【作者】 罗焱；

【导师】 胡义德；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2013， 博士

【摘要】 研究背景和目的：线粒体是真核细胞内存在的双层膜细胞器，是细胞内代谢和能量产生的中心环节，同时还是不同细胞信号通路的通信站。实际上，线粒体的功能远远不止于此，他还是氧化磷酸化的副产物-内源性活性氧(reactive oxygen species，ROS)的主要来源。过量的ROS具有潜在毒性，可能导致线粒体内蛋白质、线粒体DNA及脂质损害，甚至可能通过活化内源性凋亡途径导致细胞死亡。此外，线粒体还与线粒体功能紊乱、心脏功能障碍、衰老和其他疾病相关。因此，线粒体的数量和功能都需要受到精确调控，以发挥维持细胞能量代谢稳态及其他细胞关键进程的作用。到目前为止，已经有证据表明自噬引起的选择性线粒体降解在调节线粒体数量和功能方面发挥着重要作用。因此，线粒体与自噬之间的关系在于自噬可以选择性清除异常增多或者受到损害的线粒体，这一过程又叫做线粒体自噬。最开始研究自噬的时候，一般认为其在细胞内是一保护性过程。基础水平的自噬通过再循环和利用细胞内成分在维持细胞稳态方面发挥着重要的作用。发展到现在，有关自噬的研究已经超出最初的定义，广泛应用于生理及病理学的研究。例如，自噬水平异常升高可能与一系列病理过程相关，如感染、神经退行性疾病、心血管疾病、衰老及肿瘤等。有关自噬与肿瘤的研究中，Beclin-1是最早发现的把自噬和肿瘤联系起来的基因，并在刚发现时将其定义为肿瘤抑制基因。进一步的研究发现，包括MAP-1-LC3和HsGSA7等在内的其他一些自噬相关基因(ATG)也与肿瘤的发生发展密切相关。与自噬相关基因相对应，自噬相关蛋白是自噬过程中的必需因子。微管相关蛋白轻链3(LC3)是哺乳动物体内酵母Atg8的同源物，通过剪切体内新合成PorLC3C端氨其酸形成LC3-Ⅰ。进而，具有E2样酶活性的Atg7再将LC3-Ⅰ C端的22个氨基酸剪切，形成LC3-Ⅱ，而LC3-Ⅱ直接参与自噬体的合成，并且是自噬现象的标志性蛋白。正常情况下，基础水平的自噬通过生理性清除受损细胞器和长寿命蛋白维持细胞稳态，而在代谢应激或能量危机如营养、生长因子或氧气剥夺，抑或线粒体损害时，自噬途径被持续激活。我们知道，线粒体在细胞生长、分裂及能量代谢等过程中发挥着非常重要的作用。显而易见，线粒体的这些功能也是肿瘤细胞增殖、存活及转移所必需的。根据文献报道，在血清饥饿条件下，自发线粒体膜电位去极化的频率是增加的，这些受损的线粒体进入酸性液泡，被自噬体和自噬溶酶体内捕获及降解。为研究肺癌细胞的能量代谢，我们通过低剂量溴化乙锭(ethidium bromide，EtBr)诱导培养的方法敲逐步敲除线粒体DNA，在线粒体DNA逐步缺失的过程中，我们研究发现线粒体数量大量减少，线粒体膜电位下降，更重要的是，在诱导培养过程中发现自噬标志物明显增高。因此，我们推测在EtBr诱导的肺癌细胞线粒体DNA敲除过程中，自噬通路被激活，并可能是线粒体降解的机制之一。因此，本文研究在使用低剂量EtBr诱导肺癌细胞线粒体DNA缺失过程中，研究mtDNA缺失程度对肺癌细胞生物学行为的影响，以及进一步探讨线粒体降解的可能机制。研究方法：1.肺癌细胞低剂量EtBr诱导培养及线粒体DNA缺失程度的鉴定人肺癌细胞系A549、SPC-A1和H322在含有10%的FBS及双抗的培养基中培养，当细胞融合度达到90%～95%时进行传代。为逐步敲除线粒体DNA，细胞培养基中加入250ng/ml的溴化乙锭，连续培养7天，培养时隔天换液。同时，培养基中额外加入50μg/ml的尿嘧啶和100μg/ml的丙酮酸钠。分别收集EtBr诱导培养前，及培养后第1、3、5和7天的肺癌细胞，通过实时定量PCR检测肺癌细胞中线粒体DNA含量及COX II的mRNA表达水平。另外鉴定EtBr诱导培养7天后肺癌细胞对尿嘧啶的营养依赖特性。2.线粒体DNA缺失程度对肺癌细胞生物学行为的影响人肺癌细胞A549、SPC-A1和H322在含EtBr的培养基中分别培养1、3、5和7天，将细胞用胰酶消化并计数，分别进行细胞增殖、克隆形成及细胞迁移实验，以检测肺癌细胞mtDNA敲除过程中细胞的增殖及迁移能力。此外，通过PI染色和Annexin V-FITC染色进行流式检测，分别测定不同mtDNA缺失程度肺癌细胞的细胞周期和早期凋亡水平的改变。同时，我们在体内研究mtDNA缺失不同程度肺癌细胞的生长情况。首先，用NOD/SCID小鼠建立肺癌细胞的皮下异种移植模型，每只小鼠在左侧皮下注射1×106个EtBr处理不同时间的细胞，待肿瘤体积肉眼可见后每周测量两次肿瘤体积，到限值时处死小鼠，取出肿瘤测量肿瘤直径及计算体积。3. EtBr诱导肺癌细胞线粒体降解的鉴定首先，用激光共聚焦验证线粒体的溶酶体降解过程。EtBr诱导培养不同时间的肺癌细胞在检测前20分钟同时加入200nM的绿色线粒体荧光探针和200nM的红色溶酶体荧光探针，孵育结束后用新鲜配置的PBS溶液洗涤三遍。随后在激光共聚焦显微镜下观察，共聚焦图像以2μm间隔进行采集。然后，使用TMRM荧光探针和JC-1荧光探针孵育细胞，分别通过激光共聚焦显微镜和流式细胞仪检测线粒体膜电位的变化情况。最后，将EtBr诱导培养后的肺癌细胞固定染色，在透射电镜下进一步研究EtBr引起的线粒体数量及超微结构的改变。4. EtBr诱导肺癌细胞线粒体降解的机制研究首先，通过LTR、MTG双色荧光探针共定位技术分析线粒体的降解途径，以及电镜下观察线粒体降解的超微结构。然后，我们采用GFP-LC3瞬时转染EtBr诱导培养的肺癌细胞，在共聚焦显微镜下观察LC3的表达水平。随后，我们用Western Blot检测EtBr诱导培养细胞里自噬相关蛋白Beclin-1和LC3的表达情况。同时使用自噬抑制剂3-MA处理，观察EtBr诱导培养引发的自噬现象能否被抑制。另外，采用免疫荧光技术检测EtBr处理肺癌细胞中Beclin-1和PINK1的表达水平。研究结果：1.低剂量EtBr诱导培养能逐步敲除肺癌细胞mtDNA我们通过低剂量EtBr诱导培养的方法，能有效地逐步敲除肺癌细胞的线粒体DNA。实时定量PCR结果显示肺癌细胞线粒体DNA的两个标志物在EtBr诱导培养后显著下降；显微镜下可见细胞形态逐渐向长梭形改变。