【作者】 刘辰；

【导师】 倪泉兴； 傅德良； 金忱； 虞先浚； 徐近；

【作者基本信息】 复旦大学 ， 外科学， 2008， 博士

【摘要】 目的:MBD1(methyl-CpG binding domain protein 1,MBD1)基因是一个重要的转录调控因子,它可通过结合甲基化位点抑制转录,导致肿瘤甲基化相关基因表达下降。本研究将通过RNA干扰技术,观察MBD1基因沉默后胰腺癌细胞蛋白质表达的差异、肿瘤细胞生物学特性的改变,以及相关基因表达的改变,探讨MBD1在胰腺癌发病机制中的角色及其参与的信号转导通路和调控网络,从而更深入地揭示胰腺癌发病的分子机制,为寻找胰腺癌的早期有效的诊断和治疗方法提供新的思路及实验基础。方法:(1)选择MBD1高表达的人胰腺癌细胞株BxPC-3作为研究对象,利用RNA干扰的方法沉默BxPC-3细胞株MBD1的表达,建立稳定的MBD1沉默表达的细胞克隆。(2)采用二维电泳(2-DE)和质谱技术(MALDI-TOF-MS),筛选与鉴定MBD1下调后胰腺癌细胞BxPC-3中差异表达的蛋白质,并用RT-PCR和Western blot方法进行验证。免疫组化方法验证胰腺癌组织中差异相关蛋白的表达及其与胰腺癌临床病理特征的关系。(3)通过裸鼠胰腺癌移植瘤动物模型,研究MBD1下调对胰腺癌移植瘤生长的影响。RT-PCR检测移植瘤中MBD1和甲基化相关基因及部分抑癌基因的表达情况。(4)四甲基偶氮唑蓝法(MTT)比较MBD1下调前后BxPC-3细胞的生长情况,“伤口愈合”(Wound healing)试验检测MBD1下调后BxPC-3细胞运动迁移能力的改变。结果:(1)设计合成了针对MBD1基因的小分子干扰RNA(small interfering RNA,siRNA),成功构建MBD1-siRNA真核表达质粒,采用脂质体介导的方法将MBD1-siRNA真核表达质粒转染胰腺癌细胞系BxPC-3,并筛选到MBD1沉默稳定表达的细胞株,经RT-PCR、Western blot证实,转染后MBD1表达水平明显下调。(2)在MBD1下调前后的人胰腺癌细胞株BxPC-3中建立了重复性良好的、可靠的二维电泳图谱。质谱鉴定得到14个差异表达蛋白:有9个相关蛋白(GRP78、Tollip、HSP A8、Vimentin、Stathmin 1、cofilin 2、hnRNP K、eIF3、ZFP-36)表达下调;5个蛋白(tubulinbeta 2、splicing factor arginine/serine-rich isoform 1、ER-60、P4hb、EFHD2)表达上调,并应用Western blot法进一步验证。(3)通过免疫组化法检测16例胰腺癌组织中Vimentin、GRP78、Stathmin的表达,发现与正常胰腺组织相比,Vimentin、Stathmin在胰腺癌中存在高表达,且Vimentin与胰腺癌淋巴结转移有关。(4)MTT试验结果显示MBD1下调后胰腺癌细胞生长明显减缓。(5)动物移植瘤实验表明,转染MBD1siRNA质粒的裸鼠移植瘤生长速度明显较对照组减慢,RT-PCR检测三组移植瘤MBD1、CDH-1、Rb基因的mRNA表达变化,发现与对照组相比,转染MBD1-siRNA移植瘤组在MBD1下调的同时,抑癌基因CDH-1与Rb的表达明显上调,RT-PCR检测移植瘤中Vimentin和GRP78的表达,结果显示MBD1-siRNA移植瘤组中Vimentin和GRP78基因的表达明显低于对照组。(6)Woundhealing试验提示MBD1下调对胰腺癌细胞的迁移能力无明显影响。结论:siRNA技术可以沉默胰腺癌细胞中MBD1基因表达,MBD1的下调可使多个与肿瘤相关蛋白的表达发生改变,并可能通过恢复抑癌基因的表达改变胰腺癌细胞的增殖生长能力及其生物学特性,MBD1相关的转录调控网络机制可能在胰腺癌的发生发展过程中起到重要的作用。更多还原

【Abstract】 Objective:Methyl-CpG binding domain protein 1(MBD1),a suppressor of gene transcription,may be involved in inactivation of tumor suppressor genes during tumorigenesis.Over-expression of MBD1 had been detected in human pancreatic carcinomas and correlated with tumor metastasis.The purpose of this study is to investigate the different expression of methyl-related proteins and genes after the silence of MBD1 in pancreatic cancer cell line BxPC-3,observe the effects of MBD1-knockdown on tumor biological characteristics and discuss the role of MBD1 as an impotant transcription regulator in carcinogenesis of pancreatic cancer.Methods:The stable MBD1-knockdown pancreatic cancer cell line(BxPC-3) was established by RNA interference.The differential proteins between control and MBD1-knock-down cells were detected by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The results were confirmed by RT-PCR and western blot.The expression of MBD1-related proteins in tumor tissues of 16 specimens of resected pancreatic cancer and 10 normal pancreatic tissues were determined by immunohistochemical staining.MTT assay and wound healing test were used to detect the changes of BxPC-3 cell growth and cell migratory ability after MBD1 knockdown respectively.In vivo,the pancreatic cancer nude mice model was established to observe the tumor growth and compare the different expression of some related tumor suppressor genes and methyl-related genes between MBD1-knockdown and control group by RT-PCR assay.Results:MBD1-siRNA recombinant plasmid were stably transfected into BxPC-3 cell line,the expression of MBD1 was significantly down-regulated after RNA interference, which was confirmed by RT-PCR and Western blot assay.By two-dimensional gel electrophoresis and mass spectrometry,five up-regulated proteins(including tubulin beta 2,splicing factor arginine/serine- rich isoform 1,ER-60,P4hb,EFHD2) and nine down-regulated proteins(including GRP78,Tollip,HSP A8,Vimentin,Stathmin 1, cofilin 2,hnRNP K,eIF3,ZFP-36) were identified clearly.Most of the identified differential proteins were involved in tumorigenesis,especially related with tumor metastasis,and some were prognostic biomarkers for human malignant tumors.The expressions of Vimentin and Stathmin in pancreatic carcinoma were significantly higher than that in normal pancreas tissues,and the positive expression of Vimentin was closely related with lymph node metastasis.The growth of BxPC-3 cell was suppressed after RNA interference targeting MBD1 while cell migratory ability remained unchanged in vivo and vitro.The mRNA expression of tumor suppressor genes CDH1 and Rb were significantly up-regulated when MBD1,Vimentin and GRP78 were down-regulated in transplanted tumor of nude mice model.Conclusion:The expression of MBD1 in pancreatic cancer cell BxPC-3 could be down-regulated by RNAi.Down-regulation of MBD1 could relieve the inhibition of some tumor suppressor genes and affect the biological features of pancreatic cancer cell BxPC-3,resulting in significant changes of some methyl-related proteins involved in carcinogenesis and metastasis.MBD1,as a transcription regulator,might play an important role in tumorigenesis of pancreatic cancer.更多还原