microRNA-214通过靶向作用QKI调控干细胞向血管平滑肌细胞分化研究

发布时间：2018-07-09 13:56

本文选题：microRNA-214 + QKI　；参考：《浙江大学》2016年博士论文

【摘要】：研究背景及目的:以动脉粥样硬化为基本组织病理改变的血管损伤性病变,其基本病理进程为:血管损伤引起血管修复和重塑,继而血管内膜增厚,导致管腔狭窄。在这一病变过程中,血管平滑肌细胞通过表型转换、增殖、迁移以及干细胞分化两种方式参与其中。既往研究已经发现microRNA-214(miR-214)调控成熟血管平滑肌细胞功能和表型转换,然而对于其在干细胞向血管平滑肌细胞分化中的作用尚不是十分清楚。本研究希望揭示miR-214在胚胎干细胞向血管平滑肌细胞分化中的作用,鉴定miR-214的靶基因,阐明其作用机制。研究方法:本研究主要利用课题组前面建立的、稳定的、体外的小鼠胚胎干细胞向平滑肌细胞分化模型,观察miR-214表达变化,并过表达或抑制miR-214表达,研究miR-214对胚胎干细胞向血管平滑肌细胞分化的作用。其次,利用生物信息学技术分析及萤光素酶基因报告系统等多种方法鉴定及确认miR-214的功能性靶基因。再者,利用免疫共沉淀(IP)、双荧光素酶基因报告系统等多种分子实验技术,证明QKI对胚胎干细胞向血管平滑肌细胞分化的作用及作用途径。最后,利用体外转染的细胞打入动物体内模拟在体环境的小鼠胚胎干细胞分化模型,研究miR-214对胚胎干细胞向平滑肌细胞分化的影响。结果:利用课题组以前建立的模型,小鼠胚胎干细胞在CollagenIV的刺激下能够稳定的分化成血管平滑肌细胞,并且随着时间的推移,平滑肌标志基因(SM-22α、SM-MHC、h1-Calponin)等表达增多。在此过程中,miR-214表达显著上升趋势。在功能研究实验中,过表达miR-214能够显著促进血管平滑肌细胞标志基因在mRNA和蛋白水平的表达,而抑制miR-214表达则降低平滑肌标志基因的表达。多个miRNA的生物信息学分析数据库分析软件预测QKI是miR-214最有可能的的靶基因之一。在分化中的小鼠胚胎干细胞上,过表达miR-214能够在mRNA和蛋白水平下调QKI表达,而抑制miR-214引起QKI表达水平升高。荧光素酶基因报告实验表明,过表达miR-214能够显著降低含有QKI3'UTR的荧光素酶活性,抑制miR-214能够相应引起QKI 3'UTR的荧光素酶活性在一定程度上升高。而对QKI 3'UTR中miR-214潜在结合位点进行突变后,miR-214对荧光素酶活性的抑制作用消失。miR-214过表达模拟物和QKI过表达质粒共同转染细胞,发现高表达QKI可消除过表达的miR-214对小鼠胚胎干细胞向血管平滑肌细胞分化的促进作用。同时,分别过表达和抑制QKI,发现过表达QKI能够降低血管平滑肌细胞标志基因的mRNA和蛋白表达,抑制QKI表达则具有相反的作用。进而,IP显示,QKI能够直接结合在平滑肌细胞分化关键转录因子的启动子序列。最后,在成功建立的小鼠胚胎干细胞在体分化模型中,发现miR-214能够促进平滑肌分化标志基因的表达,在一定程度上从体内实验层面验证了 miR-214促进鼠胚胎干细胞向血管平滑肌细胞分化。结论:本研究成功揭示了 miR-214在胚胎干细胞向血管平滑肌细胞分化中的新功能。miR-214促进胚胎干细胞向血管平滑肌细胞分化。QKI是miR-214的功能性靶基因,能够直接结合在平滑肌分化关键转录因子的启动子区域,从而抑制分化。miR-214直接结合QKI mRNA的3'UTR区域,下调QKI表达,并通过调控QKI影响干细胞向血管平滑肌细胞分化。miR-214可能是防治平滑肌细胞相关血管损伤性疾病的新靶点。
[Abstract]:Research background and purpose: the basic pathological process of vascular injury with atherosclerosis as the basic histopathological changes is that vascular injury causes vascular repair and remodeling, and then the intima thickening of the vessels leads to the stenosis of the lumen. In this process, the vascular smooth muscle cells transfer, proliferate, migrate and stem cells through the phenotype. MicroRNA-214 (miR-214) has been found to regulate the functional and phenotypic transformation of mature vascular smooth muscle cells. However, the role of microRNA-214 in the differentiation of stem cells to vascular smooth muscle cells is not very clear. This study hopes to reveal the differentiation of miR-214 from embryonic stem cells to vascular smooth muscle cells. The purpose of this study is to identify the target gene of miR-214 and to elucidate its mechanism of action. Research methods: This study mainly used the stable, stable, in vitro mouse embryonic stem cells to differentiate into the smooth muscle cells, observe the changes in the expression of miR-214, and overexpress or inhibit the expression of miR-214, and study the smooth vascular smooth of the embryonic stem cells to the blood vessels. Secondly, the functional target genes of miR-214 were identified and confirmed by bioinformatics analysis and fluoropenzyme gene reporting system. Furthermore, a variety of molecular experimental techniques, such as immunoprecipitation (IP) and double luciferase gene report system, were used to prove that QKI was fine to the vascular smooth muscle of the embryonic stem cells. Finally, the effect of miR-214 on the differentiation of embryonic stem cells to smooth muscle cells in vivo