同时EtBr诱导培养后肺癌细胞对营养依赖性的检测发现，EtBr诱导培养7天的肺癌细胞在不含尿嘧啶的培养基中表现出增殖速度减慢，贴壁能力减弱，并逐渐漂浮死亡。因此，本文研究中使用的EtBr剂量能有效敲除肺癌细胞的线粒体DNA，需要补充外源性尿嘧啶维持细胞存活。2. EtBr诱导培养显著抑制肺癌细胞在体外、体内的生长体外细胞增殖、克隆形成及迁移实验表明EtBr诱导培养导致的肺癌细胞生长和迁移抑制与mtDNA缺失程度密切相关，即线粒体DNA缺失能影响肺癌细胞的生物学行为，但是并不会明显引发细胞早期凋亡事件。细胞周期检测表明EtBr诱导培养引发细胞周期G0-G1期阻滞。肺癌异种移植模型显示，EtBr预处理的肺癌细胞比对照组生长明显减慢，且与mtDNA缺失程度相关。另外，解剖后发现，与对照组相比，EtBr诱导培养后肺癌细胞体内发生转移的机率也明显降低。3. EtBr诱导培养过程中线粒体发生降解LTR、MTG双色荧光探针标记的共聚焦显微镜检测结果显示，EtBr诱导培养肺癌细胞的LTR与MTG共定位细胞器结构显著多于对照细胞。我们在经典的血清饥饿诱导培养模型中也观察到相同的结果。但是与血清饥饿组细胞相比，EtBr诱导培养引发的LTR和MTG共定位结构增多更显著。而在电镜下观察也发现EtBr诱导培养后肺癌细胞中的线粒体数量逐渐减少，且与mtDNA的缺失程度相关。另外，TMRM共聚焦结果和JC-1流式细胞检测均证实mtDNA逐步缺失过程中线粒体的膜电位显著下降。4. EtBr诱导肺癌细胞线粒体降解通过自噬途径完成在EtBr诱导培养的肺癌细胞中，观察到MTG标记的线粒体数量明显减少而LTR结构明显增多，并且更多MTG标记的线粒体移向LTR结构中。此外，EtBr诱导培养肺癌细胞中GFP-LC3蛋白的表达明显增多，且从细胞质中的弥散分布向点状聚集改变。Western Blot实验结果也表明在线粒体降解过程中，LC3-II的表达水平明显增加，此过程能被自噬抑制剂3-MA抑制。同时，在EtBr诱导的线粒体降解过程中， Beclin-1蛋白的表达水平也有明显增高，同样能被3-MA抑制。而免疫荧光检测也发现EtBr处理肺癌细胞中Beclin-1和PINK1的表达水平较对照细胞显著增高。结论：1.低剂量EtBr诱导培养可以有效敲除肺癌细胞的线粒体DNA，随着诱导培养时间增加逐步出现对尿嘧啶的依赖性，且诱导培养后细胞形态发生改变。EtBr诱导培养后肺癌细胞的增殖、克隆形成及迁移能力均减弱，且与mtDNA的缺失程度相关；在体内，经EtBr预处理的肺癌细胞的肿瘤形成能力及生长能力均减弱，同时EtBr诱导培养后肺癌细胞的转移潜能下降。2. EtBr诱导肺癌细胞mtDNA逐步缺失过程中伴随线粒体的降解，表现为线粒体膜电位下降和线粒体数量减少。在线粒体的降解过程中，自噬信号通路被激活，该过程能被自噬抑制剂3-MA抑制，因此我们认为自噬是溴化乙锭诱导肺癌细胞线粒体降解的机制之一，并可能受PI3K-Beclin1信号通路的调控。而Beclin-1和PINK1的高表达也表明线粒体是通过自噬途径被降解。更多还原

【Abstract】 IntroductionMitochondria, a type of double membrane-bound compartments, are highly essentialand dedicated organelles present in all eukaryotic cells. Mitochondria function as chemicalfactories for key metabolic reactions and energy generation, and as communication site fordiverse signaling pathways. However, the role of mitochondria goes beyond the above, theyare also the major source of endogenous reactive oxygen species (ROS), the side productsof oxidative phosphorylation. Excessive ROS is potentially deleterious and may causedamage to mitochondrial proteins, mtDNA and lipids, even lead to cell death by promotingthe intrinsic apoptotic pathway. In addition, mitochondria have been implicated inmitochondrial disorders, cardiac dysfunction, aging process and other human diseases.From this point, the quality and quantity of mitochondria need to be accurately controlledfor energy metabolism homeostasis and other key essential cellular processes. To date,several lines of evidence suggest that the selective degradation of mitochondria byautophagy controls mitochondrial number and health. Clearly, the link between autophagyand mitochondria is the selective removal of superfluous and damaged mitochondria, aprocess termed mitophagy.Autophagy is primarily supposed to be a protective process for the cell. Basal levels ofautophagy play critical role in maintaining normal cellular homeostasis by recycling ofintracellular