利用生物信息学技术对差异表达的miRNA进行分析，对其靶基因进行预测，并对miRNA可能起作用的细胞间信号通路进行预测。
3.结合目前已有CS病因学研究成果,选取可能的目标miRNA,并在后续研究中进行验证,以期寻找与之相关的异常的调控机制,了解其在CS发病中的作用。为CS的预防、早期诊断以及治疗提供新的线索。
研究方法
1.本研究收集了3例分节障碍型、3例形成障碍型先天性脊柱侧凸患者以及3例年龄、性别匹配的正常对照人群的血清。提取血清中总RNA,经质控合格后,利用miRNA微阵列芯片技术对两种亚型CS患者与正常对照之间差异表达的miRNA进行筛选。筛选差异表达的miRNA的标准为：上调的Foldchange值>2,P值<0.05；下调的Foldchange值<0.5,P值<0.05。
2.通过查询miRbase数据库对差异的miRNA进行序列分析；查询miRbase以及Targetscan数据库进行靶基因预测；通过http://diana.cslab.ece.ntua.gr/pathways/预测差异miRNA与细胞间信号传导通路的关系；在Pumbmed以及NCBI数据库查询靶基因位点注释信息。结合目前已有CS病因学研究成果,选取可能的目标miRNA。
研究结果
1.标本经质控确认合格后,根据标准进行miRNA筛选。分节障碍型先天性脊柱侧凸患者同正常对照相比上调的miRNA有54个,下调的有42个；形成障碍型先天性脊柱侧凸患者同正常对照组相比上调的miRNA有19个,下调的有28个。
2.通过对每个miRNA靶基因的预测以及相关细胞间信号传导通路的查询,结合目前已知的CS病因学研究资料筛选出可能的目标miRNA。其中分节障碍组上调选取11个,分别为：hsa-miR-10a-3p、hsa-miR-10a-5p、hsa-miR-3915、hsa-miR-548j、hsa-miR-378g、hsa-miR-3173-5p、hsa-miR-604、hsa-miR-3913-3p、hsa-miR-4312、hsa-miR-520a-5p以及hsa-miR-378c;下调选取9个,分别为hsa-miR-4726-5p、hsa-miR-4472、hsa-miR-181c-5p、hsa-miR-1293、hsa-miR-181d、hsa-miR-138-5p、hsa-miR-144-3p、hsa-miR-30e-3p和hsa-miR-181b-5p。形成障碍组上调选取5个,分别为：hsa-miR-574-5p、hsa-miR-4796-5p、hsa-miR-548a-5p、hsa-miR-604以及hsa-miR-3913-3p;下调选取5个,分别为hsa-miR-4262、hsa-miR-10b-5p、hsa-miR-30e-3p、 hsa-miR-4483以及hsa-miR-3148。选取的miRNA的靶基因均为目前已知CS的候选基因,或表达产物为可能参与发病相关细胞间信号通路组成部分的基因；选取目标miRNA相关的细胞间信号传导通路为"Notch通路”、"Wnt通路”以及"Dorso-ventral axis formation通路”,其中Notch通路已经被证实在CS的发病中发挥重要调控作用；而另两者为胚胎早期的体节发育过程中重要的调控通路。
3. hsa-miR-548a-5p (hsa-miR-548j), hsa-miR-604以及hsa-miR-3913-3p在分节障碍组以及形成障碍组均表现为上调,涉及的与CS发病相关的靶基因有：LMX1A、DVL3以及Notch2。其相关细胞间信号传导通路为Notch通路以及Wnt通路。hsa-miR-30e-3p在两组均表现为下调,其涉及的与CS发病相关的靶基因为Notch1以及JAG2,分别编码Notch通路的受体与配体。
结论
1.本研究首次对CS患者进行血清miRNA微阵列芯片研究,发现CS患者的血清miRNA与正常人存在差异,并成功构建了CS患者与正常对照组患者血清差异miRNA资料库。
2.利用生物信息学技术对差异表达的miRNA进行分析,结合现有先天性脊柱侧凸病因学研究成果,分别在分节障碍型CS患者和形成障碍型CS患者的血清中筛选出可能的目标miRNA:其中两组存在共同的目标miRNA:3种表现为上调,1种表现为下调。目标miRNA的靶基因及这些miRNA参与调控的细胞间信号传导通路均与CS发病以及胚胎早期体节发育相关。而两组存在相同的差异miRNA提示两种亚型可能存在共同的异常代谢以及相关调控机制,需要在后续研究中进一步探讨。
Background
Congenital scoliosis is a lateral curvature of the spine caused by vertebral anomalies that result in an imbalance of the longitudinal growth of the spine. The congenital scoliosis could be divided into three types. Type I is failure of formation. Type Ⅱ is failure of segmentation. Type III is a combination of both of these conditions. Congenital anomalies occur in the phase of somitogenesis during embryogenesis. Disruptions in somitogenesis have been shown to result in vertebral malformations. Vertebral defects can arise from the disruption of genes involved in development, drugs, enviromental insults during gestation, or a combination of these factors. Many evidences suggest that CS may be multigenetic disease.
MicroRNAs (miRNAs) are classified as regulatory RNAs, and have been proved to play important roles in the development, proliferation and differentiation of various types of cells. It has been reported that miRNA played an important role in the bone development and formation. Recent studies have indicated that miRNA also circulates in serum and plasma. Levels of miRNAs in serum are stable, and consistent among individuals of the same species. For instance, disease-specific serum miRNA has been found in cancer, diabetes, and other diseases.
It has been found that both BMD (bone mineral desity) and trabecular bone microsturcture in CS patients were lower than those in normal person, suggesting abnormal bone metabolism in these patients. Until now there has been no study on miRNA in serum of patients with congenital scoliosis.
Objectives:
1. To detect differential expressed microRNAs in serum between CS and control.
2. To analyze the differential expressed microRNAs with bioinformatics analysis.
3. To find key microRNA for further research through comparative analysis combined with bioinformatic prediction.
Methods
1. Serum was collected from3patients with formation failure,3patients with segmentation failure and3age-and gender-matched normal persons. Then total RNA was isolated and tested with Exiqon miRNA Arrays. The slides were scanned and followed by data extraction. After statistical analysis we got the list of differential expressed microRNAs.
