【作者】 赵萌；

【导师】 张贵宇；

【作者基本信息】 山东大学 ， 妇产科学， 2013， 硕士

【摘要】 研究背景与目的：子宫内膜腺癌是女性生殖道三大恶性肿瘤之一,其发病率在西方发达国家位居女性道恶性肿瘤首位,在我国列女性生殖系统肿瘤第二位,而且近年来发病率呈上升趋势,严重危害了女性的生命健康。miRNA系一类非蛋白编码的小RNA,通过阻碍翻译过程或者影响mRNA的稳定性来负性调控靶基因的表达。成熟的miRNA结合到与其互补的mRNA的位点通过碱基配对,引起相应mRNA的降解或翻译过程中断,从而调节靶基因的表达.miRNA在生物进化过程中高度保守,人类三分之一的基因表达都有其参与,涉及到人体的各种生命活动中,如细胞增殖,凋亡,免疫,神经内分泌等。miRNA与肿瘤的发生发展关系密切,超过50%的miRNA基因位于与癌症相关的基因组区域或脆性位点。人类最早是在B细胞慢性淋巴性白血病(CLL)中发现存在miRNA表达水平的改变,即miR-15a和miR-16-1的表达下调被认作是导致染色体13q14缺失的罪魁祸首。随后陆续在多种肿瘤中检测到这种变化,目前已有多种肿瘤的miRNA表达谱系被测定。miRNA在某些肿瘤中被视作原癌基因而在另一些肿瘤中被视作抑癌基因。某一种miRNA在不同肿瘤中表达量及作用各不相同。miR-205是一种在不同种系中呈高度保守性的miRNA,是一段-uccuucauuccaccggagucug-编码的miRNA。人类miR-205定位于1q32.2的LOC642587基因中。目前已证实其与多种肿瘤的发生相关。我们前期研究表明在子宫内膜样腺癌中,miR-205表达明显上调。PEG3,是一种在胚胎形成时期开始表达的印记基因,在成年后的卵巢,睾丸,肌肉和脑组织中同样高表达。PEG3在亲子和性行为的发展中起到至关重要的作用,同样它调控细胞生长和凋亡,在胶质瘤中有肿瘤抑制的作用。在胶质瘤中的研究中,PEG3被认为是通过抑制WNT/β-catenin信号通路来促进胶质瘤细胞的增殖。β-catenin是胞质中的一个多功能蛋白,目前认为它是WNT信号通路中最重要的信号传递子和中转物,当其在细胞内大量累积时,可以进入核内,引发下游靶基因的表达启动,因此β-catenin表达量是WNT信号启动与否的关键。同时c-myc基因是Wnt/β-catenin信号通路的下游靶基因,在人类许多恶性肿瘤中过度表达。其表达与细胞周期密切相关,可以刺激胚胎发育,促进细胞生长。迄今为止,尚未有文献涉及PEG3在子宫内膜癌的表达情况。本文旨在探究在子宫内膜样腺癌与正常子宫内膜组织中PEG3的表达差异,以及在高中低分化子宫内膜癌细胞系中,miR-205与PEG3的直接靶向作用。研究方法：(1)免疫组化方法确定PEG3在正常子宫内膜与高中低分化子宫内膜癌组织中表达的差异。(2)RT-PCR筛选出miR-205表达量较正常子宫内膜细胞株高的三种高中低分化子宫内膜癌细胞系。(3)上调三种细胞系的miR-205表达,CCK-8及流式细胞学实验分别检测上调miR-205前后细胞系增殖及凋亡的变化；(4)Western blot,RT-PCR和双荧光素酶靶基因报告系统等实验方法验证miR-205对PEG3是否有直接的靶抑制作用。研究结果：(1)PEG3在正常子宫内膜组织中表达明显高于高中低分化子宫内膜癌组织,且分化程度不同,PEG3表达量有明显差异。(2)三种高中低分化子宫内膜癌细胞系中,miR-205上调后,细胞增殖被促进,凋亡则受到抑制；(3)western blot结果示PEG3蛋白含量降低,PEG3抑制的wnt通路中的两个关键蛋白β-catenin及c-myc也相应的表达量上调,而RT-PCR结果显示mi R-205上调前后PEG3mRNA表达水平变化无统计学意义；双荧光素酶报告系统结果显示miR-205作用于PEG3基因的3’UTR端。研究结论：(1)PEG3在子宫内膜样腺癌组织中作为一种抑癌基因存在,且与内膜样腺癌的分化程度呈负相关。(2)miR-205作用于PEG3基因的3’UTR端,在转录后水平抑制PEG3的表达,提示PEG3是miR-205的直接靶基因。更多还原