components. Today, the role of autophagy has been extended up through tohuman diseases and physiology. For example, unregulated activation of autophagy likelycontributes to a broad spectrum of pathological processes, ranging from infections,neurodegeneration, heart disease to aging and cancer. Autophagy is firstly associated withcancer through the identification and characterization of the beclin1gene, which issuggested as a tumor suppressor. Moreover, several other autophagy genes are implicated in tumorigenesis, including MAP1-LC3(ATG8homolog) and HsGSA7gene (ATG7ortholog).Accordingly, Atg proteins, the products of Atg genes, are essential factors for the process ofautophagy. LC3(microtubule associated protein light chain3), a mammalianautophagosomal ortholog of yeast Atg8, is identified to form LC3-Ⅰ by cleaving the C-terminus of newly synthesized ProLC3. Then, an E2-like enzyme Atg7cleaves22aminoacids from the C-terminus to form LC3-Ⅱ, which is recruited to form autophagosomes andserves as an autophagic marker protein.Under normal conditions, basal levels of autophagy serve to maintain cellularhomeostasis by physiologically elimination of damaged organelles and long-lived proteins,and autophagy is activated in response to metabolic stress or energy crisis, such as nutrient,growth factor, and oxygen deprivation or mitochondria damage. We know that,mitochondria plays crucial role in cellular functions, including cell growth, division, andenergy metabolism. Obviously, these functions are also necessary for cancer cellproliferation, survival and migration. According to previous study, the rate of spontaneousdepolarization of mitochondrial membrane potential is increased under serum deprivation.Then these damaged mitochondria are demonstrated to move into acidic vacuoles, besequestrated and digested in autophagosomes and autolysosomes. During our establishmentof human lung cancer cell lines lacking mtDNA, progressive depopulation of mitochondriawas indentified. What’s important, autophagy was activated in the process. In the presentstudy, we try to indentify the involvement of autophagy in mitochondrial degradation, toexplore its molecular mechanisms and the potential impacts on biological behaviors of lungcancer cells.Methods1. Establishment and identification of lung cancer cells lacking mitochondrial DNAA549, SPC-A1and H322human lung cancer cell lines were maintained in mediumsupplemented with10%FBS and100ng/ml penicillin and streptomycin. The cells werepassaged when cells were90%~95%confluent. For EtBr treatment, cells were exposed to250ng/ml ethidium bromide for7days. The medium was additionally supplemented with50μg/ml uridine and100μg/ml pyruvate. The