2. Target gene and pathway prediction was made.
3. The key miRNA was selected after comparative analysis combined with bioinformatic prediction.
Results
1. A list of differential expressed microRNAs was made by setting the hreshold of up/down regulated microRNAs as Foldchange>2and P value<0.05.54up-regulated miRNAs and42down-regulated miRNAs were detected in the segmentation failure group VS control group.19up-regulated miRNAs and28down-regulated miRNAs were detected in formation failure group VS control group.
2. The key miRNAs were selected. For the segmental failure group,11up-regulated:hsa-miR-10a-3p, hsa-miR-10a-5p, hsa-miR-3915, hsa-miR-548j, hsa-miR-378g, hsa-miR-3173-5p, hsa-miR-604, hsa-miR-3913-3p, hsa-miR-4312, hsa-miR-520a-5p and hsa-miR-378c;9down-regulated:hsa-miR-4726-5p, hsa-miR-4472, hsa-miR-181c-5p, hsa-miR-1293, hsa-miR-181d, hsa-miR-138-5p, hsa-miR-144-3p, hsa-miR-30e-3p and hsa-miR-181b-5p. For the formation failure group,5up-regulated:hsa-miR-574-5p, hsa-miR-4796-5p, hsa-miR-548a-5p, hsa-miR-604and hsa-miR-3913-3p;5down-regulated: hsa-miR-4262, hsa-miR-10b-5p, hsa-miR-30e-3p, hsa-miR-4483and hsa-miR-3148.
Conclusions
1. This is the first time to study comparative serum miRNA of CS. And differential regulated miRNAs in serum were detected between patients and normal persons.
2. The key miRNAs of the group of segmentation failure and formation failure were selected for the following research.
引文
1. Loder RT, Hernandez MJ, Lerner AL, et al. The induction of congenital spinal deformities in mice by maternal carbon monoxide exposure. J Pediatr Orthop, 2000; 20:662-666.
2. Ingalls TH, Curley FJ. Principles governing the genesis of congenital malformations induced in mice by hypoxia. N Engl J Med,1957; 257:1121-1127.
3. Farley FA, Loder RT, Nolan BT, et al. Mouse model for thoracic congenital scoliosis. J Pediatr Orthop,2001; 21:537-40.
4. Erol B KK, Lou J, Dormans JP. Etiology of congenital scoliosis. The University of Pennsylvania. Orthop J,2002; 15:37-42.
5. Conlon RA, Reaume AQ Rossant J. Notch1 is required for the coordinate segmentation of somites. Development 1995; 121:1533-1545.
6. Bessho Y, Miyoshi G, Sakata R, et al. Hes7:a bHLH-type repressor gene regulated by Notch and expressed in the presomitic mesoderm. Genes Cells,2001; 6:175-185.
7. Copley LA, Dormans JP. Cervical spine disorders in infants and children. J Am Acad Orthop Surg,1998; 6:204-214.
8. Larsen W. The fourth week. Differentiation of the somites and the nervous System; Segmental Development and Integration. In:Larsen W, ed. Human Embryology. New York, NY:Churchill Livingstone; 1997:73-104.
9. Winter RB, Moe JH, Eilers VE. Congenital scoliosis:a study of 234 patients treated and untreated. J Bone Joint Surg,1968; 50A:1.
10. Saga Y, Hata N, Koseki H, et al. Mesp2:a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Gene Dev, 1997,11(14):1827-1839.
11. Wallin J, Wilting J, Koseki H, et al. The role of Pax-1 in axial skeleton development. Development,1994,120(5):1109-1121.
12. Shinkai Y, Tsuji T, Kawamoto Y, et al. New mutant mouse with skeletal deformities caused by mutation in delta like 3(DⅡ3) gene. Development,1994, 120(5):1109-1121.
13. Takada S, Stark KL, Shea MJ, et al. Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes Dev,1994,8(2):174-189.
14. Zhang N, Gridley T. Defects in somite formation in lunatic fringe-deficient mice[J]. Nature,1998,394(6691):374-377.
15. Bessho Y, Sakata R, Komatsu S, et al. Dynamic expression and essential functions of Hes7 in somite segmentation. Genes Dev,2001,15(20):2642-2647.
16. Giampietro PF, Blank RD, Raggio CL, et al. Congenital and idiopathic scoliosis: clinical and genetic aspects. Clin Med Res,2003,1(2):125-136.
17. Giampietro PF, Raggio CL, Reynolds CE, et al. An analysis of PAX1 in the development of vertebral malformations. Clin Genet,2005,68:448-453.
18. Dietrich S, Gross P. Undulated phenotypes suggest a role of Pax1 for the development of vertebral and extravertebral structures. Dev Biol,1995, 167:529-548.
19. 费琦,吴志宏,原所茂等。中国汉族人群PAX1基因多态性与先天性脊柱侧凸遗传易感性的关联研究。中华医学杂志,2008,88：2597-2602.
20. Bergman O. Hakansson A. Westberg L, et al. Do polymorphisms in transcription factors LMX1A and LMX1B influence the risk for Parkinson's disease. J Neural Transm.2009,116:333-3338.
21. Chizhikov V. Steshina E. Roberts R. et al. Molecular definition of an allelic series of mutations disrupting the mouse Lmxla (dreher) gene. Mammalian Genome. 2006,17:1025-32.
22. Chizhikov VV. Millen KJ. Control of roof plate formation by Lmxla in the developing spinal cord. Development.2004,131:2693-2705.
23. Costa C. Harding B. Copp AJ. Neuronal migration defects in the Dreher (Lmxla) mutant mouse:role of disorders of the glial limiting membrane. Cerebral Cortex. 2001,11:498-505.
24. Qi Fei, Zhihong Wu, Hai Wang, et al. The Association Analysis of TBX6 Polymorphism With Susceptibility to Congenital Scoliosis in a Chinese Han Population. Spine,2010,35:983-988.
25. Yasuhiko Y. Haraguchi S. Kitajima S, et al. Tbx6-mediated Notch signaling controls somite-specific Mesp2 expression. Proc Natl Acad Sci USA. 2006,103:3651-3656.
26. Chen YL. Liu B. Zhou ZN, et al. Smad6 inhibits the transcriptional activity of Tbx6 by mediating its degradation. J Biol Chem.2009,284:23481-23490.
27. Yasuhiko Y. Kitajima S. Takahashi Y. et al. Functional importance of evolutionally conserved Tbx6 binding sites in the presomitic mesoderm-specific enhancer of Mesp2. Development.2008,135:3511-3519.
28. Oginuma M. Niwa Y. Chapman DL, et al. Mesp2 and Tbx6 cooperatively create periodic patterns coupled with the clock machinery during mouse somitogenesis. Development.2008,135:2555-2562.
