MiR-590-3p在人间充质干细胞成骨向分化过程中的作用及机制研究

发布时间：2018-03-26 08:22

本文选题：miR-590-3p　切入点：间充质干细胞　出处：《南方医科大学》2017年博士论文

【摘要】：本课题通过基于基因表达谱芯片的生物信息学数据挖掘间充质干细胞成骨向分化过程中表达上调的潜在研究位点,并以qRT-PCR验证,选取microRNA-590-3p作研究,结合信号通路网络预测其作用靶点。利用慢病毒转染构建稳定miR-590-3p过表达细胞株,与空载体人间充质干细胞系及lipofectamine2000瞬转miR-590-3p抑制细胞系相对比,证明miR-590-3p对间充质干细胞增殖、细胞周期调节、成骨向分化具有促进作用。并通过双荧光素酶实验验证miR-590-3p促进人间充质干细胞成骨向分化机制是通过与APC mRNA3'UTR特异序列结合进而调节Wnt/β-catenin信号通路。这对牙槽骨再生及颌骨缺损重建的骨组织工程学有一定的指导作用。第一章间充质干细胞分化过程中microRNA表达谱的生物信息学分析目的:通过生物信息学手段挖掘潜在研究靶点,并进行初步验证方法:利用生物信息学分析筛选潜在miRNAs,以qRT-PCR验证诱导间充质干细胞成骨向分化对其表达的影响,从中筛选待研究因子,进一步结合GO分析以及信号通路网络分析探索潜在靶点。结果:筛选得到miR18,miR22,miR-590-3p等miRNA与成骨向分化相关。miR-590-3p随成骨向分化诱导,在人间充质干细胞中表达明显上升。靶基因预测为APC,FZD 1,Wnt5a等Wnt通路相关蛋白。结论:miR-590-3p与间充质干细胞成骨向分化有关,其调控通路可能为Wnt信号通路。第二章 miR-590-3p过表达/抑制人间充质干细胞株的建立及生物学效应研究目的:构建并探讨miR-590-3p过表达/抑制间充质干细胞系对间充质干细胞增殖和细胞周期调节的影响。方法:构建miR-590-3p过表达细胞株、表达抑制细胞系和对照细胞系,利用MTT实验评估三组细胞增殖能力,并通过检测CyclinD1以及pRb磷酸化水平评估三组细胞对细胞周期调节的影响。结果:miR-590-3p过表达细胞株MTT染色吸光度高,CyclinD1以及pRb磷酸化水平表达增强,miR-590-3p表达抑制细胞系相反。结论:miR-590-3p对间充质干细胞增殖、细胞周期调节具有促进作用。第三章 MicroRNA-590-3p影响人间充质干细胞成骨向分化的相关机制研究目的:评价miR-590-3p对间充质干细胞成骨向分化的影响,并验证其靶基因。方法:qRT-PCR比较成骨相关因子在不同细胞系间表达水平的高低。利用茜素红染色对比不同细胞系矿化沉积活动的强弱。通过TOP/FOP实验评价miR-590-3p表达水平与wnt/β-catenin信号通路活性之间关联。利用双荧光素酶实验验证miR-590-3p与APC的mRNA序列特异性结合。结果:miR-590-3p表达水平与成骨相关因子表达水平及矿化沉积呈正相关。TOP/FOP实验提示wnt/β-catenin信号通路水平在miR-509-3p过表达细胞株出现上调,双荧光素酶实验提示APC的3'UTR是miR-590-3p特异性绑定位点。结论:miRNA-590-3p对间充质干细胞成骨向分化有促进作用,这一作用与Wnt/β-catenin信号通路相关,而miR-590-3p对于APC的转录后调节是其中重要的机制之一。
[Abstract]:Based on bioinformatics data based on gene expression microarray, the potential sites for up-regulation of expression in osteogenic differentiation of mesenchymal stem cells (MSCs) were explored in this study. MicroRNA-590-3p was selected to study by qRT-PCR validation. The stable miR-590-3p overexpression cell line was constructed by lentivirus transfection, and compared with empty vector human mesenchymal stem cell line and lipofectamine2000 transient miR-590-3p cell line, the proliferation of mesenchymal stem cells was proved by miR-590-3p. Cell cycle regulation, The mechanism of miR-590-3p promoting osteogenic differentiation of human mesenchymal stem cells is to regulate the Wnt/ 尾 -catenin signaling pathway by binding to the specific sequence of APC mRNA3'UTR. Bone tissue engineering for reconstruction of maxillary defects. Chapter 1 bioinformatics analysis of microRNA expression profile during the differentiation of mesenchymal stem cells objective: to explore potential targets by bioinformatics. The primary verification methods were as follows: bioinformatics analysis was used to screen potential miRNAs. qRT-PCR was used to verify the effect of inducing osteogenic differentiation of mesenchymal stem cells on the expression of MIRNAs.The factors to be studied were screened. Results: miRNA such as miR18 miR22miR-590-3p was found to be related to osteogenic differentiation. MiR-590-3p was induced by osteogenesis. The target genes were predicted to be Wnt transduction related proteins such as APC-FZD1Wnt5a. Conclusion the expression of Wnt pathway is related to the osteogenic differentiation of mesenchymal stem cells. The regulatory pathway may be Wnt signaling pathway. Chapter 2 Establishment and Biological effects of miR-590-3p overexpression / inhibition of Human Mesenchymal Stem Cell Lines objective: to construct and study miR-590-3p overexpression / inhibition of Mesenchymal Stem Cell Lines against Mesenchymal Stem cells. Effects of stem cell proliferation and cell cycle regulation. Methods: miR-590-3p overexpression cell lines were constructed. Expression inhibition cell line and control cell line were used to evaluate the proliferation ability of three groups of cells by MTT assay. The effects of CyclinD1 and pRb phosphorylation on cell cycle regulation were evaluated by detecting the phosphorylation level of CyclinD1 and pRb. Results the cell line MTT staining and pRb phosphorylation enhanced the expression of miR-590-3p to inhibit cell cycle regulation. Conclusion the proliferation of mesenchymal stem cells was induced by 10% miR-590-3p. Effects of MicroRNA-590-3p on osteogenic differentiation of human mesenchymal stem cells objective: to evaluate the effect of miR-590-3p on osteogenic differentiation of human mesenchymal stem cells. Methods the expression level of osteoblast-associated factors in different cell lines was compared by using the proportion of qRT-PCR. The mineralized deposition activity of different cell lines was determined by alizarin red staining. The expression level of miR-590-3p was evaluated by TOP/FOP assay. The activity of wnt/ 尾 -catenin signaling pathway was correlated. The specific binding of miR-590-3p to APC mRNA sequence was verified by double luciferase experiment. Results the expression level of miR-590-3p / MIR-590-3p was positively correlated with the expression level of osteoblast-related factors and mineralized deposition. Topp / FOP experiment showed that wnt/ 尾 -catenin was positively related to the expression of APC. Signal pathway level was up-regulated in miR-509-3p overexpression cell lines. Double luciferase assay suggested that 3'UTR of APC is a specific binding site for miR-590-3p. Conclusion: miRNA-590-3p can promote the osteogenic differentiation of mesenchymal stem cells, which is related to the Wnt/ 尾 -catenin signaling pathway. The posttranscriptional regulation of APC by miR-590-3p is one of the important mechanisms.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68

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