【作者】 冯彦斌；

【导师】 王明荣；

【作者基本信息】 中国协和医科大学 ， 细胞生物学， 2007， 博士

【摘要】 PLK1(Polo-like kinase 1)是调控中心体成熟、染色体分离和有丝分裂退出的丝氨酸/苏氨酸蛋白激酶。PLK1对于维持有丝分裂过程中基因组稳定性非常重要,在多种恶性肿瘤中发挥作用。我们实验室在前期工作中发现PLK1基因所在的16p12染色体区带在食管鳞癌中存在增益,本研究旨在探讨PLK1在食管鳞癌中的表达变化及其参与该病发生发展的分子机制。我们首先采用免疫组化方法检测了PLK1在155例食管鳞癌中的表达及与肿瘤临床病理参数之间的关系,继而通过RNAi降低PLK1的表达,观察其对食管鳞癌细胞增殖、凋亡的影响,采用免疫共沉淀和激光共聚焦方法确定PLK1的相互作用蛋白。结果表明,与相应的正常上皮相比,PLK1在71.6%的人食管鳞癌组织中表达明显升高。PLK1过表达与区域淋巴结转移(P=0.001)和肿瘤分期(P=0.033)密切相关。PLK1表达阳性患者的术后生存时间显著短于PLK1表达阴性的患者(P=0.001)。而且,多因素生存分析显示PLK1是一个独立的预后因素(RR=4.921,P=0.011)。通过荧光原位杂交(FISH)检测食管鳞癌中PLK1基因的扩增情况,发现37.5%的标本中包含PLK1基因的RP11-141E3位点发生了拷贝数的增加,说明基因扩增是PLK1在部分食管鳞癌组织中过表达的原因之一。通过RNA干扰降低PLK1的表达,能显著抑制食管鳞癌细胞增殖,并诱导细胞发生凋亡。PI染色和流式细胞仪检测表明,与对照组相比PLK1-RNAi组出现明显的凋亡峰,并呈时间依赖性增强。抑制PLK1表达导致线粒体膜电位降低、caspase-9和caspase-3活化、以及下游多聚ADP核糖聚合酶(PARP)降解,同时表现线粒体凋亡通路相关蛋白Mcl-1和Bcl-2蛋白水平下降。免疫共沉淀和激光共聚焦分析结果显示PLK1在细胞内分别与survivin和α-catenin形成复合物。PLK1敲降导致survivin蛋白表达降低。以上结果表明PLK1可能在细胞凋亡、分化、粘附和迁移中发挥作用。Survivin作为凋亡抑制蛋白,可能与PLK1的抗凋亡功能相关。PLK1可能是一个有应用价值的预后因子和潜在的食管鳞癌治疗靶点。更多还原

【Abstract】 Polo-like kinase 1(PLK1) is one of the serine/threonine kinases involved in centrosome maturation,chromosome segregation and mitotic exit.PLK1 is frequently overexpressed in various kinds of human tumors.16p12 encompassing PLK1 gene has been previously identified as a common amplified region in esophageal squamous cell carcinoma(ESCC).The present study is to investigate the expression change of PLK1 in ESCC and the underlying molecular mechanisms through which PLK1 takes part in esophageal carcinogenesis and malignant development.Immunohistochemistry in 155 ESCC and adjacent normal tissues showed that PLK1 expression was considerably increased in tumors compared with that in normal epithelia.PLK1 overexpression was significantly associated with regional lymph node metastasis(P=0.001) and tumor stage(P=0.033).The prognosis of patients with PLK1-positive tumors was significantly worse than that with PLK1-negative (P=0.001).Multivariate survival analysis showed that PLK1 was an independent prognostic factor(RP=4.921,P=0.011).Amplification of the locus RP11-141E3 containing PLK1 gene was observed in 37.5%of tumors by fluorescence in situ hybridization,suggesting that gene amplification was the mechanism underlying PLK1 overexpression in a part of esophageal carcinomas.RNAi-mediated knockdown of PLK1 dramatically suppressed cell proliferation and resulted in apoptosis.Depletion of PLK1 activated the mitochondrial apoptotic pathway,which was substantiated by loss of mitochondrial membrane potential, reduction of survivin,Mcl-1 and Bcl-2,activation of caspase-9 and caspase-3 as well as cleavage of poly(ADP-ribose) polymerase(PARP).Co-immunoprecipitation and confocal microscopy displayed that PLK1 was associated with survivin andα-catenin.These data suggest that PLK1 plays a part in adhesion,differentiation and migration of cells.Survivin is probably involved in anti-apoptotic function of PLK1. PLK1 might be a useful prognostic marker and a potential therapeutic target for ESCC.更多还原