Ezrin与胰腺癌发生、侵袭及转移关系的探讨

【作者】 孟云霄；

【导师】 陈杰； 卢朝辉；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 胰腺癌是预后最差、死亡率最高的恶性肿瘤之一。由于发病部位的特殊性及症状的隐匿性,很难进行早期诊断,绝大多数病例在诊断时已处于晚期。胰腺癌极强的侵袭和转移能力与其预后差直接相关。手术切除是目前获得治愈的唯一手段,但只有不到20%的患者适于手术切除,而不能手术的患者平均生存时间少于六个月。胰腺癌预后差的原因之一是常在出现临床症状和影像学技术改变之前就有微转移灶形成。研究胰腺癌的这种高度恶性生长扩散方式的机制,从而找到治疗胰腺癌的新方法,是目前医学研究的迫切任务之一。近年文献报道,ezrin的高表达可以促进多种肿瘤的转移,因此ezrin可能是某些肿瘤基因治疗的潜在靶点。越来越多的研究表明ezrin在肿瘤细胞中的生物学行为中发挥着重要的作用。通过干预ezrin表达探讨ezrin对胰腺癌细胞生物学行为的影响,目前尚未有研究报道。本研究旨在利用RNA干扰技术和基因过表达技术对胰腺癌细胞系MiaPaCa-2细胞中ezrin的表达进行干预,进而探讨ezrin对胰腺癌生物学行为的影响。1.利用RNA干扰技术研究ezrin在胰腺癌细胞的表达抑制后对细胞生物学行为的影响:成功确立了ezrin的有效干扰位点,构建干扰载体,并挑选出稳定干扰克隆。发现ezrin表达抑制对胰腺癌的增殖和细胞周期没有明显的影响,但ezrin表达抑制可使胰腺癌细胞的细胞突起和表面微绒毛明显减少,在软琼脂内形成克隆数明显减少,细胞迁移至Transwell Chambers下室的数量明显下降,侵袭至Matrigel Invasion Chambers下室的细胞数量明显减少。2.利用ezrin表达载体对胰腺癌细胞MiaPaCa-2进行转染,筛选获得稳定过表达克隆。发现ezrin过表达对胰腺癌细胞的增殖和细胞周期没有明显的影响,但ezrin过表达可使胰腺癌细胞的细胞突起和微绒毛明显增多,在软琼脂内形成的克隆数明显增多,细胞迁移至Transwell Chambers下室的数量增加,侵袭至Matrigel Invasion Chambers下室的细胞增多。3.利用明胶酶谱分析发现,ezrin过表达可引起基质金属蛋白酶MMP-9的表达,但ezrin敲减表达对明胶酶表达的影响并不明显;对Akt及Erk1/2信号通路的分析表明,ezrin的过表达可使Erk1/2磷酸化水平降低,而ezrin敲减表达对Erk1/2磷酸化水平的影响不明显,ezrin的表达变化对Akt的磷酸化水平,Akt总蛋白和Erk1/2总蛋白影响不明显,此结果提示ezrin可能通过下调Erk1/2的磷酸化水平,进而导致肌动蛋白细胞骨架的重现从而引起细胞运动能力的增加,进而导致细胞侵袭能力的改变;而ezrin表达下调引起细胞运动和侵袭能力的下降可能还通过其它机制。综上所述,本研究发现ezrin的表达变化可引起细胞伪足及表面微绒毛数量、细胞运动、侵袭能力的改变和细胞非锚定依赖性的生长能力的改变,表明ezrin蛋白在这些细胞生物学行为方面发挥着重要的作用。Ezrin引起肿瘤细胞侵袭和运动能力的变化部分可能是通过影响Erk1/2磷酸化水平引起肌动蛋白细胞骨架的重构,继而导致细胞运动能力的改变引起的,部分可能是通过引起MMP的表达变化导致其侵袭能力的改变所致,具体的机制还需要进一步研究。Ezrin在胰腺癌的形态学、非锚定依赖性的生长、运动以及侵袭转移过程中发挥着重要的作用。因此,针对ezrin表达的干预可能会使胰腺癌肿瘤细胞侵袭和转移能力下降,有希望成为胰腺癌侵袭转移的一个新的治疗靶点,从而为胰腺癌的治疗提供了一条新的途径。更多还原

【Abstract】 Pancreatic ductal ademocarcinoma is the eighth leading cause of cancer mortality in China and the fourth in United States. The incidence of Pancreatic ductal ademocarcinoma is increasing. So far neither an early diagnosis nor a therapeutic strategy for advanced lesions has been developed yet. The death to incidence ratio of pancreatic ductal carcinoma is approximate to 0.98-0.993. The overall 5-year survival rate of pancreatic carcinoma is less than 5%, which is largely due to the difficulty in early diagnosis. More than 85% of patients have the disease extending beyond the pancreas at the time of diagnosis, rendering surgical and medical intervention ineffective. For the 15-20% of patients who undergo potentially curative resection, the 5-year survival is only 20%. It is an urgent mission for both the clinicians and the scientists who devote themselves to pancreatic cancer to find a breakthrough using new techniques.Ezrin is a member of the ERM family, it provides a functional link between the plasma membrane and the cortical actin cytoskeleton of the cell, and participates in crucial signal transduction pathways. There are increasing evidences that it can regulate tumor progression and metastasis. However, the role of ezrin in pancreatic cancer has not clearly delineated.Ezrin expression leval was interrupted in pancreatic cancer cells MiaPaCa-2 by ezrin knock-down and overexpression, and the biological behavior changes in these cells were observed and analyzed.1. Knock-down ezrin expression in pancreatic cancer cells MiaPaca-2 by RNAi: The suppression of ezrin did not influence cells proliferation and the cell cycle in vitro; but ezrin depression can inhibit the formation of cell protrusion and cell microvilli as well as cause a decrease in cell motility and invasiveness. Furthermore, we revealed that ezrin silencing can reduce the anchorage-independent growth of the tumor cells in soft agar.2. Ezrin overexpression in pancreatic cancer cells MiaPaca-2: The overexpression of ezrin did not influence cells proliferation and the cell cycle in vitro; but it can enhance the formation of cell protrusion and cell microvilli as well as cause an increase in cell motility and invasiveness. Furthermore, we revealed that ezrin overexpression can raise the ability of the anchorage-independent growth of the tumor cells in soft agar.3. Gelatin zymography assay revealed that overexpression of ezrin can induce the expression of MMP-9, but the effect of ezrin knock-down on the expression of gelatinase is not obvious. Ezrin overexpression can also induce the activation of Erkl/2, but ezrin silencing can not influence the activation of Erkl/2. The alteration of ezrin expression could not influence the Akt activation, total Akt as well as total Erkl/2 protein level. There may be another pathway involved in ezrin induced pancreatic ductal adenocarcinoma cells motiliy and invasiness.Our experiments showed that the alteration of ezrin expression can induce the changes in cell protrusion and cell microvilli, cell motility, invasiveness as well as the ability of the anchorage-independent growth of the tumor cells. The inactivation of Erkl/2 may induce the actin cytoskeleton reappearance and contribute to alteration of cell motility and invasiveness ability. The latter may be related to induced MMP-9 expression when ezrin was overexpressed.Consistent with these results, ezrin may be an important target for inhibiting the invasion and metastasis of pancreatic ductal adenocarcinoma.更多还原