线粒体分裂融合与Notch信号交互作用调控三阴性乳腺癌细胞再生长及机制研究

发布时间：2018-09-03 18:15

【摘要】：线粒体是高度动态的细胞器,处于不断的分裂与融合的平衡动态过程中。线粒体的分裂融合除了对维持线粒体形态、钙稳态、ROS产生[1,2]等方面有重要作用外,还对细胞代谢、增殖、迁移[3-5]有重要影响。研究表明,三阴性乳腺癌(triple negative breast cancer,TNBC)的预后较普通非三阴性乳腺癌差[6],其原因可能与肿瘤细胞存活能力增强[7,8]及缺乏有效的靶向治疗有关,但具体机制尚未完全明确。本课题以线粒体分裂融合为切入点,探究线粒体分裂融合动态平衡对三阴性乳腺癌细胞存活的影响及其机制。目的本项目以三阴性乳腺癌为研究对象,选择介导线粒体分裂的分子Drp1、介导线粒体融合的分子Mfn1为切入点,探究:(1)、三阴性乳腺癌中线粒体分裂融合分布状态及其与预后的相关性;(2)、线粒体分裂融合调控三阴性乳腺癌细胞生长的作用;(3)、线粒体分裂融合调控三阴性乳腺癌细胞生长的机制。材料与方法1、用透射电子显微镜(TEM)观察三阴性乳腺癌临床标本线粒体分裂融合分布状态,统计学分析预后相关性。2、用免疫组化(IHC)、Western blot、qRT-PCR检测三阴性乳腺癌临床组织样本与细胞中Drp1和Mfn1蛋白质、mRNA水平。3、细胞转染构建Drp1介导的线粒体分裂、Mfn1介导的线粒体融合三阴性乳腺癌细胞模型。4、MTS实验检测Drp1/Mfn1介导的线粒体分裂融合细胞模型的细胞活力。5、裸鼠乳腺原位成瘤实验检测Drp1/Mfn1介导的线粒体分裂融合细胞模型的成瘤能力。6、AnnexinⅤ-FITC/PI流式细胞术、TUNEL染色检测Drp1/Mfn1介导的线粒体分裂融合对细胞凋亡的影响。7、Eud实验和IHC Ki67染色检测Drp1/Mfn1介导的线粒体分裂融合对细胞增殖的作用。8、Western blot、IHC检测Drp1、Mfn1、Notch1、NICD1蛋白水平变化、Mito Traker Green染色检测线粒体形态探究线粒体分裂融合与Notch信号的交互作用。9、AnnexinⅤ-FITC/PI流式细胞术和Edu分别检测线粒体分裂融合与Notch信号交互作用对细胞凋亡、增殖的影响。10、Western blot检测线粒体分裂融合与Notch信号交互作用对Survivin蛋白表达的调控。11、MST实验验证Survivin介导的线粒体分裂融合与Notch信号交互对细胞活力的影响。结果1、线粒体分裂在三阴性乳腺癌中增加、融合减少,并且介导线粒体分裂的Drp1表达与TNBC预后呈负相关,介导线粒体融合的Mfn1表达与预后呈正相关。2、Drp1介导的线粒体分裂促进三阴性乳腺癌细胞存活、增殖、抑制细胞凋亡;Mfn1介导的线粒体融合抑制三阴性乳腺癌细胞存活、增殖、促进细胞凋亡。3、Drp1介导的线粒体分裂促进Notch1表达和NICD1活化、Mfn1介导的线粒体融合抑制Notch1表达和NICD1活化。4、Notch信号激活促进线粒体分裂、抑制线粒体融合。5、线粒体分裂与Notch信号的交互作用抑制细胞凋亡、促进细胞增殖,并且正向调控Survivin的表达。6、Survivin介导了线粒体分裂与Notch信号的交互作用对三阴性乳腺癌细胞活力的影响。结论本课题明确了线粒体分裂与Notch信号通路的正向反馈调控作用,并且其通过survivin介导,促进了三阴性乳腺癌细胞增殖以及抑制细胞凋亡。
[Abstract]:Mitochondria are highly dynamic organelles in the process of constant division and fusion. Mitochondrial division and fusion not only play an important role in maintaining mitochondrial morphology, calcium homeostasis, ROS production [1,2], but also have an important impact on cell metabolism, proliferation and migration [3-5]. The prognosis of breast cancer (TNBC) is worse than that of non-tri-negative breast cancer (6). The reason may be related to the enhancement of tumor cell viability [7,8] and the lack of effective targeted therapy, but the specific mechanism is not fully clear. The aim of this project is to investigate the distribution of mitochondrial fission and fusion and its relationship with prognosis in triple-negative breast cancer. (2) Mitochondrial fission and fusion regulation. Materials and Methods 1. The distribution of mitochondrial fission and fusion was observed by transmission electron microscopy (TEM), and the prognostic correlation was analyzed statistically. 2. Immunohistochemistry (IHC), Western blot, and qRT-PCR were used. Drp1 and Mfn1 proteins and mRNA levels were detected in clinical tissue samples and cells of triple-negative breast cancer. Mitochondrial division mediated by Drp1 and triple-negative breast cancer cell model mediated by Mfn1 were constructed by cell transfection. 4. MTS assay was used to detect the cell viability of the triple-negative breast cancer cell model mediated by Drp1/Mfn1. Tumor formation assay was used to detect the tumorigenic ability of Drp1/Mfn1-mediated mitochondrial division and fusion cell model.6, Annexin V-FITC/PI flow cytometry, TUNEL staining to detect the effect of Drp1/Mfn1-mediated mitochondrial division and fusion on cell apoptosis.7, Eud test and IHC Ki67 staining to detect the effect of Drp1/Mfn1-mediated mitochondrial division and fusion on cell proliferation. Mito Traker Green staining was used to detect mitochondrial morphology to explore the interaction between mitochondrial fission fusion and Notch signal. 9. Annexin V-FITC/PI flow cytometry and Edu were used to detect the effects of mitochondrial fission fusion and Notch signal interaction on apoptosis and proliferation, respectively. 10. Western blot was used to detect the regulatory effect of mitochondrial mitotic fusion and Notch signaling on the expression of Survivin protein. 11. MST assay verified the effect of Survivin-mediated mitochondrial fusion and Notch signaling interaction on cell viability. Results 1. Mitochondrial division increased, fusion decreased and mitochondrial division mediated by Survivin-mediated mitochondrial fusion in triple-negative breast cancer. The expression of Drp1 was negatively correlated with the prognosis of TNBC, and the expression of MFn1 mediated mitochondrial fusion was positively correlated with the prognosis. Mitochondrial fusion mediated by Mfn1 inhibits Notch1 expression and NICD1 activation. 4. Notch signal activation promotes mitochondrial division and inhibits mitochondrial fusion. 5. The interaction of mitochondrial division and Notch signal inhibits cell apoptosis, promotes cell proliferation, and positively regulates the expression of Survivin. Conclusion This study clarifies the positive feedback regulation of mitochondrial division and Notch signaling pathway and promotes the proliferation and inhibits apoptosis of triple-negative breast cancer cells through survivin mediation.
【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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