29. Wittler L. Shin EH. Grote P, et al. Expression of Msgnl in the presomitic mesoderm is controlled by synergism of WNT signalling and Tbx6. EMBO Reports.2007,8:784-789.
30. Ghebranious N, Raggio CL, Blank RD. et al. Lack of evidence of WNT3A as a candidate gene for congenital vertebral malformations. Scoliosis,2007,2(13).
31. Maisenbacher MK, Han JS, O'Brien ML.et al. Molecular analysis of congenital scoliosis:a candidate gene approach. Hum Genet,2005,116:416-419.
32. 费琦,吴志宏,周熹等。中国汉族人群SIM2基因多态性与先天性脊柱侧凸遗传易感性的关联研究。中华医学杂志,2009,89：2888-2893.
33. Goshu E, Jin H. Fasnaeht R. et al. SIM2 mutants have developmental defects not overlapping with those of Siml mutants. Mol Cell Biol.2002, 22:4147-4157.
34. Bulent R, Tracy MR, John DP, et al. Congenital Scoliosis and Vertebral Malformations. J Pediatr Orthop,2004,24:674-682.
35. Dunwoodie SL, Clements M, Sparrow DB, et al. Axial skeletal defects caused by mutation in the spondylocostal dysplasia pudgy gene D113 are associated with disruption of the segmentation clock within the presomitic mesoderm. Development,2002,129:1795-1806.
36. Turnpenny PD, Whittock N, Duncan J,et al. Novel mutations in DLL3, a somitogenesis gene encoding a ligand for the Notch signalling pathway, cause a consistent pattern of abnormal vertebral segmentation in spondylocostal dysostosis. J Med Genet,2003,40:333-339.
37. Taku Y, Hidetoshi K, Hideaki O, et al. Association of positional and functional candidate genes FGF1, FBN2, and LOX on 5q31 with intracranial aneurysm. J Hum Genet,2003,48:309-314.
38. Feng Y, Hoggatt AM, Zhou JL, et al.130-kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse mylk gene. Am J Physiol Cell Physiol,2006,290:1599-1609.
39. Riley MF, Bochter MS, Wahi K, et al. mir-125a-5p-Mediated Regulation of Lfng Is Essential for the Avian Segmentation Clock. Dev Cell,2013,24(5):554-561.
40. Lee RC, Feinbaum RL, Ambros V. The C. Elegans heterochmnic gene lin-4 antodes small RNAs withantisense complementarity to lin-14. Cell,1993, 75(5):843-854.
41. Nakajima N, Takahashi T, Kitamura R, et al. MicroRNA-1 facilitates skeletal myogenic differentiation without affecting osteoblastic and adipogenic differentiation. Biochem Biophys Res Commun,2006,350(4):1006-1012.
42. 孙铁为,王瑾,吴德全,等。微小RNA对肿瘤影响的研究进展。中华实验外科杂志,2007,24：1607-1608。
43. 刘长征,蔡磊,刘卫,等。微RNA-21在肝细胞癌中的表达及其靶基因研究。中华实验外科杂志,2009,26：964-966。
44. Kobayashi T, Lu J, Cobb BS, et al. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc Natl Acad Sci USA,2008, 105(6):1949-1954.
45. Watanabe T. Sato T. Amano T. et al. Dnm30s. a non-coding RNA, is required for normal growth and skeletal development in mice. Dev Dyn,2008,237(12):3738-3748.
46. Oskowitz AZ, Lu J, Penfornis P, et al. Human multipotent stromal cells from bone marrow and microRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci U S A,2008,105:18372-18377.
47. Harfe B, McManus M, Mansfield J, et al. The RNase enzyme Dicer is required for morpho-genesis but not patterning of the vertebrate limb. Proceedings of the National Academy of Sciences of the United States of America,2005,102:10898.
48. Sugatan T, Hruska KA. Impaired MicroRNA pathways diminish osteochist diflerentiation and function. J Biol Chem,2009,284(7):4667-4678.
49. Han JW, Yang T, Gao J, etal. Specific mieroRNA expression during chondrogenesis of human mesenchymal stem cells. Int J Mol Med,2010, 25(3):377-284.
50. Lawrie CH, Gal S, Dunioph M, et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br Hacematol,2008,141(5):672-675.
51. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood based maskers for cancer detection. Proc Natl Acad Sci USA,2008,105:10513-10518.
52. Chen X, Ba Y, Ma L, et al. Characterization of microRNAs in serum:a Novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res,2008, 18(10):997-1006.
53. Sisco KL. Is RNA in serum bound to nucleopmtein complexes. Clin Chem,2001, 47(9):1744-1745.
54. Hunter MP, Ismail N, Zhang X, et al. Detection of microRNA expression in human peripheral blood microvesicles. PIOS ONE,2008,3(11):e3694.
55. Ai J, Zhang R, Li Y, et al.Circulting microRNA-1 as a potential novel biomarker for acute myocardial infarction. Biachem Biophys Res Commun,2010,391:73-77.
56. Valadi H, Ekstrom K, Bossios A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol,2007,9:654-659.
57. 朱锋,邱勇,杨晓恩,等。青少年特发性和先天性脊柱侧凸患者的骨微结构及骨密度比较。中华骨科杂志,2005,25(9)：541-545。
58. Larabell CB, Tortes M, Rowning BA, et al. Establishment of the dorso-vertral axis in Xenopus embryosis presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway. J Cell Biol,1997,136(5):1123-1136.
59. Hasson P, Paroush Z. Crosstalk between the EGFR and other signaling pathways at te level of the global transcriptional corepressor Groucho/TLE. Br J Cancer,2006,94(6):771-775.
60. Dornseifer P, Takke C, Campos-Ortega JA. Over expression of a zebrafish homologue of the Drosophila neurogenic gene Delta perturbs differentiation of primary neurons and somite development. Mech Dev,1997,63(2):159-171.
61. Jen WC, Gawantka V, Pollet N. Periodic repression of Notch path way genes governs the segmentation of XenoPus embryos. Genes Dev,1999,13(11):1486-1499.
62. Takke C, Campos-Ortega JA. her1, a zebrafish pair-rule like gene, acts downstream of notch signaling to control somite development. Development, 1999,126(13):3005-3014.
63. Bhanot P, Fish M, Jemison JA, etal. Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development. Development,1999,126(18):4175-4186.
