【作者】 周勇；

【导师】 应可净；

【作者基本信息】 浙江大学 ， 内科学呼吸病， 2007， 硕士

【摘要】 研究背景和目的：肺癌是目前世界上最常见的男性恶性肿瘤，占全部恶性肿瘤的16％，恶性肿瘤死亡的28％，在大多数国家已经成为癌症死因的首位。近三年来，肺癌的分子靶向治疗发展迅速，为肺癌的治疗带来了新的曙光。如最近获准美国FDA认证并上市的EGFR(表皮生长因子受体)酪氨酸激酶抑制剂IRESSA和TARCEVA，通过高选择性地与EGFR结合，抑制酪氨酸激酶活性，影响肿瘤细胞生长、增殖和分化。这种针对异常的信号传导通路的药物比传统药物具有更好的疗效和更低的毒性，使之成为抗肿瘤治疗的靶点药物。但是Ⅱ期临床试验的有效率分别仅为11.8％-18.4％和12.5％。所以，仍然需要寻找更为有效和特异的治疗方法。PLK1(polo-like kinase 1)是人体内类似于黑腹果蝇(Drosophila melanogaster)体内的polo、啤酒酵母体内的CDC5的丝氨酸／苏氨酸激酶家族，目前研究表明，它与有丝分裂过程中的纺锤体形成及染色体的分离有密切的关系，已经成为细胞周期检控点(cell cycle checkpoint)的主要调节者，许多细胞周期关键的调节分子，如Cdc25C、cyclinB、APC、p53、有丝分裂动力蛋白，都是PLK1的直接作用对象，PLK1的高表达和肿瘤的发生、发展及预后呈现显著的相关性。许多研究表明，抑制PLK1的表达可以显著的抑制肿瘤的生长，促进肿瘤细胞凋亡，而对正常细胞的增殖影响极少。因此，抑制PLK1的功能将成为肿瘤治疗的一项重要手段。短(小)干扰RNA(short or small interfering RNA，siRNA)技术已被认为是近年来生物技术中最重大的发现，成为一种抑制哺乳动物特定的基因表达研究的重要方法。其最令人振奋和感兴趣的是它在抑制肿瘤“关键基因”显示的治疗前景。阻止癌细胞增殖和促进凋亡是肿瘤基因治疗的最基本规则，目前研究表明将siRNA转入肿瘤细胞内，通过使靶基因mRNA沉默，从而达到定向“敲除”与肿瘤细胞发生发展相关的基因，在实践中是可行的。本研究的目的就是探索靶向性的siRNA对肺癌A549细胞有丝分裂调控基因PLK1基因表达的沉默效果。本实验采用siRNA干预肺癌A549细胞PLK1基因的表达，观察其抑制效果，从而探寻具高度活性的针对A549细胞PLK1的siRNA及其最佳转染途径，为下一步siRNA用于体内抑制肺癌生长研究奠定实验基础。研究方法：根据siRNA设计原则，设计并人工化学合成针对肺癌A549细胞PLK1 mRNA的高纯度siRNA，用阳离子脂质体(lipofectamine2000)包裹，以不同浓度转染A549细胞。转染后24小时，48小时，72小时分别采用实时定量PCR同时检测PLK1 mRNA和GAPDH mRNA，并以内参照GAPDH的mRNA相对定量PLK1 mRNA表达水平；Western-blot同时观察PLK1和GAPDH蛋白表达的变化。统计学方法两组间的比较采用two-way ANOVA检验，所有数据统计在stata 8.0 for windows软件上完成。结果：siRNA可不同程度的抑制PLK1基因的表达，在分别以25nmol／L和50nmol／L浓度转染后24小时即可检测到mRNA的丰度明显下降到25％和23.1％，48和72小时继续下降到17.5％和19.5％，13.4％和8.4％；转染后48小时开始可检测到PLK1蛋白表达明显降低，转染后72小时仍有持续的抑制效果，其表达水平低于检测低限，而GAPDH蛋白表达水平不受影响。采用nonsense siRNA的对照组PLK1基因表达亦不受抑制。结论：1) siRNA干预PLK1基因表达后，PLK1 mRNA水平明显下降，而GAPDH mRNA无明显变化，提示siRNA具有靶向性；2) siRNA在25nmol浓度即可显著抑制PLK1 mRNA达91.6％(72小时)，提示siRNA的作用具有高效性和一定的持久性；脂质体(lipofectamine2000)作为一种siRNA转染载体，转染肺癌A549细胞效率高；3)本研究的siRNA对肺癌A549细胞沉默效果优于其他RNAi(RNA interference)文献的研究结果(最高抑制83％)，可能与靶位点的选择和转染载体的差异有关；4) 25nmol和50nmol两组抑制效果无统计学差异，提示可进一步降低浓度观察观察和比较干预效果；5)干预后PLK1 mRNA仅表现为表达水平的下调，但蛋白水平已低于检测低限，可能与siRNA的存在翻译水平的抑制机制(translation gene silencing，TGS)有关。6)本实验的siRNA可作为肺癌A549细胞荷瘤动物体内RNAi研究的候选siRNA，在进行相应甲基化等修饰增强血清稳定性后，经整体应用，观察其对体内肿瘤生长抑制效果。更多还原

