【作者】 张兰兰；

【导师】 李玉花； 隋广超；

【作者基本信息】 东北林业大学 ， 发育生物学， 2021， 博士

【摘要】 富含鸟嘌呤(G-rich)的DNA和RNA能够折叠形成一种特殊的结构,G-四链体(G-quadruplex)。该结构广泛存在于人类的基因组中,能够调节包括DNA复制、端粒维持、基因表达与调控以及遗传稳定性在内的多个生物学过程。AKT又称蛋白激酶B(PKB),是一种丝氨酸/苏氨酸激酶,参与多种重要的细胞信号转导途径,包括细胞生存、增殖、侵袭、凋亡和血管生成。AKT1在细胞生长和存活中起着关键作用,其在肿瘤中的激活是由不同的机制介导的,目前所知的主要包括基因表达升高和翻译后蛋白磷酸化。本研究通过生物信息学和生物技术从mRNA水平及DNA/RNA结构的角度探讨AKT1在癌症中上调的机制,研究了人的AKT1启动子及其mRNA 3’-UTR中多个鸟嘌呤簇(G-tracts)之间形成G-四链体结构的可能性。本研究主要实验结果如下:1.利用生物信息学的方法系统展示了 AKT1在多种癌症中的表达上调,探索其高表达指标用于乳腺癌、卵巢癌、肺癌、胃癌和肝癌等临床预后因子的可能性,同时探讨了AKT1基因表达在多种癌症中与免疫浸润细胞的相关性。2.圆二色谱分析表明,基于AKT1启动子G-tracts区域序列合成的寡聚核苷酸在G-四链体结构的特异波长处呈现摩尔椭圆峰。利用非变性聚丙烯酰胺凝胶电泳、凝胶迁移实验和G-四链体特异性抗体免疫染色实验,也证明了这些寡核苷酸在钾离子或锂离子存在的退火条件下形成G-四链体结构。硫酸二甲酯(DMS)足迹保护试验鉴定了参与G-四链体形成的核苷酸。染色质免疫共沉淀和PCR阻滞试验也验证了 AKT1启动子区存在G-四链体结构。在荧光素酶报告试验中,AKT1启动子的G-tracts突变降低了这些G-rich区域介导的报告基因的表达,表明G-四链体结构正向调控AKT1基因表达。实验结果还显示,AKT1启动子区G-四链体结构的形成可能受转录因子SP1调控。此外,DNA解旋酶RECQL与AKT1的表达呈负相关,因此可能调节AKT1 G-四链体的形成。3.利用圆二色谱分析、凝胶迁移实验和DMS足迹保护试验,证明了 AKT1 mRNA 3’-UTR区域存在G-四链体结构。进一步的实验结果表明,该G-四链体结构的形成可能影响了miR-193b-3p与AKT1 mRNA 3’-UTR的结合和该区域对AKT1基因表达的调控作用。4.利用已知的人MYC基因启动子的G-四链体晶体结构及分子对接技术对小分子化合物石蒜碱进行了分子对接,显示该小分子药能够很好的和G-四链体结合。非变性聚丙烯凝胶电泳及Taq聚合酶延伸阻滞试验的方法,验证了石蒜碱可以诱导形成并且稳定G-四链体结构。因此,石蒜碱代表了一类新的G-四链体结合配体,可能成为潜在的抗癌药物。综上所述,本论文的研究证明G-四链体结构存在于人AKT1的启动子和mRNA 3’-UTR区域,此结构对AKT1基因转录具有正调控作用,其机制可能是所形成的G-四链体分别影响SP1对AKT1启动子的结合和miR-193b-3p对AKT1 3’-UTR的识别。同时,RECQL可能通过解旋AKT1启动子和/或其3’-UTR中的G-四链体而介导AKT1基因表达。因此,AKT1所形成的G-四链体可能成为癌症治疗的有效靶点。另外,筛选出的小分子配体石蒜碱能够稳定G-四链体,具有开发为抑制癌症药物的潜能。更多还原

