NF-κB信号通路在胶质瘤放疗耐受中的作用及机制研究

发布时间：2018-01-26 21:36

本文关键词： 胶质瘤 NF-κB通路 DNA损伤治疗抵抗　出处：《大连医科大学》2014年博士论文　论文类型：学位论文

【摘要】：脑胶质瘤是最常见的中枢神经系统恶性肿瘤,具有高发病率、高度侵袭性、高死亡率的特点。该肿瘤通过对整个大脑的广泛入侵表现出了持续的恶性进展特点,并对传统的放疗具有抵抗力,故临床上治疗十分棘手。近年研究表明,术后肿瘤细胞对放疗不敏感与NF-κB信号通路活性增高有关。肿瘤的放射治疗一般是通过损伤DNA,诱导细胞凋亡,阻断细胞增殖从而消除肿瘤细胞。DNA损伤激活的NF-κB信号通路诱导细胞凋亡是放疗的主要作用机制之一。但是NF-κB通路在DNA损伤诱导的凋亡反应中是一把双刃剑,具有双重作用,其促进细胞凋亡的同时,还将诱导抗凋亡基因的表达。一旦NF-KB活性异常增高或者持续活化,则表现出抗凋亡作用,通过诱导抗凋亡及促进细胞增殖相关靶基因的表达,包括Bcl-2家族,Survivin,COX-2,Cyclin D1,AKT及EGFR等,导致肿瘤细胞周期进程加快,抗凋亡能力增强,侵袭及转移能力增强,从而对放疗产生抗性。由于NF-κB介导的下游靶基因有很大一部分是细胞正常生命进程所需要的,因此广谱阻断NF-κB意味着很多不良反应的产生。理想的NF-κB抑制剂应是能特异性作用于肿瘤细胞,而非正常细胞,从而达到精确清除肿瘤细胞的目的。不同组织来源的肿瘤细胞对DNA损伤的耐受性及反应性不同,对放疗的敏感性也不同。DNA损伤诱导的NF-KB信号通路起抗凋亡作用还是促凋亡作用具有一定的组织特异性。因此深入探讨特定条件诱导NF-KB通路的分子机理、调控机制及其组织特异性,有利于我们针对NF-κB在肿瘤放疗耐受中的作用,提出特异而有效的个体化治疗方案。本研究着眼于胶质瘤对放疗不敏感这一临床问题,拟在胶质瘤细胞系T98G及U87MG中探讨NF-KB通路与基于DNA损伤诱导的胶质瘤放疗不敏感的作用机制,以期为有效的胶质瘤放疗提供理论依据。实验用细胞辐照仪处理人胶质瘤细胞系T98G及U87MG,以模拟DNA损伤环境。研究发现胶质瘤细胞系T98G及U87MG对DNA损伤诱导的细胞凋亡及增殖抑制不敏感,但抑制NF-κB活性则增强其对DNA损伤的敏感性。分子机制研究发现,DNA损伤能显著增强胶质瘤细胞中NF-κB的活性,导致NF-KB介导的靶基因,包括Bcl-κL, Cyclin Dl, Survivin,及IL-8等高表达,从而增强肿瘤细胞抗凋亡活性,促进肿瘤生长。利用分子生物学研究手段,发现DNA损伤可增强NEMO的SUMO化修饰。NEMO的SUMO化修饰是细胞核内的DNA损伤信号激活细胞核外的NF-κB信号通路传导的限速步骤,因此增强NEMO的SUMO化修饰对于DNA损伤诱导的NF-KB活化至关重要。进一步研究发现,NF-κB活化可促进miR-181b表达升高,而miR-181b可靶向去SUMO化酶SENP2,使其表达下降,降低了SENP2催化的NEMO去SUMO化修饰,从而促使NF-KB保持持续活化状态。而miR-181b有望成为提高胶质瘤放射治疗效果的新靶点。第一部分：DNA损伤激活人脑胶质瘤细胞NF-κB信号通路目的：探讨人胶质瘤细胞T98G,U87MG对DNA损伤诱导的细胞凋亡及增殖抑制是否敏感,及其与NF-κB信号通路的关系。方法：应用电离辐射处理T98G及U87MG细胞诱导DNA损伤。免疫荧光检测DNA损伤标志物H2AX-γ来判断DNA损伤效果。划痕实验检测细胞划痕愈合能力；流式细胞仪检测细胞凋亡；MTT检测细胞增殖；Real-time PCR检测NF-KB调控的与肿瘤生长及转移相关的靶基因的表达水平；双荧光素酶报告系统检测NF-κB的活性；Western blot检测p-IκBα、IKBα、p50及p65水平；免疫荧光检测DNA损伤处理后T98G细胞中p65是否入核。结果：电离辐射处理T98G细胞,能造成DNA损伤；DNA损伤不影响胶质瘤细胞T98G的划痕愈合能力,不诱导其凋亡；用Bay11抑制NF-κB活性后,T98G细胞的划痕愈合能力下降,细胞增殖受抑制；抑制NF-κB活性能下调T98G细胞中DNA损伤诱导的肿瘤抗性基因,包括CyclinD1、Bcl-xL、Survivin及IL-8的表达；DNA损伤能激活胶质瘤细胞T98G及U87MG细胞中NF-κB信号通路传导,促进IκBα磷酸化及降解,从而释放NF-KB使其入核。结论：1、人胶质瘤细胞系T98G及U87MG对DNA损伤不敏感。抑制NF-κB活性后,敏感性增强。 2、DNA损伤激活人胶质瘤细胞系T98G及U87MG中NF-KB信号通路。第二部分：miR-181b通过靶向SENP2正调控DNA损伤诱导的NF-κB通路活性目的：探讨DNA损伤增强人胶质瘤细胞T98G及U87MG中NF-KB通路活性的分子机制。方法：应用免疫沉淀技术检测DNA损伤后NEMO的SUMO化修饰；利用软件预测NEMO的去SUMO化酶SENP2上microRNA的作用靶点；双荧光素酶报告系统及Western blot检测SENP23'UTR是否含有miR-181b的作用靶点；MicroRNA特异的real-time PCR检测DNA损伤处理前后U87MG细胞中microRNA表达量的变化；双荧光素酶报告系统检测miR-181b对NF-κB通路活性的调控。结果：DNA损伤能促进U87MG细胞中NEMO的SUMO化修饰；软件预测显示去SUMO化酶SENP2的3’UTR含有miR-181b作用位点；双荧光素酶报告系统及Western blot证实miR-181b可以靶向SENP2,使其表达降低：DNA损伤后miR-181b表达升高；过表达的miR-181b会进一步增强DNA损伤诱导的NF-κB通路活性。结论：1、DNA损伤能促进胶质瘤细胞中NEMO的SUMO化修饰。 2、DNA损伤后,miR-181b表达升高,miR-181b可靶向于去SUMO化酶SENP2,使其表达降低,而不能抑制NEMO的SUMO化修饰,从而进一步增强NF-κB舌性。