64. Huelsken S, Vogel R, Brinkmann V, et al. Requirement for beta-catenin in anterior-posterior axis formation in mice. Cell Biol,2000; 148(3):567-578.
65. Agirre X, Jimenez-Velasco A, San Jose-Eneriz E, et al. Down-regulation of hsa-miR-lOa in chronic myeloid leukemia CD34+ cells increases USF2-mediated cell growth. Mol Cancer Res,2008,6(12):1830-1840.
66. Blanco G, Goulton GR, Biggin A, et al. The kyphoscoliosis(ky) mouse is deficient in hypertrophic responses and is caused by a mutation in a novel muscle-specific protein. Hum Mol Genet,2001,10(1):9-16.
67. 原所茂。汉族人群基因多态性与先天性脊柱侧凸遗传易感性的关联性的研究。清华大学医学部,北京协和医学院,北京协和医院博士论文,2009。
68. Millonig JH. Millen KJ. Hatten ME. The mouse Dreher gene Lmxla control formation of the roof plate in the vertebrate CNS. Nature,2000,403(6771):764-769.
69. 费琦,吴志宏,王以朋等。LMX1A基因多态性与中国汉族人群先天性脊柱侧凸遗传易感性的关联。中国组织工程研究与临床康复,2011,12(38)：6632-6638。
70. Simpson MA, Irving MD, Asilmaz E, et al. Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss. Nature Genetics,2011,43(4):303-305.
71. Chapman DL, Papaioannou VE. Three neural tubes in mouse embryos with mutations in the T-box geneTbx6. Nature,1998,391(6668):695-697.
72. Hamblet NS, Lijam N, Ruiz-Lozano P, et al. Dishevelled 2 is essential for cardiac outflow tract development, somite segmentation and neural tube closure. Development,2002,129 (24):5827-5838.
73. Wang B, Li W, Guo K, et al. miR-181b promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients. Biochem Biophys Res Commun,2012,421(1):4-8.
74. Chen L, Yang Q, Kong WQ, et al. MicroRNA-181b targets cAMP responsive element binding protein 1 in gastric adenocarcinomas.IUBMB Life,2012, 64(7):628-635.
75. Visone R, Veronese A, Balatti V, et al. MiR-181b:new perspective to evaluate disease progression in chronic lymphocytic leukemia. Oncotarget,2012, 3(2):195-202.
76. Rio P, Agirre X, Garate L, et al. Down-regulated expression of hsa-miR-181c in Fanconi anemia patients:implications in TNFa regulation and proliferation of hematopoietic progenitor cells..Blood,2012,119(13):3042-3049.
77. Geekiyanage H, Chan C. MicroRNA-137/181 c regulates serine palmitoyltransferase and in turn amyloid β, novel targets in sporadic Alzheimer's disease.J Neurosci,2011,31(41):14820-14830.
78. Sand M, Skrygan M, Sand D, et al. Expression of microRNAs in basal cell carcinoma. Br J Dermatol,2012,167(4):847-855.
79. Kang JG, Majerciak V, Uldrick TS, et al. Kaposi's sarcoma-associated herpesviral IL-6 and human IL-6 open reading frames contain miRNA binding sites and are subject to cellular miRNA regulation. J Pathol,2011,225(3):378-389.
80. Eskildsen T, Taipaleenmaki H, Stenvang J, et al. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo. ProcNatl Acad Sci USA,2011,108(15):6139-6144.
81. Sureban SM, May R, Mondalek FG, et al. Nanoparticle-based delivery of siDCAMKL-1 increases microRNA-144 and inhibits colorectal cancer tumor growth via a Notch-1 dependent mechanism. J Nanobiotechnology,2011,9:40.
82. Wang H, Meng K, Chen Wj, et al. Serum miR-574-5p:a prognostic predictor of sepsis patients. Shock,2012,37(3):263-267.
83. Foss KM, Sima C, Ugolini D, et al. miR-1254 and miR-574-5p:serum-based microRNA biomarkers for early-stage non-small cell lung cancer. J Thorac Oncol, 2011,6(3):482-488.
84. Chen W, Cai F, Zhang B, et, al. The level of circulating miRNA-10b and miRNA-373 in detecting lymph node metastasis of breast cancer:potential biomarkers. Tumour Biol,2013,34(1):455-62.
85. Gabriely G, Yi M, Narayan RS, et al. Human glioma growth is controlled by microRNA-lOb. Cancer Res,2011,71(10):3563-72.
86. Nakata K, Ohuchida K, Mizumoto K, et al. MicroRNA-10b is overexpressed in pancreatic cancer, promotes its invasiveness, and correlates with a poor prognosis. Surgery,2011,150(5):916-22.
87. Fendler A, Jung M, Stephan C, et al. miRNAs can predict prostate cancer biochemical relapse and are involved in tumor progression. Int J Oncol,2011, 39(5):1183-92.
1. Kobayashi T, Lu J, Cobb BS, et al. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc Natl Acad Sci USA,2008,105(6):1949-1954.
2. Watanabe T, Sato T, Amano T, et al. Dnm30s, a non-coding RNA, is required for normal growth and skeletal development in mice. Dev Dyn,2008,237(12):3738-3748.
3. Lee RC, Feinbaum RL, Ambros V. The C. Elegans heterochmnic gene lin-4 antodes small RNAs withantisense complementarity to lin-14. Cell,1993,75(5):843-854.
4. Lee Y, Ahn C, Han J, et al. The nuclear RNaseⅢ Drosha initiates miRNA processing. Nature,2003,425(6956):415-419.
5. Han JJ, Lee Y, YeomKH, et al. Molecular basis for the recognition of primary micoRNAs by the Dmsha-DGCR8 complex. Cell,2006,125(5):887-901.
6. Zeng Y, Cullen BR. Sequence requirements for miRNA processing and function in human cells. RNA,2003,9(1):112-123.
7. Du T, Zamore PD. MicroPrimer:the biogenesis and function of miRNA. Development,2005,132(21):4645-4652.
8. Nakajima N, Takahashi T, Kitamura R, et al. miRNA-1 facilitates skeletal myogenic differentiation without affecting osteoblastic and adipogenic differentiation. Biochem Biophys Res Commun,2006,350(4):1006-1012.
9. Sugatan T, Hruska KA. Impaired miRNA pathways diminish osteochist diflerentiation and function. J Biol Chem,2009,284(7):4667-4678.
10. Mizuno Y, TOkuzawa Y, Ninomiya Y, et al. miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b. FEBS Lett,2009,583(13):2263-2268.
