miRNA在萎缩性骨不连中的调控作用及其分子生物学机制

发布时间：2018-03-13 21:51

  本文选题：萎缩性骨不连　切入点：芯片　出处：《中国人民解放军医学院》2011年博士论文　论文类型：学位论文

【摘要】：目的 miRNA (micro ribonucleic acid,微小核糖核酸)是一种单链非编码小分子RNA(ribonucleic acid,核糖核酸),在基因表达调控方面具有广泛和普遍的作用,但目前对其在骨折愈合与不愈合中的调控作用尚知之甚少。本研究拟首先筛选萎缩性骨不连修复组织中异常表达的miRNA种类,并通过生物信息学方法预测表达上调miRNA的成骨功能相关靶基因,再从细胞水平验证萎缩性骨不连组织中表达上调miRNA对预测靶基因的调控作用,从而阐明miRNA在萎缩性骨不连发病过程中的调控作用及其分子生物学机制,为骨不连的基础和临床研究提供理论和实践依据。 方法 (1)以临床患者萎缩性骨不连修复组织(A组,3例)与正常骨折愈合后内固定钢板取出时钢板周围骨痂组织(B组,3例)为研究对象,采用Exiqon miRCURYTM LNA microRNA芯片(11.0版)对各组织中提取的RNA进行miRNA筛选分析。在筛选出萎缩性骨不连组织中异常表达的miRNA后,采用荧光qRT-PCR(quantitative real-time polymerase chain reaction,实时定量聚合酶链反应)对芯片结果中A组表达上调的miRNA在两种组织的表达水平进行验证,并采用浏览计算机预测数据库的生物信息学方法预测其可能的靶基因,探寻miRNA可能参与的骨折愈合和萎缩性骨不连相关病理生理过程。 (2)上调miRNA与预测靶基因关系的验证：取人骨髓全血,离心分离hBMSCs (human bone mesenchymal stem cells,人骨髓基质干细胞),传代培养。将人工合成的四种表达上调miRNA的双链小RNA转染入第四代hBMSCs,48小时后提取细胞总RNA,采用qRT-PCR、WB (Western Blotting,蛋白印迹)、双荧光素酶报告基因检测等多种方法,验证miRNA对预测靶基因的作用,确认miRNA在萎缩性骨不连中的具体调控作用和分子生物学机制。 (3) miRNA和相应靶基因在诱导hBMSCs成骨分化过程中表达水平及功能的相关研究：采用成骨诱导培养液诱导hBMSCs成骨分化的方法,观察在诱导hBMSCs成骨分化过程中不同时间点(0,12h,1d,2d,4d,7d,14d)相应miRNA、靶基因mRNA(messenger ribonucleic acid,信使核糖核酸)与蛋白表达水平的变化趋势,进一步验证这些上调miRNA在诱导成骨分化中的调控作用,并探讨成骨诱导培养液的诱导成分在此过程中促进成骨分化的作用。 结果 (1)萎缩性骨不连修复组织相对钢板周围骨痂组织有9种miRNA发生1.5倍以上表达上调(hsa-miR-149*, hsa-miR-221, hsa-miR-628-3p, hsa-miR-654-5p,以及5种hsa-miRPlus),9种miRNA发生1.5倍以上表达下调(hsa-let-7b*, hsa-miR-220b, hsa-miR-513a-3p, hsa-miR-551a,hsa-miR-576-5p,hsa-miR-1236,kshv-miR-K12-6-5p,以及2种hsa-miRPlus).qRT-PCR结果提示,数据库中收录并在A组表达上调的四种miRNA在组织水平的表达变化趋势与芯片结果一致。生物信息学分析发现,四种表达上调miRNA的预测靶基因包括多种成骨功能相关基因。 (2)分离培养的hBMSCs具有典型的干细胞形态特征,贴壁时间短,细胞融合快,体外扩增容易,可以向成骨细胞诱导,适合作为种子细胞用于后续的实验研究。在成功将上调miRNA的合成双链小RNA转染进第四代hBMSCs48h后,提取细胞总RNA,qRT-PCR检测表明,三种预测靶基因(hsa-miR-149*的预测靶基因ALPL、 hsa-miR-221的预测靶基因PDGFA和hsa-miR-654-5p的预测靶基因BMP2)的mRNA表达量受到明显抑制,其余预测靶基因的mRNA无明显下降；WB则表明上述三种预测靶基因的蛋白表达量均受到明显抑制。双荧光素酶报告基因检测则证明三种预测靶基因的预测靶位点分别直接受到hsa-miR-149*、hsa-miR-221和hsa-miR-654-5p的靶向调控,其中ALPL的两个预测靶位点只有第二个受到hsa-miR-149*的直接调控。 (3)诱导hBMSCs成骨分化过程中,得到确认的成骨性靶基因ALPL.PDGFA和BMP2的mRNA和蛋白表达量在多数时间点有明显增加,尤其以诱导分化后2～7天变化最为显著(ALPL蛋白为1～7天),第14天时又有所下降。不同时间点的miRNA.靶基因mRNA和蛋白水平存在表达差异,其中hsa-miR-149*和hsa-miR-654-5p的变化趋势与各自靶基因ALPL和BMP2的mRNA及蛋白表达水平总体上呈负相关,而hsa-miR-221与PDGFA的变化趋势无明显负相关关系,两者的关系有待进一步研究。 结论 (1)萎缩性骨不连修复组织中存在miRNA的异常表达；四种在该组织中上调的miRNA(hsa-miR-149*、hsa-miR-221、hsa-miR-628-3p和hsa-miR-654-5p)有可能通过抑制多种成骨功能相关靶基因的表达,在调控骨折向萎缩性骨不连发生发展中发挥重要作用。 (2)细胞水平验证表明：四种在组织水平上调的miRNA中,hsa-miR-149*与其预测的靶基因ALPL、hsa-miR-221与其预测的靶基因PDGFA、hsa-miR-654-5p与其预测的靶基因BMP2之间存在直接调控作用,异常上调的miRNA通过抑制成骨功能相关靶基因ALPL、PDGFA和BMP2的表达而限制其生物学功能,引起了萎缩性骨不连的发生发展。 (3)多种成骨功能相关靶基因的mRNA和蛋白表达水平在诱导hBMSCs成骨分化过程中大多数时间点发生了表达上调,hsa-miR-149*和hsa-miR-654-5p与其相应靶基因ALPL和BMP2的mRNA及蛋白表达变化趋势呈负相关,说明这两种miRNA在诱导成骨分化过程中与其靶基因mRNA和蛋白表达水平的调控密切相关。 简述之,本研究从萎缩性骨不连修复组织中找到具有关键调控作用的miRNA,发现有三种表达异常上调的miRNA通过抑制成骨功能相关靶基因引起了萎缩性骨不连的发生发展,其中两种miRNA的表达水平与成骨功能相关靶基因的mRNA和蛋白表达水平密切相关。本研究可能将为萎缩性骨不连的诊断和治疗提供新的理念。