mtDNA content and the mRNA expressionof cytochrome c oxidase subunit II (COX II) were measured by quantitative real-time PCR.2. The effects of EtBr on tumor growth in vitro and in vivo Cells treated with EtBr for1,3,5or7days were trypsinized and counted. Cellproliferation assay, clonogenic assay and cell migration assay were carried out to evaluatethe ability of cell proliferation and migration after mtDNA knocked out. Additionally, cellcycle and cell apoptosis were investigated by PI staining and Annexin V-FITC flowcytometry, respectively. Finally, In vivo analysis of tumor growth was performed.NOD/SCID mice (five mice per group) were injected subcutaneously in the left flank with-treated cells suspended in200μl phosphate-buffered saline (PBS). Tumor volume wasmeasured with calipers twice a week for6weeks, after which the mice were sacrificed.Tumors were removed and photographed.3. Investigation of mitochondrial degradation after EtBr treatmentFirstly, confocal microscopy was used to identify the mitochondrial degradation bylysosome. The treated cells were co-loaded with200nM MitoTracker Green and200nMred-fluorescing LTR for20min. After fluorescence loading, cells were washed thrice withfresh PBS. Confocal images were collected at2μm intervals. Secondly, confocalmicroscopy and flow cytometry were employed to verify loss of mitochondrial membranepotential. Lastly, mitochondrial degradation during EtBr treatment was further studied withtransmission electron microscopy.4. Mechanism study of EtBr-caused mitochondrial degradationConfocal microscopy and transmission electron microscopy were carried out toidentify the autophagic structures in EtBr treated cells. The cells were transfected with GFP-LC3, and then the distribution pattern was studied by confocal microscopy. Then autophagyrelated protein expression of Beclin-1and LC3was detected. We also examined whether3-MA, an autophagy inhibitor, could inhibit EtBr-induced autophagy or not, which could helpus understand better of the EtBr caused mitochondrial degradation pathways. Finally, weuse immuno-fluoresence to detect the expression level of Beclin-1and PINK1.Results1. Success in establishing lung cancer cells lacking mtDNAWe have succeeded in establishing the lung cancer cells lacking mitochondrial DNA.The PCR results demonstrated that these two markers significantly decreased in a time-dependent manner after EtBr treatment. Confocal of TMRM loading, used for detectingpolarized mitochondria, showed that EtBr-caused decrease of mitochondrial membrane potential (MMP) was in a time-dependent manner, which was consistent with the PCRresults. In other words, EtBr can knock down the mitochondrial gene.2. EtBr