11. Sun S. Bone disease drug discovery:examining the interactions between osteoblast and Oeteoclast. Expert Opin Ther Targets,2008,12(2):239-251.
12. Oskowitz AZ, Lu J, Penfornis P, et al. Human multipotent stromal cells from bone marrow and miRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci U S A,2008,105:18372-18377.
13. Harfe B, McManus M, Mansfield J, et al. The RNase III enzyme Dicer is required for morpho-genesis but not patterning of the vertebrate limb. Proceedings of the National Academy of Sciences of the United States of America,2005,102:10898.
14. Mizuno Y, Vagi K, Tokuzawa Y, etal. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun,2008, 368(2):267-272.
15. Zhang J, Tu Q, Bonewald LF, et al. Effects of miR-335-5p in modulating osteogenic differentiation by specifically down-regulating Wnt antagonist DKK1. J Bone Miner Res,2011.
16. Kapinas K, Kessler C, Ricks T, et al. miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop. J Biol Chem,2010,285:25221-25231.
17. Lian JB, Stein GS, Stein JL, et al. Networks and hubs for the transcriptional control of osteoblastogenesis. Rev Endocr Metab Disord,2006,7(1-2):1-16.
18. Li Z, Hassan MQ, Volinia S, et al. A miRNA signature for a BMP2-induced osteoblastlineage commitment program. PNAS,2008,105(37):13906-13911.
19. Huang J, Zhao L, Xing L, et al. miRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010,28:357.
20. ICCBH5, Fifth International Conference on Childrens Bone Health. Bone,2009.
21. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are miRNA targets. Cell,2005,120(1):15-20.
22. Luzi E, Marini F, Sala SC, etal. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 trans factor. J Bone Miner Res,2008,23(2):287-295.
23. Kim YJ, Bae SW, Yu SS, et al. miR-196a regulatesproliferation and osteogenic differentiation in mesenchymal stem cells derived from human adipose tissue. J Bone Miner Res,2009,24(5):816-825.
24. Haynes DR, Crotti TN, Zreiqat H, et al. Regulation of osteoclast activity in peri-implant issues. Biomaterials,2004,25(20):4877-4885.
25. Miyaki S, Sato T, Inoue A, et al. miRNA-140 plays dual roles in both cartilage development and homeostasis. Gene Dev,2010,24(11):1173-1185.
26. Kim D, SongJ, Jin EJ. MicmRNA-221 regulates chondrogenic differentiation through promoting proteosomal degradation of slug by targeting Mdm2. J Bid Chem2010,285(35):26900-26907.
27. Han JW, Yang T, Gao J, etal. Specific mieroRNA expression during chondrogenesis of human mesenchymal stem cells. Int J Mol Med,2010,25(3):377-284.
28. Yang B, Gun H, Zhang Y, et al. The miRNA expression profiles of mouse mesen-chymal stem cell during chondrogenic differentiation. BMB Rep,2011,44(1):28-33.
29. Araldi E, Sehipani E. miRNA-140 and the silencing of osteotarthritis. Genes Dev, 2010,24(11):1075-1080.
30. Tardif C, Hum D, Pelletier JP, et al. Regulation of the IGFBP-5 and MMP-13 genes by the miRNAs miR-140 and miR-27a in human osteoarthritis chondrocytes.BMC Musculoskelet Disord,2009,10:148.
31. Jones SW, Watkins G, Le Good N, et al. The identification of differentially expressed miRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13. Osteoarthritis Cartilage,2009,17(4):464-472.
32. SumiyoshiK, Kubota S, Ohgawara T, et al. Identification of miR-1 as a micro RNA that supports late-stage differentiation of growth cartilage cells. Bio ehem Biophys Res Commun,2010,402(2):286-290.
33. Kongeharoensombat W, Nakasa T, Iishikawa M, et al. The effect of miRNA-21 on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel. Fame Surg Sports Traumatol Arthrosc,2010,18(12):1679-1684.
34. Abouheif MM, Nakasa T, Shibuya H, et al. Silencing miRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro. Rheumatology(Oxford), 2010,49(11):2054-2060.
2. Ingalls TH, Curley FJ. Principles governing the genesis of congenital malformations induced in mice by hypoxia. N Engl J Med,1957; 257:1121-1127.
3. Farley FA, Loder RT, Nolan BT, et al. Mouse model for thoracic congenital scoliosis. J Pediatr Orthop,2001; 21:537-40.
4. Erol B KK, Lou J, Dormans JP. Etiology of congenital scoliosis. The University of Pennsylvania. Orthop J,2002; 15:37-42.
5. Conlon RA, Reaume AQ Rossant J. Notch1 is required for the coordinate segmentation of somites. Development 1995; 121:1533-1545.
6. Bessho Y, Miyoshi G, Sakata R, et al. Hes7:a bHLH-type repressor gene regulated by Notch and expressed in the presomitic mesoderm. Genes Cells,2001; 6:175-185.
7. Copley LA, Dormans JP. Cervical spine disorders in infants and children. J Am Acad Orthop Surg,1998; 6:204-214.
8. Larsen W. The fourth week. Differentiation of the somites and the nervous System; Segmental Development and Integration. In:Larsen W, ed. Human Embryology. New York, NY:Churchill Livingstone; 1997:73-104.
9. Winter RB, Moe JH, Eilers VE. Congenital scoliosis:a study of 234 patients treated and untreated. J Bone Joint Surg,1968; 50A:1.
10. Saga Y, Hata N, Koseki H, et al. Mesp2:a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Gene Dev, 1997,11(14):1827-1839.
11. Wallin J, Wilting J, Koseki H, et al. The role of Pax-1 in axial skeleton development. Development,1994,120(5):1109-1121.
12. Shinkai Y, Tsuji T, Kawamoto Y, et al. New mutant mouse with skeletal deformities caused by mutation in delta like 3(DⅡ3) gene. Development,1994, 120(5):1109-1121.
13. Takada S, Stark KL, Shea MJ, et al. Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes Dev,1994,8(2):174-189.
14. Zhang N, Gridley T. Defects in somite formation in lunatic fringe-deficient mice[J]. Nature,1998,394(6691):374-377.
15. Bessho Y, Sakata R, Komatsu S, et al. Dynamic expression and essential functions of Hes7 in somite segmentation. Genes Dev,2001,15(20):2642-2647.
16. Giampietro PF, Blank RD, Raggio CL, et al. Congenital and idiopathic scoliosis: clinical and genetic aspects. Clin Med Res,2003,1(2):125-136.
