【作者】 张玲；

【导师】 韩俊庆；

【作者基本信息】 山东大学 ， 肿瘤学（专业学位）， 2017， 博士

【摘要】 研究背景及意义乳腺癌是乳腺上皮细胞发生癌变的恶性肿瘤,在世界上严重威胁人类健康。在发达国家及地区,患乳腺癌的概率及其死亡率均居女性恶性肿瘤之首。近年来乳腺癌治疗方式不断进展,由手术、化疗、放疗、内分泌治疗、分子生物靶向治疗和中医药治疗结合的综合治疗已成为当前治疗乳腺癌的主流模式,乳腺癌的治疗疗效不断提高,已成为预后较好的实体肿瘤之一。但是,目前全球乳腺癌的发病率每年都在增加,在中国,乳腺癌的发病率在女性疾病中占据第一位,并且有年轻化的趋势,成为危害女性身心健康的头号杀手。因此防治乳腺癌具有重要的意义。近年来,在乳腺癌分子代谢重编方面的研究有了一些进展。例如,在乳腺癌中,丙酮酸脱氢酶激酶1(PDK1)能通过调节低氧诱导因子1 α来调节乳腺癌的增殖。支链氨基酸(BCAAs),主要有亮氨酸、异亮氨酸及缬氨酸,是重要的营养信号,对机体活动有直直接和间接的影响。血浆中BCAAs水平和心血管疾病的发生有关,并且血清中高水平BCAAs和人类未来糖尿病有极大的联系,这可能是因为BCAAs能影响胰岛素耐受性。BCAAs分解代谢缺陷也会引起疾病。例如,在血脑屏障期受损的氨基酸运输能降低脑链氨基酸的正常水平,并且诱导支链氨基酸的代谢缺陷。另外,支链氨基酸分解代谢缺陷是心力衰竭的代谢标志,能够促进心力衰竭。支链氨基酸还参与癌症的发生及发展。血浆中,支链氨基酸的高水平增加胰腺癌发生的两倍风险。支链氨基酸转氨酶1(BCAT1)能够激发支链氨基酸的分解代谢,在携带异柠檬酸脱氢酶1的人类神经胶质瘤中,BCAT1能够通过氨基酸代谢促进神经胶质瘤细胞增殖。在非小细胞肺癌中,BCAT1和BCAT2的损失能够破坏非小细胞肺癌的形成。BCAT1可能通过调控线粒体的合成及功能影响乳腺癌的发生和发展,线粒体的生物合成和功能与许多因子有关,如过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1 α)、呼吸因子(NRF-1)、线粒体转录因子A(TFAM)、超氧化物歧化酶(SOD)、过氧化氢酶以及活性氧(ROS)。然而,支链氨基酸分解代谢在乳腺癌血液及组织中的作用仍未知。本次研究支链氨基酸代谢在人类乳腺癌中的潜在作用。本研究共分为四个部分,第一部分,收集山东大学附属省立医院2010到2015年共计200例乳腺癌患者及100例正常人。应用LC-MS、实时定量PCR检测支链氨基酸及核心酶在肿瘤患者血浆及肿瘤组织中的表达。第二部分,采用shRNA和siRNA技术干扰人乳腺癌细胞系MCF-7/T47D中BCAT1的表达,观察干扰BCAT1对MCF-7/T47D细胞增殖、克隆形成的影响。第三部分,采用实时定量PCR、分光光度法、mitoSOX染色法等检测线粒体生物合成的调节因子表达、柠檬酸合酶活性、细胞ATP水平、线粒体ROS及抗氧化剂表达,来探究BCAT1对线粒体生物合成及功能的影响。第四部分,通过检测mTOR的蛋白表达、线粒体DNA含量、线粒体ROS,探究BCAT1影响乳腺癌细胞增殖速度及克隆能力的分子机制。第一部分支链氨基酸及核心酶在乳腺癌中的表达研究目的:1、探究支链氨基酸在乳腺癌病人血液及癌组织中的水平。2、探究影响支链氨基酸代谢的核心酶表达。研究方法:1.选取在2010到2015年于山东大学附属省立医院进行手术的200例乳腺癌病人以及100例健康志愿者。收集其血浆、肿瘤组织和癌旁无肿瘤组织,-80℃保存组织标本以备检测;2.采用液相色谱-质谱联用的方法测定支链氨基酸在乳腺癌患者及健康志愿者中的血浆水平图:3.检测乳腺癌患者和正常人的血浆中、乳腺癌患者肿瘤组织和癌旁无肿瘤组织标本中亮氨酸、异亮氨酸和缬氨酸的表达;4.采用实时定量 PCR检测支链氨基酸转氨酶1在乳腺癌患者及健康志愿者中的表达;研究结果:1.与正常捐赠者相比,亮氨酸、异亮氨酸和缬氨酸在乳腺癌患者血浆中的表达明显上调,差异具有统计学意义(p<0.05);2.乳腺癌患者肿瘤组织中的亮氨酸、异亮氨酸和缬氨酸水平显著高于癌旁非肿瘤组织(p<0.05);3.乳腺癌患者肿瘤组织中的支链氨基酸转氨酶1表达显著高于癌旁非肿瘤组织(p<0.05);结论:总的来说,支链氨基酸水平及支链氨基酸降解酶在乳腺癌患者中的表达水平显著升高(p<0.05),这表明在乳腺癌组织中,支链氨基酸代谢被激活,从而有助于制定乳腺癌的治疗方案及预后。第二部分干扰BCAT1表达对乳腺癌细胞增殖及克隆能力的影响研究目的:1.探究BCAT1过表达对乳腺癌细胞增殖及克隆形成的影响;2.探究干扰BCAT1表达对乳腺癌细胞增殖及克隆形成的影响;研究方法:1.将购买好的转染质粒到转染到乳腺癌细胞中,空白表达的质粒作为对照组,构建BCAT1沉默组(shBCAT1组)、对照组(shCtrl组)、BCAT1过表达组及对照组(Ctrl组),采用蛋白印记验证质粒转染成功;2.采用CCK-8比色法探究BCAT1过表达和沉默对人乳腺癌细胞系MCF-7和T47D细胞增殖能力的影响。3.采用平板克降形成实验探索BCAT1过农达和沉默对MCF-7和T47D细胞系的细胞克降形成能力的影响;研究结果:1.蛋白印迹显示,转染72小时后,与对照组相比,shBCAT1组人乳腺癌细胞系MCF-7和T47D中BCAT1的蛋白表达水平显著降低(P<0.05),而BCAT1组人乳腺癌细胞系MCF-7和T47D中BCAT1的蛋白表达水平显著升高(P<0.05)。2.CCK-8增殖实验显示,与shCtrl组相比,从24h开始,shBCAT1组增殖活性开始明显下降,差异具有统计学意义(P<0.05)。克隆形成实验显示,与shCtrl组相比,sh BCAT1组的克隆细胞数显著降低,差异具有统计学意义(P<0.05)。3.CCK-8增殖实验显示,与Ctrl组相比,从24h开始,BCAT1组增殖活性开始明显升高,差异具有统计学意义(P<0.05)。克隆形成实验显示,与Ctrl组相比,BCAT1组的克隆细胞数显著增加,差异具有统计学意义(P<0.05)。结论:1.病毒及质粒感染能够有效上调和下调BCAT1的表达;2.BCAT1过表达能够提高乳腺癌细胞的增殖及克隆形成,BCAT1沉默能够降低乳腺癌细胞的增殖及克隆形成;第三部分BCAT1调节线粒体的生物合成及功能研究目的:1.探究BCAT1在调节线粒体生物合成中的作用2.探究BCAT1对线粒体生物功能的影响研究方法:1.通过测定人乳腺癌细胞系MCF-7和T47D中线粒体DNA含量,探究BCAT1能否调节线粒体生物合成及功能;2.采用实时定量 PCR检测Ctrl组、BCAT1组、shCtrl组、shBCAT1组中线粒体生物合成关键基因-过氧化酶体增生物激活受体、核呼吸因子1、线粒体转录因子 A、β3-F1型ATP合成酶-的mRNA表达;3.采用分光光度法检测shCtrl组、shBCAT1组人乳腺癌细胞系MCF-7和T47D中的柠檬酸合酶活性;4.采用mitoSOX染色检测shCtrl组、shBCAT1组人乳腺癌细胞系MCF-7和T47D中线粒体ROS;5.采用实时定量 PCR检测shCtrl组、shBCAT1组中超氧化物歧化酶1.超氧化物歧化酶2.