was studied by using in vitro transfected cells to simulate the mouse embryonic stem cell differentiation model in vivo. Results: the mouse embryonic stem cells could be stimulated by CollagenIV. It is stable enough to differentiate into vascular smooth muscle cells, and the expression of the smooth muscle marker gene (SM-22, SM-MHC, h1-Calponin) increases with time. In this process, the expression of miR-214 is significantly increased. In the functional study, overexpression of miR-214 can significantly promote the level of vascular smooth muscle cell marker genes in mRNA and protein levels. The expression of miR-214 decreased the expression of the smooth muscle marker gene. The multi miRNA bioinformatics analysis database analysis software predicted that QKI was one of the most likely target genes of miR-214. In the differentiated mouse embryonic stem cells, the overexpression of miR-214 could reduce the QKI expression at the level of mRNA and protein, and inhibit miR-214. The expression level of QKI increased. The luciferase gene report showed that overexpression of miR-214 could significantly reduce the activity of luciferase containing QKI3'UTR, and the inhibition of miR-214 could increase the luciferase activity of QKI 3'UTR to a certain extent. While miR-214 latent at the binding site in QKI 3'UTR, miR-214 to fluorescein The inhibition of enzyme activity disappears.MiR-214 overexpressed and QKI overexpressed plasmids co transfected cells. It is found that high expression of QKI can eliminate the effect of overexpressed miR-214 on the differentiation of mouse embryonic stem cells to vascular smooth muscle cells. At the same time, the expression and inhibition of QKI, respectively, can be found to reduce the expression of QKI to reduce vascular smooth muscle cells. The mRNA and protein expression of the marker gene and the inhibition of the expression of QKI have the opposite effect. Then, IP shows that QKI can directly bind the promoter sequence of the key transcription factor of the smooth muscle cells. Finally, in the successfully established mouse embryonic stem cells in the body differentiation model, the expression of miR-214 can promote the expression of the marker gene of the smooth muscle differentiation. To a certain extent, the miR-214 promotes the differentiation of mouse embryonic stem cells to vascular smooth muscle cells. Conclusion: This study successfully revealed that the new function of miR-214 in the differentiation of embryonic stem cells to vascular smooth muscle cells (.MiR-214) promotes the differentiation of.QKI from embryonic stem cells to smooth muscle cells of the vascular smooth muscle cells, which is the function of miR-214. The target gene, which can directly bind to the promoter region of the key transcription factor of smooth muscle differentiation, inhibits the direct binding of the differentiated.MiR-214 to the 3'UTR region of the QKI mRNA, and down regulates the expression of QKI, and may be a new disease against vascular smooth muscle cells associated with vascular smooth muscle cell differentiation by regulating QKI to affect the differentiation of.MiR-214 into vascular smooth muscle cells. Target.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5

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