Wntl通过TCF4和PPAR-γ上调巨噬细胞CD36表达和MicroRNA-29a通过靶向QKI促进巨噬细胞SRA的表

发布时间：2018-02-03 08:16

本文关键词： Wnt1 β-catenin TCF4 PPAR-γ CD36 分化 QKI SRA microRNA-29a 脂质吞噬　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：论文一：wntl通过促进TCF4和PPAR-γ上调巨噬细胞清道夫受体CD36的表达研究背景清道夫受体家族B类成员CD36调控了巨噬细胞对氧化型低密度脂蛋白的吞噬,同时也在巨噬细胞的生理活动中发挥了重要作用。但是,CD36在巨噬细胞中本地表达的调控机制还未明了。目的本研究主要探索wntl在巨噬细胞的生理作用,并深入研究了wntl调控巨噬细胞清道夫受体CD36表达的分子机制以及wntl在单核细胞向巨噬细胞分化过程中的表达机制。方法本研究中,我们探讨了由单核细胞加GM-CSF刺激分化而来的巨噬细胞中wntl与CD36之间的联系。转染wntl的siRNA或是wntl过表达质粒,巨噬细胞中CD36表达分别呈现下调或上调。通过使用β-catenin的抑制剂,转染PPAR-γ的siRNA或TCF4的siRNA,我们证实了wntl通过促进PPAR-γ和TCF4上调CD36表达。免疫共沉淀,染色质免疫共沉淀及免疫荧光的方法进一步发现β-catenin可以和PPAR-γ发生互作,而PPAR-γ又可以与TCF4在核内发生共定位。同时,转录因子pax3在单核向巨噬分化过程中调控wntl表达也通过western blot被证实。结果我们的研究证实了由单核细胞加GM-CSF刺激分化而来的巨噬细胞中,wnt1通过激活PPAR-γ和TCF4转录因子促进了CD36的表达。结论我们的发现提示了wntl通过激活wnt经典通路,在巨噬细胞生理过程中发挥了重要作用。论文二：MicroRNA-29a通过靶向QKI (quaking)促进了单核细胞向巨噬细胞分化过程中SRA的表达背景在单核细胞向巨噬细胞分化过程中,miR-29a和清道夫受体-SRA上调,而RNA结合蛋白QKI下调。由于巨噬细胞表达的SRA受体在动脉粥样硬化疾病中的重要性加上我们之前的工作已发现miR-29a可以上调SRA,探讨SRA调控的内在机制显得尤为重要。已有工作分别联系了miR-29a与SRA和QKI同单核-巨噬分化。但三者之间在单核-巨噬分化时是否存在着相互联系还未知晓。目的深入研究miR-29a上调SRA的详细机制,进一步发现单核向巨噬分化过程中,miR-29a, SRA和QKI三者之间的内在联系。方法本研究中,我们首先运用qRT-PCR和western blot的方法验证了在单核细胞向巨噬细胞分化时miR-29a升高,QKI表达下降。在巨噬细胞中分别转染miR-29a的模拟物和抑制物后检测了QKI的蛋白表达变化,结合荧光素酶报告基因技术验证了QKI是miR-29a的靶基因。信息学分析发现SRAmRNA的3'UTR上存在一个QKI结合靶位(QRE区)。因此我们深入探讨了QKI与SRA的关系。通过在巨噬细胞中过表达或敲低QKI,我们发现QKI在转录水平上抑制了SRA。借助于荧光素酶报告基因技术,我们验证了QKI可以和SRAmRNA上QRE区直接结合。另外,油红试验的开展进一步证实QKI可以通过作用于SRA抑制巨噬细胞脂质吞噬功能。结果我们发现miR-29a通过结合于QKI的3'UTR抑制了QKI的表达,而QKI通过作用于SRAmRNA的3'UTR上的QRE区促进了SRAmRNA的降解。结论miR-29a通过靶向RNA结合蛋白-QKI对单核细胞向巨噬细胞分化过程中SRA的上调发挥了重要作用。
[Abstract]:Paper I:. Wntl upregulated the expression of scavenger receptor CD36 in macrophages by promoting TCF4 and PPAR- 纬 background CD36, a member of the scavenger receptor family B, regulates macrophage response to oxidative low density. The phagocytosis of lipoproteins. It also plays an important role in the physiological activities of macrophages. The mechanism of local expression of CD36 in macrophages is not clear. Objective to explore the physiological role of wntl in macrophages. The molecular mechanism of wntl regulating the expression of scavenger receptor CD36 in macrophages and the mechanism of wntl expression in the process of monocyte differentiation into macrophages were studied. Methods in this study. We investigated the relationship between wntl and CD36 in macrophages stimulated by monocytes and GM-CSF. SiRNA or wntl overexpression plasmids transfected with wntl were investigated. The expression of CD36 in macrophages was down-regulated or up-regulated, and the siRNA of PPAR- 纬 or the siRNA of TCF4 were transfected with 尾 -catenin inhibitor. We confirmed that wntl upregulated CD36 expression and immunoprecipitation by promoting PPAR- 纬 and TCF4. Chromatin immunoprecipitation and immunofluorescence further revealed that 尾 -catenin could interact with PPAR- 纬. PPAR- 纬 can co-locate with TCF4 in nucleus. Transcription factor pax3 regulates the expression of wntl during mononuclear macrophage differentiation through western. Blot was confirmed. As a result, our study confirmed that macrophages derived from monocytes and GM-CSF stimulated differentiation. Wnt1 promotes the expression of CD36 by activating PPAR- 纬 and TCF4 transcription factors. Conclusion our findings suggest that wntl activates the classical wnt pathway. It plays an important role in the physiological process of macrophages. Promote the expression of SRA in the process of monocyte differentiation to macrophage. MiR-29a and scavenger receptor -SRA were up-regulated. The RNA binding protein QKI is down-regulated because of the importance of SRA receptors expressed by macrophages in atherosclerotic diseases and our previous work has shown that miR-29a can up-regulate SRA. It is particularly important to explore the intrinsic mechanism of SRA regulation. Previous work has linked miR-29a with SRA and QKI with monocyte-macrophage differentiation, but whether the three exist in monocyte-macrophage differentiation. Objective to study the detailed mechanism of miR-29a upregulation of SRA. The relationship among miR-29a, SRA and QKI in the process of mononuclear macrophage differentiation was further found. Methods in this study. At first, we used qRT-PCR and western blot methods to verify the increase of miR-29a when monocytes differentiated into macrophages. The expression of QKI was decreased and the expression of QKI protein was detected after transfection of miR-29a mimics and inhibitors in macrophages. Combined with luciferase reporter gene technique, QKI is the target gene of miR-29a. Therefore, we explored the relationship between QKI and SRA by overexpressing or knocking down QKI in macrophages. We found that QKI inhibited SRAs at the transcriptional level. With the help of luciferase reporter gene technology, we confirmed that QKI could bind directly to the QRE region on SRAmRNA. The oil red test further confirmed that QKI could inhibit the lipid phagocytosis of macrophages by acting on SRA. Results We found that miR-29a inhibited QK by binding to QKI 3 UTR. I expression. QKI can promote the degradation of SRAmRNA by acting on the QRE region of SRAmRNA. Conclusion miR-29a can promote the degradation of mononuclear SRAmRNA by targeting RNA binding protein -QKI. The up-regulation of SRA plays an important role in the differentiation of macrophages.
【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】：R392

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