过氧化氢酶和谷胱甘肽过氧化物酶抗的mRNA表达;研究结果:1.BCAT1沉默降低MCF-7/T47D细胞系中的线粒体DNA含量,而BCAT1过表达增加了MCF-7/T47D细胞系中的线粒体DNA含量(p<0.05);2.BCAT1沉默降低过氧化酶体增生物激活受体、核呼吸因子1、线粒体转录因了A、β-F1型ATP合成酶的mRNA水平,BCAT1过表达导致相反的结果;3.BCAT1沉默降低柠檬酸合酶活性水平,抑制超氧化物歧化酶1,超氧化物歧化酶2.过氧化氢酶和谷胱甘肽过氧化物酶mRNA水平,并且诱导线粒体ROS的增加;结论:1.BCAT1能够通过增加线粒体数目、提高影响线粒体生物合成的调节因子来影响线粒体的生物合成;2.BCAT1能通过提高柠檬酸合酶活性、抗氧化剂-超氧化物歧化酶1,超氧化物歧化酶2,过氧化氢酶和谷胱甘肽过氧化物酶-的mRNA水平以及抑制线粒体ROS来影响线粒体功能;第四部分BCAT1对乳腺癌细胞系mTOR的影响研究目的:1.探究BCAT1对乳腺癌组织中关键蛋白AMPK和mTOR磷酸化水平的影响。2.探究受BCAT1影响的mTOR对线粒体功能及乳腺癌细胞增殖的影响。研究方法:1.采用蛋白印迹法检测shCtrl组、shBCAT1组p-AMPK、AMPK、SIRT1的蛋白表达,并使用蛋白印迹法测定shCtrl组、shBCAT1组及Ctr1组、BCAT1组 p-mTOR、mTOR、p-S6K1 和 S6K1 的蛋白表达;2.使用雷帕霉素抑制乳腺癌细胞系中的mTOR,并采用实时定量PCR检测BCAT1过表达乳腺癌细胞系中 PGC1α、NRF-1、Tfam、β-F1-ATPase、SOD1、SOD2、过氧化氢酶和Gpx1的mRNA水平;3.测定 Ctrl+Vechicle 组、BCAT1+Vechicle 组、Ctrl+Rapamycin 组、BCAT1+Rapamycin组乳腺癌细胞系中的线粒体DNA含量、线粒体ROS、细胞克隆数和细胞增殖情清况。研究结果:1.BCAT1沉默不会影响AMPK和SIRT1的蛋白表达、mTOR信号,BCAT1过表达激活mTOR信号;2.雷帕霉素能抑制mTOR信号,而mTOR通路被抑制的人乳腺癌细胞中,BCAT1过表达不会影响PGC1α、NRF-1、Tfam、β-F1-ATP酶、SOD1、SOD2、过氧化氧酶和Gpx1的mRMA水平;3.mTOR通路被抑制能够阻止BCAT1对线粒体生物合成及线粒体ROS的影响;4.mTOR通路被抑制能够减弱BCAT1对乳腺癌细胞增殖速度及克隆形成能力的促进作用。结论:1.BCAT 1通过调节mTOR信号影响线粒体功能;2.mTOR有助于BCAT1对乳腺癌细胞增殖的促进作用。更多还原

【Abstract】 Background and objectiveBreast cancer is a malignant tumor and resulted from the canceration of mammary gland epithelial cells.It is one of the malignant tumors that seriously threating the health of women in the world.In developed countries and regions,the morbidity and mortality of breast cancer are among the top of the female malignant tumors.In recent years,treatments for breast cancer have a perpetual progress,and molecular targeted therapy and comprehensive treatment combined by traditional Chinese medicine have become the mains pattern for the treatment of breast cancer.The continuous improvement of treatment on breast cancer has a good prognosis.In china,the incidence of breast cancer has become the first female malignant tumor and has become the top killer of threat to the health of women.Therefore,prevention and treatment on breast cancer is of great significance.Recently,the studies of metabolic reprogramming in human breast cancer have achieved some progress.For instance,pyruvate dehydrogenase kinase-1(PDK1)regulates hypoxia-inducible factor-la(HIF-1α)to trigger metabolic reprogramming and dictates metastatic potential in breast cancer.Branched-chain amino acids(BCAAs),including leucine,isoleucine,and valine,are important nutrient signals that have direct and indirect effects.Plasma BCAAs level is associated with the incident of cardiovascular diseases.In addition,the high serum level of BCAAs has highly significant associations with future diabetes in human,which may due to the effects of BCAAs on insulin resistance.Defects in BCAAs catabolism also causes diseases.For instance,impaired amino acid transport at the blood-brain barrier decreases normal levels of brain BCAAs and induces the metabolic defect in BCAAs.In addition,the BCAA catabolic defect is a metabolic hallmark of failing heart.BCAAs catabolic defect imposes a significant contribution to heart failure.BCAAs also