【Abstract】 Backgrounds and Objective:Lung cancer ranks the most common malignant disease in male patients, it takes about 16% of all malignant disease, and 28% of all death of malignancy. It’ s the first cause of cancer in most countries in the world.In recent years, molecular targeting therapy of lung cancer has been developed rapidly, such as IRESSA and TARCEVA. They can selectively combine to EGFR and inhibit protein tyrosine kinase(PTK) activities, thus inhibit cancer growth. Compared with traditional drugs, they are more effective and have lower toxicity. But in the phase II clinical trail, the efficacy ratio was only 11.8%-18. 4% and 12. 5%. Therefore more researches are badly needed.Polo and Polo-like kinases (Plks) are a family of conservedregulators of multiple events during cell division. The founding member of this family, Polo, was originally identified in the fruit fly(Drosophila melanogaster) and shown to be a serine - threonine kinase that is required for mitosis. Protein kinases play a pivotal role in execution of cell division. Polo and Polo-like kinases have emerged as major regulators for various cell cycle checkpoints. Plks localize primarily to the centrosome during interphase and the mitotic apparatus during mitosis. Many key cell cycle regulators such as p53, Cdc25C,cyclin B, components of the anaphase-promoting complex(APC), and mitotic motor proteins are directly targeted by Plks. Plks are important mediators for various cell cycle checkpoints that monitor centrosome duplication, DNA replication, formation of bipolar mitotic spindle, segregation of chromosomes, and mitotic exit, thus protecting cells against genetic instability during cell division. Many researches have proven that when PLK1 gene expression is inhibited, malignant tumors stop growing to apoptosis, while normal cells growth rarely affected. In conclusion, inhibiting PLK1 gene expression may be an important method of malignancy cure. SiRNA(short or small interfering RNA) is believed to be the most important discovery in biotechnological progresses of recent years, and has now been adopted as a standard methodology for silencing the expression of specific genes in mammalian cells. The potential of siRNA based cancer therapy, that is, inhibiting cancer proliferation via targeting and silencing "key genes" of malignancy, is the most exciting and interesting one. Many studies have used siRNAs as an experimental tool to dissect the cellular pathways that lead to inhibit cell proliferation and RNAi has been proposed as a potential treatment for cancer.We aim at studying the knockdown effect of small interfering RNA(siRNA) targeting PLK1 (Polo-like kinase1) mRNA in lung cancercell line A549, and identifying the hyperactive siRNA targeting PLK1 and noval transfection ways for future in vivo RNAi experiments targeting PLK1 mRNA and inhibiting lung cancer growth in animal.Methods:Special siRNA molecules were designed targeting PLK1 mRNA sequence and chemically synthesized, that were transfected into A549 via lipofectamine2000. PLK1 and house keeping GAPDH mRNA levels were assayed by Real-Time PCR 24hours, 48hours and 72hours after transfection, and the expression levels of PLK1 mRNA were quantified based on the GAPDH mRNA levels. The changes of PLK1 and GAPDH protein levels, were checked by Western-blot.Statistical methods:Two-way ANOVA was adopted for comparation between two groups. All data were subjected to stata 8.0 for windows.Results:The siRNA molecules, on concerntrations of 50nmol/L and 25nmol/L, knocked PLK1 mRNA down at different levels, remaining 25-23. 1%, 17. 5-19. 5% and 13. 4%-8. 4%, 24hous, 48hours and 72hours after transfection respectively. 48hours after transfection, Western-blot detected dominantly decreased levels of PLK1 protein, but the GAPDH protein levels remained intact. Nonsense siRNA , as a negative control, didn’ t affect PLK1 mRNA levels.Conclusions:1) SiRNA down regulated PLK1 mRNA apparently, but not GAPDH mRNA, suggesting siRNA’ s targeting affects.2) SiRNA at 25nmol/L knocked down PLK1 mRNA level by 91.6% 72 hours after transfection, proving it’ s hyperactivity and durability; Lipofectamine2000, as a transfection vehicle forA549 cell RNAi, has high efficacy.3) The study got the highest knocking down RNAi efficacy targeting A549 cell PLK1 mRNA, while compared with published literature(83%),which probably is due to the differences of targets selection and transfection vehicles.4) There was no knocking down efficacy difference between 25nmol/L and 50nmol/L groups,which calling for further experiments on RNAi at lower concentrations.5) We observed down regulation of PLK1 mRNA after RNAi,but the PLK1 protein level was undetectable, which probably due to the translation gene silencing(TGS).6) This siRNA can be a candidate, after methylation modifications to increase its serum stability, for in vivo RNAi research against tumour growth of A549 cell bearing nude mouse.更多还原