[Abstract]:Glioma is the most common malignant tumors of the central nervous system, with high incidence, highly invasive, high mortality. The tumor through extensive invasion of whole brain showed malignant progression characteristics continuously, and the traditional radiotherapy resistant, so clinical treatment is very difficult.
Recent studies show that postoperative tumor cells to radiation and NF- B pathway activity increased. Radiotherapy is usually through injury DNA cell apoptosis induced by blocking cell proliferation, thereby eliminating tumor cell.DNA damage and activation of NF- B signaling pathway induced apoptosis is one of the main mechanisms of radiotherapy. But the NF- B pathway in apoptosis induced by DNA damage response is a double-edged sword, has a dual role, which promote cell apoptosis at the same time, will also induce the expression of anti apoptotic genes. Once the abnormal increase of NF-KB activity or sustained activation, showed anti apoptosis effect, through the expression of anti apoptosis and promote cell proliferation induced by the relevant target genes, including Bcl-2 family, Survivin, COX-2, Cyclin, D1, AKT and EGFR, leading to tumor cell cycle progression and enhance the anti apoptosis ability, enhance the ability of invasion and metastasis, thus Resistant to radiotherapy. As the downstream target genes of NF- kappa B mediated by a large part of the required normal cellular processes, thus blocking the NF- spectrum K B means a lot of adverse reactions. NF- B inhibitors ideal should be specific to tumor cells, but not normal cells. To achieve the precise clear tumor cells. Different tolerance and reactivity of various tumor cells to DNA damage, sensitivity to radiotherapy is different NF-KB signaling pathway.DNA damage induced by the anti apoptosis or apoptosis is tissue specific. So to explore molecular mechanism of NF-KB pathway induced by specific conditions the regulatory mechanisms and tissue specificity, we can according to the role of NF- K B in the radiotherapy of tumor tolerance, put forward specific and effective individualized treatment plan.
This study focuses on the clinical problem of glioma which is not sensitive to radiotherapy. We intend to explore the mechanism of NF-KB pathway and insensitivity to DNA damage induced glioma radiotherapy in glioma cell line T98G and U87MG, in order to provide a theoretical basis for effective glioma radiotherapy.