17. Giampietro PF, Raggio CL, Reynolds CE, et al. An analysis of PAX1 in the development of vertebral malformations. Clin Genet,2005,68:448-453.
18. Dietrich S, Gross P. Undulated phenotypes suggest a role of Pax1 for the development of vertebral and extravertebral structures. Dev Biol,1995, 167:529-548.
19. 费琦,吴志宏,原所茂等。中国汉族人群PAX1基因多态性与先天性脊柱侧凸遗传易感性的关联研究。中华医学杂志,2008,88：2597-2602.
20. Bergman O. Hakansson A. Westberg L, et al. Do polymorphisms in transcription factors LMX1A and LMX1B influence the risk for Parkinson's disease. J Neural Transm.2009,116:333-3338.
21. Chizhikov V. Steshina E. Roberts R. et al. Molecular definition of an allelic series of mutations disrupting the mouse Lmxla (dreher) gene. Mammalian Genome. 2006,17:1025-32.
22. Chizhikov VV. Millen KJ. Control of roof plate formation by Lmxla in the developing spinal cord. Development.2004,131:2693-2705.
23. Costa C. Harding B. Copp AJ. Neuronal migration defects in the Dreher (Lmxla) mutant mouse:role of disorders of the glial limiting membrane. Cerebral Cortex. 2001,11:498-505.
24. Qi Fei, Zhihong Wu, Hai Wang, et al. The Association Analysis of TBX6 Polymorphism With Susceptibility to Congenital Scoliosis in a Chinese Han Population. Spine,2010,35:983-988.
25. Yasuhiko Y. Haraguchi S. Kitajima S, et al. Tbx6-mediated Notch signaling controls somite-specific Mesp2 expression. Proc Natl Acad Sci USA. 2006,103:3651-3656.
26. Chen YL. Liu B. Zhou ZN, et al. Smad6 inhibits the transcriptional activity of Tbx6 by mediating its degradation. J Biol Chem.2009,284:23481-23490.
27. Yasuhiko Y. Kitajima S. Takahashi Y. et al. Functional importance of evolutionally conserved Tbx6 binding sites in the presomitic mesoderm-specific enhancer of Mesp2. Development.2008,135:3511-3519.
28. Oginuma M. Niwa Y. Chapman DL, et al. Mesp2 and Tbx6 cooperatively create periodic patterns coupled with the clock machinery during mouse somitogenesis. Development.2008,135:2555-2562.
29. Wittler L. Shin EH. Grote P, et al. Expression of Msgnl in the presomitic mesoderm is controlled by synergism of WNT signalling and Tbx6. EMBO Reports.2007,8:784-789.
30. Ghebranious N, Raggio CL, Blank RD. et al. Lack of evidence of WNT3A as a candidate gene for congenital vertebral malformations. Scoliosis,2007,2(13).
31. Maisenbacher MK, Han JS, O'Brien ML.et al. Molecular analysis of congenital scoliosis:a candidate gene approach. Hum Genet,2005,116:416-419.
32. 费琦,吴志宏,周熹等。中国汉族人群SIM2基因多态性与先天性脊柱侧凸遗传易感性的关联研究。中华医学杂志,2009,89：2888-2893.
33. Goshu E, Jin H. Fasnaeht R. et al. SIM2 mutants have developmental defects not overlapping with those of Siml mutants. Mol Cell Biol.2002, 22:4147-4157.
34. Bulent R, Tracy MR, John DP, et al. Congenital Scoliosis and Vertebral Malformations. J Pediatr Orthop,2004,24:674-682.
35. Dunwoodie SL, Clements M, Sparrow DB, et al. Axial skeletal defects caused by mutation in the spondylocostal dysplasia pudgy gene D113 are associated with disruption of the segmentation clock within the presomitic mesoderm. Development,2002,129:1795-1806.
36. Turnpenny PD, Whittock N, Duncan J,et al. Novel mutations in DLL3, a somitogenesis gene encoding a ligand for the Notch signalling pathway, cause a consistent pattern of abnormal vertebral segmentation in spondylocostal dysostosis. J Med Genet,2003,40:333-339.
37. Taku Y, Hidetoshi K, Hideaki O, et al. Association of positional and functional candidate genes FGF1, FBN2, and LOX on 5q31 with intracranial aneurysm. J Hum Genet,2003,48:309-314.
38. Feng Y, Hoggatt AM, Zhou JL, et al.130-kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse mylk gene. Am J Physiol Cell Physiol,2006,290:1599-1609.
39. Riley MF, Bochter MS, Wahi K, et al. mir-125a-5p-Mediated Regulation of Lfng Is Essential for the Avian Segmentation Clock. Dev Cell,2013,24(5):554-561.
40. Lee RC, Feinbaum RL, Ambros V. The C. Elegans heterochmnic gene lin-4 antodes small RNAs withantisense complementarity to lin-14. Cell,1993, 75(5):843-854.
41. Nakajima N, Takahashi T, Kitamura R, et al. MicroRNA-1 facilitates skeletal myogenic differentiation without affecting osteoblastic and adipogenic differentiation. Biochem Biophys Res Commun,2006,350(4):1006-1012.
42. 孙铁为,王瑾,吴德全,等。微小RNA对肿瘤影响的研究进展。中华实验外科杂志,2007,24：1607-1608。
43. 刘长征,蔡磊,刘卫,等。微RNA-21在肝细胞癌中的表达及其靶基因研究。中华实验外科杂志,2009,26：964-966。
44. Kobayashi T, Lu J, Cobb BS, et al. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc Natl Acad Sci USA,2008, 105(6):1949-1954.
45. Watanabe T. Sato T. Amano T. et al. Dnm30s. a non-coding RNA, is required for normal growth and skeletal development in mice. Dev Dyn,2008,237(12):3738-3748.
46. Oskowitz AZ, Lu J, Penfornis P, et al. Human multipotent stromal cells from bone marrow and microRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci U S A,2008,105:18372-18377.
47. Harfe B, McManus M, Mansfield J, et al. The RNase enzyme Dicer is required for morpho-genesis but not patterning of the vertebrate limb. Proceedings of the National Academy of Sciences of the United States of America,2005,102:10898.
48. Sugatan T, Hruska KA. Impaired MicroRNA pathways diminish osteochist diflerentiation and function. J Biol Chem,2009,284(7):4667-4678.
49. Han JW, Yang T, Gao J, etal. Specific mieroRNA expression during chondrogenesis of human mesenchymal stem cells. Int J Mol Med,2010, 25(3):377-284.