[Abstract]:objective
MiRNA (micro ribonucleic acid, microRNA) is a single chain non encoding small molecule RNA (ribonucleic acid, ribonucleic acid), has extensive role in gene expression regulation, but its role in the regulation of fracture healing and nonunion is still poorly understood. Abnormal expression of this study firstly screening of atrophic nonunion tissue in miRNA type, and through the method of bioinformatics to predict bone functional target genes into upregulating the expression of miRNA, up-regulated the expression of miRNA in prediction of regulation of target genes from the cell level verification of atrophic bone nonunion tissue, so as to clarify the miRNA in atrophic bone nonunion control the role and molecular mechanism of the pathogenesis, provide theoretical and practical basis for nonunion and clinical research.
Method
(1) in patients with atrophic nonunion tissue (3 cases of group A) and remove the plate normal after fracture healing in the bone callus around the plate (group B, 3 cases) as the research object, using the Exiqon miRCURYTM LNA microRNA chip (11 Edition) miRNA screening analysis of each tissue extract the abnormal expression of RNA. In the screening of atrophic nonunion tissue in miRNA, qRT-PCR (quantitative real-time polymerase by fluorescence chain reaction and real-time quantitative polymerase chain reaction) to validate the expression level of A group was up-regulated in miRNA chip results in two kinds of tissues, and the biological information database browsing computer prediction the prediction of the possible target genes to explore the methodology, miRNA may be involved in the healing of fracture and nonunion related pathophysiological processes.
(2) to verify the relationship between the upregulation of miRNA and prediction of target genes: the human bone marrow blood, centrifugal separation (human bone mesenchymal hBMSCs stem cells, human bone marrow stromal stem cells), subculture. The artificial synthesis of four kinds of small double stranded RNA by upregulating the expression of miRNA in the fourth generation hBMSCs, 48 hours after the extraction of total cell RNA, by qRT-PCR WB (Western, Blotting, Western blot), dual luciferase reporter gene assay and other methods, to verify the miRNA target prediction function, confirm the miRNA in atrophic bonenonunion regulation and molecular mechanism.
