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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
Feedback Loop between Mitochondrial Fission/Fussion and Notch Signaling Regulates Survivin-mediated Survival of TNBC Cells
【作者】 陈丽;
【导师】 姜鹤群;
【作者基本信息】 成都医学院 , 病理学与病理生理学, 2017, 硕士
【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,which are in constant process of fission and fusion.Mitochondrial fission and fusion not only have an important effect on maintaining mitochondrial morphology,calcium homeostasis,ROS production and so on,but also affect cell metabolism,proliferation and migration.Studies have shown that triple negative breast cancer(TNBC)has a worse prognosis than non triple negative breast cancer.The reason may be associated with enhanced tumor cell survival ability and the lack of effective therapeutic targets.But the mechanism is not yet fully clear.In this study,mitochondrial fission and fusion was chosen as a breakthrough point to explore the effect of mitochondrial dynamics on the survival of triple negative breast cancer cells and the mechanism.ObjectiveIn this study,we chose triple negative breast cancer as the research object,Drp1 and Mfn1 as the breakthrough point(the molecules of which mediated mitochondrial fission and fusion respectively),to explore:(1),the distribution state of mitochondrial fusion and fission in triple negative breast cancer and its correlation with prognosis;(2),the regulatory role of mitochondrial fission and fusion involved in triple negative breast cancer cell growth;(3),the mechanism of mitochondrial fission and fusion involved in triple negative breast cancer cell growth.Materials and Methods1.Transmission electron microscope(TEM)was used to observe the distribution state of mitochondrial fission and fusion in triple negative breast cancer patients,and statistical analysis was used to assess prognosis.2.Immunohistochemical staining(IHC),Western blot and qRT-PCR were used to detect the levels of Drp1 and Mfn1 protein and mRNA in the tissue samples and cells of triple negative breast cancer.3.Transfection was used to construct Drp1 mediated mitochondrial fission and Mfn1 mediated mitochondrial fusion triple negative breast cancer cell models.4.MTS assay was used to detect the cell viability of the cell models of Drp1/Mfn1 mediated mitochondrial fission/fusion.5.Situ tumor formation in nude mice was used to detect the ability of Drp1/Mfn1 mediated mitochondrial fission/fusion cell models.6.Annexin V-FITC/PI/flow cytometry and TUNEL staining were used to detect cell apoptosis in Drp1/Mfn1 mediated mitochondrial fission/fusion cells.7.Eud assay and IHC Ki67 staining were used to detect the cell proliferation of Drp1/Mfn1 mediated mitochondrial fission/fusion cells.8.Western blot,IHC were used to detect Drp1,Mfn1,Notch1,NICD1 protein levels,and mitochondrial morphology staining was used to explore the interaction of mitochondrial fission/ fusion and Notch signal.9.Annexin V-FITC/PI/flow cytometry and Edu were used to detect the effect of the interaction between mitochondrial fission/fusion and Notch signaling on cell apoptosis and proliferation.10.Western blot was used to detect the expression of survivin regulated by the interaction between mitochondrial fission/fusion and Notch signal.11.MST experiment demonstrated that survivin mediated the effect of mitochondrial fission/fusion and Notch signal on cell viability.Results1.Mitochondrial fission increased in triple negative breast cancer and the expression of Drp1 was negatively correlated with prognosis;mitochondrial fusion decreased and the expression of Mfn1 was positively correlated with prognosis in TNBC cells.2.Drp1 mediated mitochondrial fission promoted cell survival,proliferation and inhibited apoptosis in TNBC cells;Mfn1 mediated mitochondrial fusion inhibited cell survival,proliferation and promoted apoptosis.3.Drp1 mediated mitochondrial fission promoted Notch1 expression and NICD1 activation;Mfn1mediated mitochondrial fusion inhibited Notch1 expression and NICD1 activation.4.Activation of Notch signal promoted mitochondrial fission and inhibited mitochondrial fusion.5.The interaction of mitochondrial fission and Notch signaling inhibited cell apoptosis and promoted cell proliferation,and positively regulated the expression of survivin.6.Survivin mediated the effect of the interaction of mitochondrial fission and Notch signaling on cell viability of triple negative breast cancer cells.ConclusionIn this study,the positive feedback regulation of mitochondrial fission and Notch signaling pathway was identified.And it’s regulating effect on promoting cell survival and inhibiting cell apoptosis in triple negative breast cancer cells was mediated by survivin.