participates in the development of cancer.Elevated plasma levels of BCAAs in humans are associated with a greater than two-fold increased risk of future pancreatic cancer diagnosis,which was confirmed in rodent models.Branched-chain amino acid transaminase 1(BCAT1)is the enzyme that initiates the catabolism of BCAAs.In human gliomas carrying wild-type isocitrate dehydrogenase 1,BCAT1 promotes cell proliferation through amino acid catabolism.In non-small cell lung carcinoma(NSCLC),tumors incorporate free BCAAs into tissue protein and use BCAAs as a nitrogen source.Loss of BCAT1 and BCAT2 impairs NSCLC tumor formation.However,the role of BCAAs metabolism in human breast cancer remains unknown.In this paper,we aimed to explore the potential role of BACC metabolism in human breast cancer.The whole dissertation can be divided into four parts.In the first part,we collected twenty human patients with breast cancer and ten healthy donors from 2010～2015 at Shandong Provincial Hospital Affiliated to Shandong University.We performed LC-MS and RT-PCR to explore the expression of BACC and key enzymes in serum and tissues of breast cancer patients.In the second part,we used shRNA and siRNA transfection technique to disturb the expression of BCAT1 in breast cancer cell lines MCF-7 and T47D.The influence of BCAT1 after transfection on the proliferation and cloning formation was observed.In the third part,we used RT-PCR.spectrophotometry and mitoSOX staining to determine the expression of regulators of mitochondrial biogenesis,the activity of citrate synthase,cellular ATP level,mitochondrial ROS and antioxidants to explore the influence of BCAT1 on mitochondrial biogenesis and function.In the fourth part,we explored the mechanism about the effect of BCAT1 on the proliferation and cloning formation of breast cancer cells by determining the expression of mTOR,mitochondrial DNA content and mitochondrial ROS.Part IExpression of BACC and core enzyme in breast cancerObjective:1.To explore the level of BACC in serum and tissues of breast cancer patients.2.To explore the expression of core enzymes involved in BCAAs metabolism.Methods:1.Two hundred cases of breast cancer patients and one hundred of healthy volunteers were collected from 2010 to 2015 at the Provincial Hospital Affiliated to Shandong University.The plasma,tumor tissues and the adjacent tissues were collected from breast cancer patients and then are kept at-80℃.2.Liquid chromatography-mass spectrometry(LC-MS)was used to determine the profile of plasma level of amino acids in breast cancer patients and normal people.3.The expression of leucine,isoleucine,and valine in plasma of breast patients and the healthy volunteers,and in tumor tissues and the adjacent tissues of breast cancer patients.4.RT-PCR was used to determine the expression of BCAT1 in breast cancer patients and healthy volunteers.Results:1.Compared with the normal,the expression of leucine,isoleucine,and valine in breast cancer patients was significantly upregulated and the difference is statistically