【作者】 谭力铭；

【导师】 王兵；

【作者基本信息】 南华大学 ， 临床医学， 2024， 硕士

【摘要】 研究背景:近年来,越来越多的证据表明,环状RNA(circRNA)在胶质瘤中扮演着关键角色。作为一种重要的非编码RNA,在细胞内部形成环状结构的circRNA已被广泛研究,并被发现在多种生物学过程中发挥重要功能。尤其是在癌症中,circRNA的异常表达和功能异常已成为研究的热点。然而,至今仍然缺乏关于circXRCC5在胶质瘤中的研究报道,以及其潜在的分子机制。XRCC5是一种编码X射线交联修复蛋白Ku80的基因,其参与DNA损伤修复和细胞周期调控等多个生物学过程。因此,circXRCC5可能通过调节细胞内的DNA损伤修复机制和细胞周期,影响胶质瘤的发生和发展。此外,IGF2BP2作为一种RNA结合蛋白,在肿瘤中的表达和功能异常已被广泛关注。然而,circXRCC5与IGF2BP2在胶质瘤中的相互作用及其对肿瘤生长的影响尚未被报道。因此,本研究旨在探索circXRCC5在胶质瘤中的表达水平及其潜在的作用机制,以期为深入理解胶质瘤的发病机制和开发新的治疗策略提供理论依据。研究目的:本研究旨在探究circXRCC5在胶质瘤中的表达情况及其潜在的作用机制。具体目的包括:1.分析circXRCC5在胶质瘤组织中的表达水平;2.研究circXRCC5对胶质瘤细胞增殖和侵袭的影响;3.探讨circXRCC5可能通过调节DNA损伤修复机制和细胞周期来影响胶质瘤发生和发展;4.研究circXRCC5与IGF2BP2在胶质瘤中的相互作用及其对肿瘤生长的影响。通过实现这些目标,我们的研究有望深入理解circXRCC5在胶质瘤中的作用机制,为未来开发针对胶质瘤的治疗策略提供理论基础。研究方法:在本研究中,我们采用了多种方法来检测RNA转录本和蛋白水平,以及评估细胞增殖、炎症小体形成和细胞因子释放等生物学过程。首先,我们利用定量逆转录聚合酶链反应(qRT-PCR)、免疫组化(IHC)和原位杂交(ISH)等技术检测RNA转录本和蛋白的表达水平。这些技术可以提供对目标分子在组织或细胞水平上的定量和定位信息。其次,为了评估细胞增殖能力,我们采用了细胞计数试剂盒(CCK-8)和克隆形成实验。通过这些实验,我们可以对细胞增殖速率和克隆形成能力进行准确的定量和定性分析。为了研究炎症小体的形成,我们利用了免疫荧光(IF)和免疫印迹(Western blot)技术来检测NLRP3炎症小体的形成。这些技术能够清晰地显示炎症小体在细胞内的定位和数量,并确定相关蛋白的表达水平。此外,为了确定细胞因子的释放水平,我们使用了免疫吸附测定(ELISA)和免疫印迹技术。这些方法能够快速、准确地检测细胞因子的含量,并且可以用于定量和定性分析。最后,为了验证分子间的相互作用,我们进行了RNA免疫共沉淀(RIP)和下拉实验。这些实验能够有效地鉴定目标RNA与蛋白之间的直接相互作用,从而验证分子间的特定结合关系。研究结果:circXRCC5在胶质瘤中呈现明显的高表达,与患者的生存率较短、病情晚期(TNM分期)和肿瘤体积增大呈正相关。进一步的实验表明,circXRCC5的敲除抑制了胶质瘤细胞的增殖能力,同时也减弱了NLRP3介导的炎症小体的激活。我们的研究揭示了circXRCC5通过与IGF2BP2结合,维持了CLC3的mRNA稳定性。此外,我们发现CLC3通过PI3K/PDK1/AKT信号通路加速了SGK1的表达。进一步的挽救实验表明,CLC3或SGK1的过表达显著缓解了circXRCC5敲除引起的胶质瘤细胞增殖抑制和NLRP3介导的炎症小体激活。在体内实验证实,circXRCC5通过调节CLC3/SGK1轴加速了胶质瘤的生长。研究结论:circXRCC5与IGF2BP2形成复合物,通过CLC3/SGK1轴调控炎性小体激活和促进肿瘤生长。更多还原

