【作者】 杜颖；

【导师】 姒健敏； 周天华；

【作者基本信息】 浙江大学 ， 临床专业， 2009， 博士

【摘要】 微小RNA(microRNAs,miRNAs)是近年来发现的一类长约21-25个核苷酸的内源性单链非编码RNA,在动植物中均有表达。miRNAs通常与靶mRNAs的3’-非翻译区(3’-untranslated region,3’-UTR)互补结合,在转录后水平抑制靶基因的表达。miRNAs调节细胞增殖、凋亡、分化以及迁移等重要的生物学过程。miRNAs表达和代谢的异常还与多种疾病,比如肿瘤密切相关。一些miRNAs在肿瘤组织中表达异常,促进了肿瘤的发生和进展,其作用相当于癌基因或抑癌基因。目前,已有证据提示miRNAs可应用于肿瘤的基因诊断和治疗。应用高通量的生物芯片技术,科学家已在多种人类肿瘤,如肺癌、乳腺癌、肝癌、食管癌和前列腺癌等中筛选出表达异常的miRNAs。其中,Hsa-miR-141(miR-141)作为miR-200家族的成员之一,在乳腺癌、结直肠癌细胞株和胆管癌细胞株中表达增高,而在前列腺癌、肝细胞肝癌和肾细胞癌中表达降低。异常表达的miR-141参与了胆管癌细胞的增殖和肾癌细胞的侵袭活动,提示miR-141与多种肿瘤的发病机制相关。胃癌是全球高发的恶性肿瘤之一,是男性肿瘤的第二死因,女性肿瘤的第三死因。2008年,美国新增的胃癌患者估计有21,500例,新增的胃癌死亡患者约10,880例。在中国每年新增约36-40万胃癌患者,死亡率占据我国肿瘤死因的第二位。但是,我们对胃癌发病机制尚缺乏全面和深入的了解。最近研究发现miR-106b-25簇、miR-21和miR-27a在胃癌中的表达增加;同时,肠型胃癌和未分化型胃癌均有特征性的miRNAs表达谱,提示miRNAs与胃癌的发病机制有密切的联系。在本课题中,我们利用生物芯片技术筛选在胃癌组织和癌远端非肿瘤组织中差异表达的miRNAs,并通过定量Real-time PCR技术检测miR-141在胃癌患者肿瘤组织和胃癌细胞株中的表达状态;同时采用现代细胞与分子生物学技术,通过体外胃癌细胞模型研究miR-141在胃癌发病机制中的作用,为胃癌早期诊断和寻求药物靶点提供科学的依据。1材料和方法1.1收集胃癌患者手术组织样本,通过miRNAs芯片筛选在胃癌组织及癌远端非肿瘤组织中差异表达的miRNAs,并用定量Real-time PCR验证miR-141的表达情况。然后使用独立样本t检验和单因素方差检验分析miR-141表达水平与各临床病理参数之间的相关性。1.2使用定量Real-time PCR分析5株胃癌细胞株中miR-141的表达情况,结合细胞的增殖和分化特征,筛选合适的胃癌细胞株。1.3使用miR-141 precursor molecule转染方法在MGC-803胃癌细胞内过量表达miR-141,通过相差显微镜观察以及MTT检测研究过量表达miR-141对细胞增殖的影响。1.4使用生物信息学方法(如miRanda,Pictar,Targetscan等)预测miR-141靶基因,结合既往文献筛选与胃癌发病机制相关的靶基因,并进行初步验证。2主要结果2.1生物芯片结果发现,与癌远端非肿瘤组织相比,胃癌组织中的miR-141下调了大约12.0倍。使用定量Real-time PCR发现在80%的胃癌组织样本中miR-141的表达水平下调,平均下降2.1倍(P<0.01)。但miR-141的表达水平与各临床病理参数之间无显著相关性。2.2筛选出来源于胃癌的MGC-803细胞株为合适的体外细胞模型。用miR-141 precursor转染MGC-803细胞后,细胞中miR-141的表达上调了大约7×10~4倍;而pre-miR negative control转染的细胞在荧光显微镜下几乎均显示为红色,提示miR-141 precursor在MGC-803细胞中转染成功且过量表达了miR-141。2.3用相差显微镜和MTT法观察miR-141 precursor过量表达48小时和72惺焙蟮腗GC-803细胞。与pre-miR negative control转染组相比,两种方法均提示miR-141 precursor转染组细胞的增殖受到显著抑制(48小时,P<0.05;72小时,P<0.01)。2.4使用生物信息学方法分析,预选出miR-141的靶基因是FGFR2和ZEB2。但定量Real-time PCR检测未显示过量表达miR-141的MGC-803细胞内FGFR2和ZEB2的mRNA水平有显著改变。3结论3.1基因芯片和定量Real-time PCR发现在胃癌患者的肿瘤组织中,miR-141的表达水平与癌远端非肿瘤组织相比显著减少,提示miR-141的表达下调可能与胃癌的发病机制有关。3.2过量表达miR-141后MGC-803细胞增殖受到显著抑制,显示miR-141表达水平可能参与细胞增殖的分子调控,有可能成为胃癌治疗的分子靶点。3.3关于FGFR2和ZEB2是否为miR-141与胃癌相关的靶基因,还需要继续扩大研究,深入探索并验证之。更多还原