significant(p<0.05).2.The plasma levels of leucine,isoleucine,and valine in breast cancer patients was significantly higher than the normal(p<0.05).3.The expression of leucine,isoleucine,and valine in tumor tissues was higher than the adjacent tissues of breast cancer patients(p<0.05).4.The expression levels of BCAT1 in tumor tissues was higher than the adjacent tissues of breast cancer patients(p<0.05).Conclusions:In conclusion,the expression of BACC and BCAT1 was increased markedly in breast cancer patients(p<0.05),which indicated BCAAs metabolism was activated in human breast cancer tissues.Part ⅡInterference on the expression BCAT1 influences the growth and cloning formation of breast cancer cellsObjective:1.To explore the influence of BCAT1 overexpression on the proliferation and cloning formation in breast cancer.2.To explore the influence of interference on BCAT1 expression on the proliferation and cloning formation in breast cancer.Methods:1.Purchased transfection plasmid was transfected to breast cancer cells and blank plasmid was taken as the control.We constructed BCAT1 silent group(shBCAT1),control group(shCtrl),BCAT1 overexpression group(siCtrl)and control group(Ctrl).Western blotting was used to determine the transfection result.2.CCK-8 assay was used to determine the effect of BCAT1 overexpression and knockdown on the proliferation of breast cancer cells.3.Cloning formation assay was used to determine the influence of BCAT1 overexpression and knockdown on cloning formation of’ MCF-7 and T47D cells.Results:1.Results of western blotting showed the protein expression level of BCAT1 in MCF-7and T47D cell lines in shBCAT1 group was effectively decreased compared with control group(P<0.05)after transfection for 72h.However.the protein expression level of BCAT1 in MCF-7and T47D cell lines in BCAT1 group was effectively increased(P<0.05)2.Results of CCK-8 showed the proliferation capacity of MCF-7and T47D cell lines began to decrease dramatically at 24h compared with shCtrl group(P<0.05).Results of cloning formation assay showed the number of cloning cells in shBCAT1 group reduced significantly compared with shCtrl group(P<0.05).3.Results of CCK-8 showed the proliferation capacity of breast cancer cells in BCAT1 group began to increased dramatically at 24h compared with Ctrl group(P<0.05).Results of cloning formation assay showed the number of cloning cells in BCATI group increased significantly compared with Ctrl group(P<0.05).Conclusions:1.Lentivirus and plasmid transfection could effectively upregulate or downregulate the expression of BCAT1.2.BCAT1 overexpression could promote the proliferation and cloning formation of breast cancer cells,while BCAT1 knockdown could inhibit the proliferation and cloning formation of breast cancer cells.Part ⅢBCAT1 regulates mitochondrial biogenesis and functionObjective:1.To explore the role of BCAT1 on mitochondrial biogenesis.2.To explore the role of BCAT1 on mitochondrial function.Methods:1.To explore the role of BCAT1 on mitochondrial biogenesis and function by determining