50. Lawrie CH, Gal S, Dunioph M, et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br Hacematol,2008,141(5):672-675.
51. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood based maskers for cancer detection. Proc Natl Acad Sci USA,2008,105:10513-10518.
52. Chen X, Ba Y, Ma L, et al. Characterization of microRNAs in serum:a Novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res,2008, 18(10):997-1006.
53. Sisco KL. Is RNA in serum bound to nucleopmtein complexes. Clin Chem,2001, 47(9):1744-1745.
54. Hunter MP, Ismail N, Zhang X, et al. Detection of microRNA expression in human peripheral blood microvesicles. PIOS ONE,2008,3(11):e3694.
55. Ai J, Zhang R, Li Y, et al.Circulting microRNA-1 as a potential novel biomarker for acute myocardial infarction. Biachem Biophys Res Commun,2010,391:73-77.
56. Valadi H, Ekstrom K, Bossios A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol,2007,9:654-659.
57. 朱锋,邱勇,杨晓恩,等。青少年特发性和先天性脊柱侧凸患者的骨微结构及骨密度比较。中华骨科杂志,2005,25(9)：541-545。
58. Larabell CB, Tortes M, Rowning BA, et al. Establishment of the dorso-vertral axis in Xenopus embryosis presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway. J Cell Biol,1997,136(5):1123-1136.
59. Hasson P, Paroush Z. Crosstalk between the EGFR and other signaling pathways at te level of the global transcriptional corepressor Groucho/TLE. Br J Cancer,2006,94(6):771-775.
60. Dornseifer P, Takke C, Campos-Ortega JA. Over expression of a zebrafish homologue of the Drosophila neurogenic gene Delta perturbs differentiation of primary neurons and somite development. Mech Dev,1997,63(2):159-171.
61. Jen WC, Gawantka V, Pollet N. Periodic repression of Notch path way genes governs the segmentation of XenoPus embryos. Genes Dev,1999,13(11):1486-1499.
62. Takke C, Campos-Ortega JA. her1, a zebrafish pair-rule like gene, acts downstream of notch signaling to control somite development. Development, 1999,126(13):3005-3014.
63. Bhanot P, Fish M, Jemison JA, etal. Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development. Development,1999,126(18):4175-4186.
64. Huelsken S, Vogel R, Brinkmann V, et al. Requirement for beta-catenin in anterior-posterior axis formation in mice. Cell Biol,2000; 148(3):567-578.
65. Agirre X, Jimenez-Velasco A, San Jose-Eneriz E, et al. Down-regulation of hsa-miR-lOa in chronic myeloid leukemia CD34+ cells increases USF2-mediated cell growth. Mol Cancer Res,2008,6(12):1830-1840.
66. Blanco G, Goulton GR, Biggin A, et al. The kyphoscoliosis(ky) mouse is deficient in hypertrophic responses and is caused by a mutation in a novel muscle-specific protein. Hum Mol Genet,2001,10(1):9-16.
67. 原所茂。汉族人群基因多态性与先天性脊柱侧凸遗传易感性的关联性的研究。清华大学医学部,北京协和医学院,北京协和医院博士论文,2009。
68. Millonig JH. Millen KJ. Hatten ME. The mouse Dreher gene Lmxla control formation of the roof plate in the vertebrate CNS. Nature,2000,403(6771):764-769.
69. 费琦,吴志宏,王以朋等。LMX1A基因多态性与中国汉族人群先天性脊柱侧凸遗传易感性的关联。中国组织工程研究与临床康复,2011,12(38)：6632-6638。
70. Simpson MA, Irving MD, Asilmaz E, et al. Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss. Nature Genetics,2011,43(4):303-305.
71. Chapman DL, Papaioannou VE. Three neural tubes in mouse embryos with mutations in the T-box geneTbx6. Nature,1998,391(6668):695-697.
72. Hamblet NS, Lijam N, Ruiz-Lozano P, et al. Dishevelled 2 is essential for cardiac outflow tract development, somite segmentation and neural tube closure. Development,2002,129 (24):5827-5838.
73. Wang B, Li W, Guo K, et al. miR-181b promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients. Biochem Biophys Res Commun,2012,421(1):4-8.
74. Chen L, Yang Q, Kong WQ, et al. MicroRNA-181b targets cAMP responsive element binding protein 1 in gastric adenocarcinomas.IUBMB Life,2012, 64(7):628-635.
75. Visone R, Veronese A, Balatti V, et al. MiR-181b:new perspective to evaluate disease progression in chronic lymphocytic leukemia. Oncotarget,2012, 3(2):195-202.
76. Rio P, Agirre X, Garate L, et al. Down-regulated expression of hsa-miR-181c in Fanconi anemia patients:implications in TNFa regulation and proliferation of hematopoietic progenitor cells..Blood,2012,119(13):3042-3049.
77. Geekiyanage H, Chan C. MicroRNA-137/181 c regulates serine palmitoyltransferase and in turn amyloid β, novel targets in sporadic Alzheimer's disease.J Neurosci,2011,31(41):14820-14830.
78. Sand M, Skrygan M, Sand D, et al. Expression of microRNAs in basal cell carcinoma. Br J Dermatol,2012,167(4):847-855.
79. Kang JG, Majerciak V, Uldrick TS, et al. Kaposi's sarcoma-associated herpesviral IL-6 and human IL-6 open reading frames contain miRNA binding sites and are subject to cellular miRNA regulation. J Pathol,2011,225(3):378-389.
80. Eskildsen T, Taipaleenmaki H, Stenvang J, et al. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo. ProcNatl Acad Sci USA,2011,108(15):6139-6144.
81. Sureban SM, May R, Mondalek FG, et al. Nanoparticle-based delivery of siDCAMKL-1 increases microRNA-144 and inhibits colorectal cancer tumor growth via a Notch-1 dependent mechanism. J Nanobiotechnology,2011,9:40.
82. Wang H, Meng K, Chen Wj, et al. Serum miR-574-5p:a prognostic predictor of sepsis patients. Shock,2012,37(3):263-267.
83. Foss KM, Sima C, Ugolini D, et al. miR-1254 and miR-574-5p:serum-based microRNA biomarkers for early-stage non-small cell lung cancer. J Thorac Oncol, 2011,6(3):482-488.
84. Chen W, Cai F, Zhang B, et, al. The level of circulating miRNA-10b and miRNA-373 in detecting lymph node metastasis of breast cancer:potential biomarkers. Tumour Biol,2013,34(1):455-62.