【Abstract】 Background And Objectives:Endometrial carcinoma is the most common gynecologic malignancy in western countries and the second gynecologic cancer in China.MicroRNAs (miRNA) are small non-coding RNAs which function as negative regulators of gene expression by blocking the translation or decreasing the stability of mRNAs. It has been shown that miRNAs form a complex named miRNAs-containing ribonucleo-protein particles to the complementary site in the3’-untranslated region(3-UTR) of the target mRNA. If it is partially matched or completely matched, translation blockage or mRNA degradation will happen to the target gene. MiRNA is highly conserved in biological evolution, and it is involved in one third of gene expression in human vital movement, like cell proliferation, apoptosis, immunity and neuroendocrine. MicroRNAs are reported to involve in many human cancers, and they served as either oncogenes or tumor suppressor genes. MiRNAs are initially found in chronic lymphocytic leukemia (CLL), which showed that down-expression of miR-15a and miR-16-1caused the13q14deletion. Now, it has been found in many other carcinomas and the expression files were detected followed. MiRNAs are considered to be oncogenes in some tumors but tumor suppressor genes in the others. One miRNA had different expression level in different cancers.Human miR-205is located in the second intron of LOC642587in chromosome1. Our previous study clearly demonstrated that miR-205is up-regulated in endometrial carcinoma.PEG-3(paternally expressed gene3) is an imprinted gene that expresses primarily during embryogenesis, as well as in adult ovary, testis, muscle, and brain. PEG-3invovles in parenting and sexual behaviors, regulates growth and apoptosis, and exhibits tumor suppressor activity in glioma cell line. The down-expression of PEG-3in giloma cells promotes proliferations by inhibiting WNT/β-catenin pathways.β-catenin is a kind of multifunction protein in cytoplasma. And it is known to play the most important role in WNT pathway. The accumulation of β-catenin can incur the expression of the genes in downstream. C-myc is a kind of downstream gene in the Wnt/β-catenin pathway and overexpressed in many kinds of human malignant tumors. So far, there is no report about PEG3in endometrial cancer.This study is aim to explore the differences of expression level between the endometrial cancer tissues and normal endometrial tissue. And further more, the relationship between micro-205and PEG3(Paternally Expressed Gene3) in endometrial cancer celllines.Methods:(1).Immunohistochemical to detect the different expression of PEG3in normal endometrial tissue and endometrioid endometrial carcinoma tsues.(2).Three diffenrent differtiation kinds of endometrial cancer celllines were chosen which were highly expressed miR-205than normal endometrial cellline confirmed by RT-PCR.(3).Then, CCK8, flow cytometry anlyasis were used to detect the cell proliferation and apoptosis after the miR-205eleveted by miR-205mimics.(4).Western blot, RT-PCR, and dual luciferase assay are used to explore the target relationship between micro-205and PEG3.Results:(1) Immunohistometrical to detect the different expression of PEG3in normal endometrial tissue and endometrioid endometrial carcinoma tsues.(2) Three diffirent differtiation kinds of endometrial cancer celllines were chosen which were highly expressed miR-205than normal endometrial cellline confirmed by RT-PCR.(3) Then, CCK8, flow cytometry anlyasis were used to detect the cell proliferation and apoptosis after the miR-205eleveted by miR-205mimics.(4) Western blot, RT-PCR, and dual luciferase assay are used to explore the target relationship between micro-205and PEG3.Conclusion:(1).PEG3act as an tumor suppressor gene in endometrial cancer. And in different stage of the cancer, the PEG3expression level are consequently different.(2)MiR-205directly target PEG3by acting on the3’UTR of PEG3mRNA.更多还原