mitochondrial DNA content in breast cancer cell lines.2.RT-PCR was used to determine the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC1α),nuclear respiratory factor 1(NRF-1),mitochondrial transcription factor A(TFAM).and β-F1-ATPase in Ctrl,BCAT1,shCtrl and shBCAT1 group.3.Spectrophotometry was used to determine the activity of citrate synthase in shCtrl and shBCAT1 group.4.mitoSOX staining was used to determine mitochondrial ROS in breast cancer cell lines MCF-7/T47D in shCtrl and shBCAT1 group.5.RT-PCR was used to determine the mRNA levels of antioxidants(SOD1,SOD2,catalase,and Gpxl)in shCtrl and shBCAT1 group.Results:1.BCAT1 knockdown decreased mitochondrial DNA content,while BCAT1 overexpression increased mitochondrial DNA content in MCF-7and T47D cell lines2.BCAT1 knockdown decreased the mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC1α),nuclear respiratory factor 1(NRF-1),mitochondrial transcription factor A(TFAM),and P-F1-ATPase,while BCAT1 overexpression has the opposing results.3.BCAT1 knockdown decreased the activity of citrate synthase,reduced cellular ATP level,repressed the mRNA level of antioxidants(SOD1,SOD2,catalase,and Gpx1),and induced mitochondrial ROS.Conclusions:1.BCAT1 could affect mitochondrial biogenesis by increasing mitochondrial number and enhancing regulators.2.BCAT1 could affect mitochondrial function by increasing the activity of citrate synthase,cellular ATP level,mitochondrial ROS,antioxidants(SOD1,SOD2,catalase,and Gpx1)and mitochondrial ROS.Part IVInfluence of BCAT1 on mTOR in breast cancer cellsObjective:1.To explore the influence of BCAT1 on phosphorylation of AMPK and mTOR.2.To explore the role of mTOR affected by BCAT1 on the proliferation and cloning formation of breast cancer cells.Methods:1.Western blotting assay was used to determine the protein expression of AMPK,SIRT1,mTOR and S6K1 in shCtrl and shBCAT1 group.We also used western blotting assay to determine the protein expression of ρ-mTOR.mTOR.ρ-S6K1and S6K1 in shCtrl,shBCAT1,Ctrl and BCAT1 group.2.Rapamycin was used to inhibit the expression of mTOR in breast cancer cell lines.RT-PCR was used to determine the mRNA levels of PGC1α,NRF-1,Tfam,β-F1-ATPase.SOD1,SOD2,catalase,and Gpx1.3.Mitochondria DNA content,mitochondria ROS.number of cloning cells and situation of cell proliferation in breast cancer cell lines in Ctrl+Vechic.BCAT1+Vechicle,Ctrl+Rapamycin and BCAT1+Rapamycin group was determined.Results:1.BCAT1 knockdown had no influence on the expression of AMPK.SIRT1,mTOR and S6K1.2.Rapamycin could inhibit mTOR signaling,while BCAT1 overexpression had no influence on PGC1α,NRF-1,Tfam,β-F1-ATPase.SOD1,SOD2,catalase,and Gpx1,.3.Inhibition on mTOR could prevent the effect of BCAT1 on mitochondrial biogenesis and mitochondrial ROS.3.Inhibition on mTOR could weaken the promotion of BCAT1 on the proliferation and cloning formation of breast cancer cells.Conclusions:1.BCAT1 regulated mitochondrial biogenesis by mTOR signaling.2.mTOR contributed to the effect of BCAT1 on breast cancer cell growth.更多还原