85. Gabriely G, Yi M, Narayan RS, et al. Human glioma growth is controlled by microRNA-lOb. Cancer Res,2011,71(10):3563-72.
86. Nakata K, Ohuchida K, Mizumoto K, et al. MicroRNA-10b is overexpressed in pancreatic cancer, promotes its invasiveness, and correlates with a poor prognosis. Surgery,2011,150(5):916-22.
87. Fendler A, Jung M, Stephan C, et al. miRNAs can predict prostate cancer biochemical relapse and are involved in tumor progression. Int J Oncol,2011, 39(5):1183-92.
1. Kobayashi T, Lu J, Cobb BS, et al. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc Natl Acad Sci USA,2008,105(6):1949-1954.
2. Watanabe T, Sato T, Amano T, et al. Dnm30s, a non-coding RNA, is required for normal growth and skeletal development in mice. Dev Dyn,2008,237(12):3738-3748.
3. Lee RC, Feinbaum RL, Ambros V. The C. Elegans heterochmnic gene lin-4 antodes small RNAs withantisense complementarity to lin-14. Cell,1993,75(5):843-854.
4. Lee Y, Ahn C, Han J, et al. The nuclear RNaseⅢ Drosha initiates miRNA processing. Nature,2003,425(6956):415-419.
5. Han JJ, Lee Y, YeomKH, et al. Molecular basis for the recognition of primary micoRNAs by the Dmsha-DGCR8 complex. Cell,2006,125(5):887-901.
6. Zeng Y, Cullen BR. Sequence requirements for miRNA processing and function in human cells. RNA,2003,9(1):112-123.
7. Du T, Zamore PD. MicroPrimer:the biogenesis and function of miRNA. Development,2005,132(21):4645-4652.
8. Nakajima N, Takahashi T, Kitamura R, et al. miRNA-1 facilitates skeletal myogenic differentiation without affecting osteoblastic and adipogenic differentiation. Biochem Biophys Res Commun,2006,350(4):1006-1012.
9. Sugatan T, Hruska KA. Impaired miRNA pathways diminish osteochist diflerentiation and function. J Biol Chem,2009,284(7):4667-4678.
10. Mizuno Y, TOkuzawa Y, Ninomiya Y, et al. miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b. FEBS Lett,2009,583(13):2263-2268.
11. Sun S. Bone disease drug discovery:examining the interactions between osteoblast and Oeteoclast. Expert Opin Ther Targets,2008,12(2):239-251.
12. Oskowitz AZ, Lu J, Penfornis P, et al. Human multipotent stromal cells from bone marrow and miRNA:regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci U S A,2008,105:18372-18377.
13. Harfe B, McManus M, Mansfield J, et al. The RNase III enzyme Dicer is required for morpho-genesis but not patterning of the vertebrate limb. Proceedings of the National Academy of Sciences of the United States of America,2005,102:10898.
14. Mizuno Y, Vagi K, Tokuzawa Y, etal. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun,2008, 368(2):267-272.
15. Zhang J, Tu Q, Bonewald LF, et al. Effects of miR-335-5p in modulating osteogenic differentiation by specifically down-regulating Wnt antagonist DKK1. J Bone Miner Res,2011.
16. Kapinas K, Kessler C, Ricks T, et al. miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop. J Biol Chem,2010,285:25221-25231.
17. Lian JB, Stein GS, Stein JL, et al. Networks and hubs for the transcriptional control of osteoblastogenesis. Rev Endocr Metab Disord,2006,7(1-2):1-16.
18. Li Z, Hassan MQ, Volinia S, et al. A miRNA signature for a BMP2-induced osteoblastlineage commitment program. PNAS,2008,105(37):13906-13911.
19. Huang J, Zhao L, Xing L, et al. miRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells,2010,28:357.
20. ICCBH5, Fifth International Conference on Childrens Bone Health. Bone,2009.
21. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are miRNA targets. Cell,2005,120(1):15-20.
22. Luzi E, Marini F, Sala SC, etal. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 trans factor. J Bone Miner Res,2008,23(2):287-295.
23. Kim YJ, Bae SW, Yu SS, et al. miR-196a regulatesproliferation and osteogenic differentiation in mesenchymal stem cells derived from human adipose tissue. J Bone Miner Res,2009,24(5):816-825.
24. Haynes DR, Crotti TN, Zreiqat H, et al. Regulation of osteoclast activity in peri-implant issues. Biomaterials,2004,25(20):4877-4885.
25. Miyaki S, Sato T, Inoue A, et al. miRNA-140 plays dual roles in both cartilage development and homeostasis. Gene Dev,2010,24(11):1173-1185.
26. Kim D, SongJ, Jin EJ. MicmRNA-221 regulates chondrogenic differentiation through promoting proteosomal degradation of slug by targeting Mdm2. J Bid Chem2010,285(35):26900-26907.
27. Han JW, Yang T, Gao J, etal. Specific mieroRNA expression during chondrogenesis of human mesenchymal stem cells. Int J Mol Med,2010,25(3):377-284.
28. Yang B, Gun H, Zhang Y, et al. The miRNA expression profiles of mouse mesen-chymal stem cell during chondrogenic differentiation. BMB Rep,2011,44(1):28-33.
29. Araldi E, Sehipani E. miRNA-140 and the silencing of osteotarthritis. Genes Dev, 2010,24(11):1075-1080.
30. Tardif C, Hum D, Pelletier JP, et al. Regulation of the IGFBP-5 and MMP-13 genes by the miRNAs miR-140 and miR-27a in human osteoarthritis chondrocytes.BMC Musculoskelet Disord,2009,10:148.
31. Jones SW, Watkins G, Le Good N, et al. The identification of differentially expressed miRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13. Osteoarthritis Cartilage,2009,17(4):464-472.
32. SumiyoshiK, Kubota S, Ohgawara T, et al. Identification of miR-1 as a micro RNA that supports late-stage differentiation of growth cartilage cells. Bio ehem Biophys Res Commun,2010,402(2):286-290.
33. Kongeharoensombat W, Nakasa T, Iishikawa M, et al. The effect of miRNA-21 on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel. Fame Surg Sports Traumatol Arthrosc,2010,18(12):1679-1684.
34. Abouheif MM, Nakasa T, Shibuya H, et al. Silencing miRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro. Rheumatology(Oxford), 2010,49(11):2054-2060.