【Abstract】 MicroRNAs(miRNAs),a new class of endogenous,noncoding and single-stranded RNAs,were recently discovered in both animals and plants.They trigger translational repression and/or mRNA degradation mostly through complementary binding to the 3’-untranslated regions of target mRNAs.Studies have shown that miRNAs can regulate a wide array of biological processes such as cell proliferation,differentiation and apoptosis.Aberrant expression and metabolism of miRNAs are associated with human diseases including malignancy.Alterations of miRNAs expression may play various roles in the pathogenesis of many human cancers.Some miRNAs have been shown to possess oncogenic or tumor suppressor activity,influencing cell transformation,tumor progression or metastasis.Accumulating evidences suggest that miRNAs could potentially be widely used in the diagnosis,prognosis and therapy of human cancer.High-throughput techniques have been used to screen miRNAs differentially expressed between human nonmalignant and malignant samples and found a number of miRNAs deregulated in numerous human tumors including lung,breast,liver, esophageal,colon and prostate cancers.Among these miRNAs,miR-141,a member of the miR-200 family,is over-expressed in ovarian cancer,colorectal cancer cells and cholangiocarcinoma cells,and down-regulated in prostate,hepatocellular and renal cell carcinoma.Recent studies suggested that miR-141 might play a role in the proliferation of cholangiocarcinoma cells and the invasiveness of renal carcinoma cells.Gastric cancer is the second leading cause of male cancer-related death and the third leading cause for female cancer-related death worldwide.In year 2008,the estimated new cases of stomach cancer is 21,500 and the estimated death reaches 10,880 in United States.However,little is known about the role of miRNAs in gastric cancer.Recently,it was reported that miR-106b-25 cluster,miR-21 and miR-27a were up-regulated in gastric tumor tissues.Moreover,intestinal-type and undifferentiated gastric cancers had distinct miRNAs expression profiles,suggesting that miRNAs were involved in the gastric carcinogenesis.In this study,we screened differentially expressed miRNAs between gastric tumor tissue and adjacent non-tumor tissue through microarray technology,and found that miR-141 was one of the most substantially down-regulated miRNAs.Therefore,we aimed to examine the expression of miR-141 in stomach cancer tissue and cell lines using quantitative Real-time PCR;and explore the effect of miR-141 on cancer progression through gastric cancer cell model in vivo.We hope our study could provide convincing evidence on miR-141’s association with gastric tumorigenesis,and facilitate the search of suitable candidates for cancer targeting therapy.1 Materials and Methods1.1 Tissue samples were obtained from 35 patients undergoing gastrectomy for gastric cancer in Sir Run Run Shaw Hospital,Zhejiang University School of Medicine, Hangzhou,P.R.China.Written informed consent was obtained before collection.Tumor samples were resected at surgery.Non-tumor samples from the macroscopic tumor margin were isolated at the same patients and used as the matched adjacent non-neoplastic tissue.Microarray technology was applied to screen differentially expressed miRNAs between gastric tumor tissue and adjacent non-tumor tissue.The expression of miR-141 was analyzed using Real-time PCR.Then,we investigated the association of clinical and pathological characteristics of cancer samples with miR-141 expression through independent sample t test and one-way ANOVA. 1.2 Expression of miR-141 in 5 gastric cancer cell lines originated from various differentiation stages was analyzed using Real-time PCR.The proliferation of these cells was also evaluated for the selection of suitable cell lines.1.3 MiR-141 in MGC-803 cell lines was over-expressed by miR-141 precursor molecule transfection.Phase contrast microscopy and MTT analyses were performed to investigate the effect of miR-141 over-expression on cell proliferation.1.4 Potential target genes of miR-141 were predicted by several bioinformatic programs,and their association with gastric pathogenesis was preliminarily investigated.2 Results2.1 Our microarray results showed that compared with the adjacent non-tumor tissue,miR-141 expression in tumor tissue was down-regulated by approximately 12.0 folds.Real-time PCR analysis displayed that the decrease of miR-141 was clearly found in 80%out of the 35 cases with a median change about 2.1 folds(P<0.01).However, we failed to observe any association of miR-141 expression with clinical and pathological parameters of gastric cancer.2.2 Gastric cancer cell line MGC-803 was found to be a suitable in vivo cell model. The expression of miR-141 in MGC-803 cells transfected with miR-141 precursor was up-regulated by approximately 7×10~4 folds compared with that in cells transfected with pre-miR negative control oligonucleotides.Under the observation of fluorescence microscopy,almost all cells transfected with pre-miR negative control showed red color, suggesting the miR-141 precursor transfection was successful and it over-expressed miR-141 level in MGC-803 cells.2.3 The phase-contrast microscopy and MTT assays showed that 48 h and 72 h after transfection,the proliferation of MGC-803 cells transfected with miR-141 precursor was significantly inhibited relative to that of cells transfected with pre-miR negative control(48 h,P<0.05;72 h,P<0.01).2.4 Using bioinformatic algorithms,we predicted FGFR2 and ZEB2 as potential targets of miR-141 involved in gastric carcinogenesis.However,quantative Real-time PCR analysis failed to show any significant changes of FGFR2 and ZEB2 mRNA expression in MGC-803 cells with miR-141 over-expression.3 Conclusions3.1 MiRNA microarray and quantitative Real-time PCR analyses showed that miR-141 was down-regulated in gastric tumor tissue compared with adjacent non-tumor tissue,suggesting that down-regulation of miR-141 is associated with the pathogenesis of stomach cancer.3.2 MiR-141 over-expression significantly inhibited the proliferation of MGC-803 cells,suggesting that the down-regulation of miR-141 may play a role in gastric cancer cell proliferation.3.3 Whether FGFR2 and ZEB2 were truly target genes of miR-141 which were associated with gastric tumorigenesis remains unclear.Further investigations are warranted to elucidate the inhibitory and targeting relations between these two genes with miR-141.更多还原