inhibits lung cancer cells growth in vitro and in vivo.In vitro cell proliferation, clonogenic and migration assays demonstrated that EtBrinhibited lung cancer cell growth and migration in a time-dependent manner, but there wasno significant increase in apoptotic events in EtBr-treated cells. The PI staining resultsdemonstrated that EtBr-treated cells underwent cell cycle arrest. In vivo, EtBr-treated cellsgrew more slowly than untreated control cells in lung cancer xenograft models.3. Confocal microscopy and TEM revealed mitochondrial degradationDual-labeled confocal microscopy revealed that LTR and MTG co-localizationstructures significantly increased in EtBr-treated lung cancer cells, in contrast to the controlgroup. Similar observation was evidenced in cells under starvation. Compared withstarvation, EtBr treatment more dramatically increased the co-localization of MTG-andLTR-positive structures. In addition, the numbers of mitochondria in cells treated with EtBrwere discovered to be less compared to the control cells with TEM.4. Mitochondrial degradation in the presence of EtBr was completed via autophagyAfter EtBr treatment, an increasing of LTR was observed with the decreasing ofmitochondria. Also, more MTG-labeled mitochondria were identified to move into the LTRstructures after EtBr treatment. Further, GFP-LC3expression level was dramaticallyincreased after the treatment, which distributed as green dots. Then the recruitment of LC3-II to autophagosomes during mitochondrial degradation was further identified by westernblot analysis, and the process could be inhibited by3-MA. Besides, an obvious increased ofBeclin-1protein was verified by WB. Besides, the expression level of Beclin-1and PINK1were shown to be increased via immuno-fluorescence.Conclusions1. We have succeeded in establishing lung cancer cells lacking mtDNA with a lowconcentration of EtBr, which was identified by PCR results of mtDNA content and mRNAexpression level of cytochrome c oxidase subunit II. EtBr treatment led to decreased cellproliferation, colony formation and migration in vitro. Tumor suppression effect of EtBrtreatment was also observed in lung cancer xenograft model. 2. Imaging method identified progressive depopulation of mitochondria during EtBrtreatment, accompanied by increased LTR uptake and co-localization of LTR-and MTG-positive structures. We conclude that autophagy is responsible for mitochondrialdegradation when exposed to a low concentration of EtBr, most likely through the PI3K-Beclin1pathway. The high expersson level of Beclin-1and PINK1detected by immuno-fluorescence also demonstrated they were involved in the process of autophagic degradationof mitochondria.更多还原