With the treatment of human glioma cell line T98G and U87MG cell irradiator experiment to simulate the damage environment. DNA glioma cell lines T98G and U87MG on DNA damage insensitive cell apoptosis and proliferation induced by the discovery, but the inhibition of NF- kappa B activity enhances their sensitivity to DNA damage. The molecular mechanism study found that DNA damage can significantly enhance the glioma cells NF- kappa B activity leads to target genes mediated by NF-KB, including Cyclin Dl, Bcl- kappa L, Survivin, and high expression of IL-8, so as to enhance the anti apoptosis activity of tumor cells, promote tumor growth. The use of molecular biology research methods, found that DNA damage can be enhanced SUMO modification of SUMO modification of.NEMO NEMO is the rate limiting step in DNA damage signaling within the nucleus activation of NF- kappa B signaling pathway of the cell nucleus, thus enhance the NEMO modification of SUMO is essential for activation of DNA damage induced by NF-KB. Further studies showed that NF- B activation can promote the expression of miR-181b increased, while miR-181b can be targeted to the SUMO enzyme SENP2, which decreased the expression of SENP2, reduce the catalytic NEMO to SUMO modification, so as to promote the sustained activation of NF-KB. MiR-181b is expected to become a new target to improve the treatment effect of radial glia.
Part one: DNA injury activates NF- kappa B signaling pathway in human glioma cells
Objective: To investigate whether human glioma cells T98G, U87MG are sensitive to apoptosis and proliferation inhibition induced by DNA damage, and their relationship with the NF- kappa B signaling pathway.
Methods: application of ionizing radiation treated T98G and U87MG cells induced by DNA damage. Immunofluorescence detection of DNA injury markers to determine H2AX- gamma DNA damage effect. Cell scratch scratch assay. Healing; cell apoptosis was detected by flow cytometry to detect the cell proliferation; MTT; detection of NF-KB regulation of Real-time PCR and tumor growth and metastasis related gene the level of expression of the dual luciferase reporter assay; NF- kappa B activity; Western blot detection of p-I kappa B alpha, alpha IKB, P50 and p65; immunofluorescence detection of DNA damage in p65 treated T98G cells into the nucleus.
Results: T98G cells were treated with ionizing radiation, can cause DNA damage; DNA damage does not affect the T98G glioma cells scratch healing ability, does not induce apoptosis; inhibition of NF- kappa B activity by Bay11 after T98G cell wound healing ability, inhibition of cell proliferation; inhibition of NF- kappa B induced down-regulation of DNA activity tumor resistance gene in T98G cells, including CyclinD1, Bcl-xL, expression of Survivin and IL-8; DNA injury can activate T98G glioma cells and U87MG cells in NF- kappa B signaling pathway, promoting I kappa B alpha phosphorylation and degradation, thereby releasing the NF-KB into the nucleus.
Conclusion: 1, human glioma cell lines T98G and U87MG are insensitive to DNA damage. The sensitivity of NF- kappa B activity is enhanced.
2, DNA damage activates the NF-KB signaling pathway in the human glioma cell line T98G and U87MG.
The second part: miR-181b regulates the activity of NF- kappa B pathway induced by DNA damage through target SENP2
Objective: To investigate the molecular mechanism of DNA injury to enhance the activity of NF-KB pathway in human glioma cells, T98G and U87MG.
Methods: using immunoprecipitation technique to detect SUMO modification of NEMO DNA after injury; prediction of NEMO to target SUMO SENP2 microRNA enzyme by software; target whether dual luciferase reporter system and Western blot detection of SENP23'UTR containing miR-181b and MicroRNA; specific real-time PCR to detect DNA damage in U87MG cell the expression of microRNA; dual luciferase reporter assay miR-181b regulation of NF- B pathway activity.
Results: DNA injury can promote the modification of SUMO NEMO in U87MG cells; software predictive display to SUMO enzyme SENP2 3 UTR containing miR-181b binding sites; dual luciferase reporter system and Western blot confirmed that miR-181b can target SENP2, which decreased the expression of DNA: the expression of miR-181b increased after injury; overexpression of miR-181b would further enhanced NF- B pathway activity induced by DNA.
Conclusion: 1, DNA damage can promote the SUMO modification of NEMO in glioma cells.
2, after DNA injury, the expression of miR-181b is increased. MiR-181b can target SUMO de SENP2, reduce its expression, but not inhibit SUMO modification of NEMO, thereby further enhance the tongue nature of NF- kappa B.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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