【作者】 杨晓军；

【导师】 张有成； Prof.Lin Zhang；

【作者基本信息】 兰州大学 ， 外科学， 2010， 博士

【摘要】 背景与研究目的甲胎蛋白(Alpha-fetoprotein, AFP)发现至今已有40年的历史,目前仍作为肿瘤尤其是肝细胞癌(Hepatocellular carcinoma, HCC)的重要分子标记用于临床诊断、治疗及预后判断。近年的研究表明,在HCC的发生发展过程中,AFP绝非仅仅是一种伴随的癌胚蛋白,而是在HCC的增殖和凋亡中发挥着主要作用,但目前对AFP的具体功能和作用机制仍存在较多争议。本研究设计合成靶向AFP基因的StealthTM RNAi,转染高表达AFP的肝癌细胞株Huh7,有效沉默AFP基因的表达,研究AFP在肝癌细胞中的促增殖作用和凋亡抵抗效应,并探讨其凋亡抵抗效应的相关机制,为HCC的基因靶向治疗提供理论支持。方法1.电化学发光法,放免法以及ELISA检测AFP在肝癌细胞系HepG2, Huh7, SMMC7721以及肝正常细胞株Changliver中的表达水平。2.设计合成三条靶向AFP基因的StealthTM RNAi,采用脂质体转染肝癌细胞系Huh7。采用RT-PCR检测mRNA水平,ELISA和Western blot检测蛋白水平AFP基因的表达水平变化,以确定转染条件和筛选最佳干扰序列。3.选择最佳StealthTM RNAi沉默Huh7中AFP基因的表达,采用MTT比色法检测各组细胞的增殖活性；Hochest染色法和透射电镜检测细胞的凋亡情况；并用流式细胞仪进行凋亡率检测。4. Western blot检测凋亡信号通路的相关蛋白水平,探讨沉默AFP基因后诱导细胞凋亡的潜在机制。结果1.AFP在肝癌细胞系HepG2, Huh7中高表达,SMMC7721中低表达,正常细胞株Changliver中未检测到AFP的表达。2.三条靶向AFP基因的StealthTM RNAi在mRNA水平和蛋白水平均可明显抑制Huh7中AFP基因的表达；其中AFP-HSS304和三条StealthTM RNAi的cocktail对AFP基因的表达具有最强抑制效应,两者转染后72小时在mRNA水平的抑制率分别为97.4%和98.5%,蛋白水平的抑制率分别为94.57%和94.84%.3. AFP-HSS304和三条StealthTM RNAi的cocktail沉默Huh7细胞中AFP基因的表达后,与对照组相比,可以明显抑制细胞的增殖,两者转染后72小时对细胞增殖的抑制率分别为65.63%和67.72%；并可以诱发凋亡,Hochest染色法和透射电镜可以观察到明显的凋亡小体,转染后72小时流式细胞仪检测到早期凋亡率分别为60.73%和69.23%。4.Western blot检测结果显示,沉默AFP基因后Huh7细胞中突变型P53,Bcl-2, Procaspase-3以及线粒体中的Cytochrome C水平显著降低；而Bax以及细胞浆中的Cytochrome C水平明显升高。沉默AFP基因后诱导Huh7细胞凋亡可能是P53/Bax/ Cytochrome C/ Caspase-3凋亡信号通路所介导。结论1.靶向AFP基因的StealthTM RNAi可明显抑制Huh7中AFP基因在mRNA水平和蛋白水平的表达。2.靶向沉默Huh7细胞中AFP基因的表达后,可明显抑制细胞的增殖,并可以诱发凋亡,以早期凋亡为主。3.沉默AFP基因诱导Huh7细胞凋亡可能是P53/Bax/Cytochrome C/Caspase-3凋亡信号通路所介导。4.AFP基因可以做为肝癌基因治疗的潜在靶点进行深入研究。更多还原