【Abstract】 Background: In recent years,increasing evidence has indicated that circular RNA(circRNA)plays a crucial role in gliomas.As an important type of non-coding RNA,circRNA with a circular structure formed inside cells has been extensively studied and found to exert significant functions in various biological processes.Particularly in cancer,the abnormal expression and function of circRNA have become a research hotspot.However,there is still a lack of research reports on circXRCC5 in gliomas and its potential molecular mechanisms.XRCC5 is a gene encoding the X-ray cross-complementing repair protein Ku80,which participates in multiple biological processes including DNA damage repair and cell cycle regulation.Therefore,circXRCC5 may influence the occurrence and development of gliomas by modulating intracellular DNA damage repair mechanisms and the cell cycle.Additionally,IGF2BP2,as an RNA-binding protein,has been widely studied for its abnormal expression and function in tumors.However,the interaction between circXRCC5 and IGF2BP2 in gliomas and their effects on tumor growth have not been reported.Thus,this study aims to explore the expression level of circXRCC5 in gliomas and its potential mechanisms,in order to provide theoretical basis for a deeper understanding of the pathogenesis of gliomas and the development of new therapeutic strategies.Objective: This study aims to investigate the expression pattern and potential mechanisms of circXRCC5 in gliomas.Specific objectives include: 1.Analyzing the expression level of circXRCC5 in glioma tissues;2.Investigating the impact of circXRCC5 on glioma cell proliferation and invasion;3.Exploring the potential role of circXRCC5 in gliomagenesis and progression by modulating DNA damage repair mechanisms and the cell cycle;4.Investigating the interaction between circXRCC5 and IGF2BP2 in gliomas and their effects on tumor growth.By achieving these objectives,our study aims to provide a deeper understanding of the role and mechanisms of circXRCC5 in gliomas,laying a theoretical foundation for the development of future therapeutic strategies targeting gliomas.Methods: In this study,we employed various methods to detect RNA transcripts and protein levels,as well as to evaluate biological processes such as cell proliferation,inflammasome formation,and cytokine release.Firstly,we utilized techniques including quantitative reverse transcription polymerase chain reaction(q RT-PCR),immunohistochemistry(IHC),and in situ hybridization(ISH)to assess the expression levels of RNA transcripts and proteins.These techniques provide quantitative and localization information of target molecules at tissue or cellular levels.Secondly,to evaluate cell proliferation,we conducted cell counting kit-8(CCK-8)assays and clone formation experiments.These experiments allowed for accurate quantitative and qualitative analysis of cell proliferation rate and clonogenic ability.In order to study inflammasome formation,we utilized immunofluorescence(IF)and Western blot techniques to detect the formation of NLRP3 inflammasomes.These techniques clearly visualize the localization and quantity of inflammasomes within cells and determine the expression levels of related proteins.Additionally,to determine the levels of cytokine release,we employed enzyme-linked immunosorbent assay(ELISA)and immunoblotting techniques.These methods enable rapid and accurate detection of cytokine levels,suitable for both quantitative and qualitative analysis.Finally,to validate molecular interactions,we conducted RNA immunoprecipitation(RIP)and pull-down experiments.These experiments effectively identify direct interactions between target RNA and proteins,thus validating specific molecular binding relationships.Results: circXRCC5 exhibits significantly high expression in gliomas,which correlates positively with shorter patient survival,advanced disease stage(TNM staging),and increased tumor volume.Further experiments revealed that silencing circXRCC5 suppressed glioma cell proliferation and attenuated NLRP3-mediated inflammasome activation.Our study elucidated that circXRCC5 maintains mRNA stability of CLC3 by binding to IGF2BP2.Additionally,we discovered that CLC3 accelerates SGK1 expression via the PI3K/PDK1/AKT signaling pathway.Rescue experiments demonstrated that overexpression of CLC3 or SGK1 significantly alleviated the inhibition of glioma cell proliferation and NLRP3-mediated inflammasome activation induced by circXRCC5 knockdown.In vivo experiments confirmed that circXRCC5 accelerates glioma growth by modulating the CLC3/SGK1 axis.Conclusion: circXRCC5 and IGF2BP2 form a complex to regulate inflammasome activation and promote tumor growth via the CLC3/SGK1 axis.更多还原