【Abstract】 Guanine-rich DNA and RNA can fold into special structures,named as G-quadruplexes.These structures are widespread in the human genome and involved in multiple biological processes,such as DNA replication,telomere maintenance,gene expression and regulation and genetic stability.AKT,also known as protein kinase B(PKB),is a serine/threonine kinase involved in a variety of important cellular signal transduction pathways,including cell survival,proliferation,invasion,apoptosis,and angiogenesis.AKT1 plays a key role in cell growth and survival.Activation of AKT1 is mediated by distinct mechanisms,including increased gene expression and post-translational protein phosphorylation.In this study,bioinformatic and biotechnological approaches were employed to investigate the mechanism of AKT1 up-regulation in cancers from the perspective of mRNA levels and DNA/RNA structures,and the potential of G-quadruplex formation by the G-tracts in the human AKT1 promoter and its mRNA 3’-UTR was studied.The results in this study were as follows:1.Bioinformatic analyses indicated that AKT1 was upregulated in various malignancies and physiologically relevant to the clinical outcomes of patients with breast,ovarian,lung,gastric,and liver cancers.In addition,we also explored the relationship between AKT1 expression and immune cell infiltration in different types of malignant tumors.2.Circular dichroism analyses showed that synthetic oligonucleotides based on these G-tract regions of the AKT1 promoter exhibited molar ellipticity peaks at specific wavelengths for G-quadruplex structures.Native polyacrylamide gel electrophoresis,gel-shift assays,and immunostaining by a G4-specific antibody were used to demonstrate the formation of G-quadruplex structures at different annealing conditions.Dimethyl sulfate(DMS)footprinting was used to elucidate the nucleotides involved in forming G-quadruplex structures.Chromatin immunoprecipitation(ChIP)assay coupled with PCR-stop assay revealed the presence of G-quadruplex structures in the AKT1 promoter.In luciferase reporter assay,mutations of the G-tracts in the AKT1 promoter reduced the expression mediated by the promoter,suggesting that G-quadruplex structures positively regulated AKT1 gene expression.Our data also suggested that G-quadruplex formation in the AKT1 promoter could likely alter the binding and regulation of transcription factor SP1.Additionally,the DNA helicase RECQL negatively correlated with AKT1 expression and potentially regulated AKT1 G-quadruplex formation.3.Circular dichroism analysis,gel-shift assay,and DMS footprinting assay were employed to demonstrate the formation of G-quadruplex structures in the AKT1 mRNA 3’-UTR region.These G-quadruplexes could likely alter the binding of miR-193b-3p to the AKT1 mRNA 3’-UTR and impact its effects on AKT1 expression.4.Using molecular docking simulation,the crystal structure of the G-quadruplex in the human MYC gene promoter could be docked to the molecule of Lycorine.The results showed that Lycorine could potentially bind to the G-quadruplex.Native polyacrylamide gel electrophoresis and PCR-stop assay were used to demonstrate that Lycorine induced the formation of the G-quadruplex structure and increased its stability.Therefore,Lycorine represents a new class of G-quadruplex binding ligands and may become a potential anticancer drug.In conclusion,our study proved that the presence of G-quadruplex structures in the promoter and mRNA 3’-UTR region of human AKT1,and these G-quadruplexes positively regulated AKT1 expression,likely through mechanisms of altering the recognition and binding of SP1 to the AKT1 promoter,and interfering with the binding of miR-193b-3p to the 3’-UTR of its mRNA.Meanwhile,RECQL could potentially mediate AKT1 gene expression by unwinding G-quadruplexes in the AKT1 promoter and/or its 3’-UTR.Therefore,the G-quadruplexes in AKT1 may serve as effective targets in cancer therapies.Additionally,Lycorine identified in this study with ability of stabilizing G-quadruplexes can be potentially developed into an anti-cancer agent.更多还原