(3) study of the expression level and function of miRNA and the corresponding target genes in hBMSCs induced osteogenic differentiation in the process of using osteogenic medium to induce hBMSCs differentiation into bone, observed at different time points in the process of osteogenic differentiation induced by hBMSCs (0,12h, 1D, 2D, 4D, 7d, 14d) corresponding miRNA, mRNA (messenger ribonucleic acid target gene, messenger ribonucleic acid) and protein expression level change trend, further validation of these induced upregulation of miRNA in the regulation of bone differentiation, and to explore the osteogenic medium to induce osteogenic differentiation of components in the process fluid.
Result
(1) atrophic nonunion relative plate callus tissues around 9 miRNA occurred more than 1.5 times up-regulated (hsa-miR-149*, hsa-miR-221, hsa-miR-628-3p, hsa-miR-654-5p, and 5 hsa-miRPlus), 9 miRNA more than 1.5 times the expression (hsa-let-7b*, hsa-miR-220b, hsa-miR-513a-3p, hsa-miR-551a, hsa-miR-576-5p, hsa-miR-1236, kshv-miR-K12-6-5p 2 hsa-miRPlus.QRT-PCR), and the results suggest that the database and expression of four miRNA up-regulated in tissue expression level change trend consistent with microarray results in the A group. The bioinformatics analysis, four up-regulated miRNA expression predicted target genes including many osteogenic genes.
(2) hBMSCs isolated from the stem cells with typical morphological characteristics, adherent cell fusion time is short, fast, easy amplification, can be induced into osteoblasts, suitable as seed cells for subsequent experimental study. The success of the upregulation of miRNA synthesis of small double stranded RNA transfected into the fourth generation of hBMSCs48h. Extraction of total RNA cells, qRT-PCR assay showed that three predicted target genes (predicted target gene of BMP2 PDGFA and hsa-miR-654-5p hsa-miR-221 predicted target gene prediction of target gene ALPL, hsa-miR-149*) mRNA expression was inhibited, the predicted target gene of mRNA decreased significantly; WB showed that the expression volume were significantly inhibited three prediction of target gene protein. Dual luciferase reporter gene assay demonstrated that the predicted target sites three predicted target genes were directly affected by hsa-miR-149*, hsa-miR-221 and hsa-miR-654-5p target Only second of the two target loci of ALPL were directly regulated by hsa-miR-149*.
(3) hBMSCs induced osteogenic differentiation process, confirmed the expression of mRNA and protein of bone target gene ALPL.PDGFA and BMP2 in most of the time points increased, especially in the differentiation of 2~7 days after the most significant changes (ALPL protein for 1~7 days), fourteenth days have declined. At different time points the target genes of miRNA. mRNA and protein levels in the differential expression of mRNA and protein, the changing trend of hsa-miR-149* and hsa-miR-654-5p and their target genes ALPL and BMP2 expression level was negatively correlated, and the changing trend of hsa-miR-221 and PDGFA no significant negative correlation, the relationship between the two needs further study.
conclusion
(1) abnormal expression of miRNA exists in the atrophic nonunion tissue; four up-regulated in the tissues of miRNA (hsa-miR-149*, hsa-miR-221, hsa-miR-628-3p and hsa-miR-654-5p) can inhibit a variety of expression of functional genes and in the regulation of fracture to atrophic bone nonunion plays an important role in the development.
(2) show that the four kinds of cells in tissue level up-regulated miRNA, ALPL hsa-miR-149* and its target gene prediction, target gene PDGFA hsa-miR-221 and forecast, there is a direct regulatory role between BMP2 hsa-miR-654-5p and its target gene prediction, abnormal upregulation of miRNA by inhibiting osteoblast function related target gene expression of PDGFA and ALPL. BMP2 the limit of its biological functions, causing atrophic nonunion.
(3) a variety of mRNA and protein in bone functional genes expression in hBMSCs induced osteogenic differentiation in most of the time point had expression of mRNA and protein expression of hsa-miR-149* and hsa-miR-654-5p and its target genes ALPL and BMP2 showed a negative correlation trend, indicating that the two miRNA in the induction of expression regulation in the process of the differentiation of bone and its target gene mRNA and protein are closely related.
Briefly, this research has found the key regulatory role of miRNA from atrophic nonunion tissue, found three abnormal expression upregulation of miRNA by inhibiting osteogenic functions of target genes causing atrophic nonunion development, closely related to the expression level of mRNA protein and the expression level of two miRNA with the osteogenic function of target genes. This study may provide new ideas for the atrophic nonunion of the diagnosis and treatment.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R683;R3411

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