【Abstract】 Effects of Silencing Alpha-fetoprotein Gene by RNAi on Proliferation and Apoptosis in Hepatocellular Cancer Cell PhD Candidate:Xiao Jun Yang Mentor: You Cheng ZhangBackground:Discovered in 1963, forty years ago, Alpha-fetoprotein (AFP) still is an important tumor marker used in diagnosis, therapy and prognosis for hepatocellular carcinoma (HCC) now. However, it is demonstrated from recent studies that AFP play a critical role in proliferation and apoptosis of HCC, not only as a tumor-associated protein. Although there have been considerable researches indicating that AFP can modulate apoptotic signals, the precise mechanism of AFP-mediated cell growth regulation and apoptosis resistance remains obscure.Objective:To study the growth promotion and apoptosis-resistant effects of AFP on hepatocellular cancer cells using RNAi, also explore the possible molecular mechanisms that underlie the apoptosis induction by silencing AFP in Huh7 cells.Methods:1.Detecting the expression levels of AFP in HepG2, Huh7, SMMC7721 and Changliver cells using ECLIA(electro-chemiluminescence immunoassay), RIA(Radioimmunoassay) and ELISA(Enzyme-Linked ImmunoSorbent Assay).2.According to the siRNA design guidelines, three stealth RNAi targeting AFP were customarily synthesized by Invitrogen and transfected into Huh7 cells using the lipofectamine RNAi Max, then detecting AFP mRNA and protein by real time RT-PCR, ELISA and Western blot to confirm the best RNAi sequences and transfection conditions.3.Determining cell proliferation rates of all groups by Dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide (MTT) assay after silencing AFP gene in Huh7 cell using the best stealth RNAi targeting AFP; We also detected cell apoptosis by Hochest staining and transmission electron microscope (TEM), the apoptosis rates were determined by flow cytometry(FCM).4. Detecting the proteins levels involved in apoptosis pathway using Western blot, such as P53, Bax, Bcl-2, Cytochrome c, Caspase-3, to explore the possible molecular mechanisms that underlie the apoptosis induction after silencing AFP in Huh7 cells.Results: 1.It was found that the expression of AFP protein in Huh7 and HepG2 cells was remarkably higher than that in SMMC7721 cell. But we didn’t find the expression of AFP protein in Changliver cell.2.The results obtained from real time RT-PCR, Western blot and ELISA assay demonstrated that AFP expression was significantly inhibited both at mRNA and protein levels in Huh7 cells after transfection of the stealth RNAi (HSS303, HSS304, HSS305) and the RNAi cocktail for 48h and 72h; Among them, AFP-HSS304 and the cocktail had the most potent effects, The reduction rates were 97.4% and 98.5% at mRNA level(P<0.01),94.57% and 94.84% at protein level(P<0.01) at 72h after transfection respectively.3. Compared to the blank control, the results came from MTT assay demonstrated that the cell viability was reduced significantly by AFP-HSS304 and RNAi cocktail at 24h,48h and 72h after transfection, the inhibition rates of cell proliferation were 65.63% and 67.72%(P< 0.05) at 72h after transfection respectively. AFP-HSS304 and RNAi cocktail also induced apoptosis in Huh7 cells, the apoptotic bodies can be observed in the two groups by Hochest staining and TEM, FCM assay demonstrated that the apoptotic rate in early stage were 60.73% and 69.23% (P< 0.05) at 72h after transfection respectively.4.The results obtained from Western blot showed that the levels of P53, Bcl-2, Procaspase-3 and Cytochrome C in mitochondrial intermembrane space were reduced distinctly(P<0.05) in Huh7 cells after AFP silenced, however, the levels of Bax and Cytochrome C in cytoplasm was elevated markedly (P<0.05). So the effect of inducing apoptosis after AFP silenced in Huh7 cell may mediated by P53/Bax/Cytochrome C/Caspase-3 pathway.Conclusions: 1.The expression of AFP gene in Huh7 can be inhibited obviously both at mRNA and protein levels by the StealthTM RNAi that targeting AFP.2. Target-silencing AFP gene can inhibited cell proliferation and induced apoptosis in Huh7 cell, the apoptotic cells were mainly at the early apoptotic stage.3. The effect of inducing apoptosis in Huh7 cell after AFP silenced may mediated by P53/Bax/Cytochrome C/caspase-3 apoptotic signaling pathway.4. AFP can be further studied as an important target for hepatocellular cancer gene therapy.更多还原