【作者】 张文辉；

【导师】 吴本俨；

【作者基本信息】 中国人民解放军军医进修学院 ， 老年医学， 2010， 博士

【摘要】 研究背景：微小RNAs (micro RNAs, miRNAs)是一类长约21～25 nt的非编码单链RNA分子,广泛存在于动植物中。它通过与靶mRNA完全或不完全的互补配对,产生基因沉默、发挥负性调控基因表达的作用。研究发现,特定的miRNA在细胞增殖、分化、凋亡、基因调控及肿瘤的发生中扮演重要的角色。其在胃癌的发生、发展中起癌基因或者抑癌基因的作用,为胃癌的诊断、治疗和预后评估提供了新的工具。但迄今为止,真正确认功能的miRNA还微乎其微,而且其发挥作用的具体机制也有待进一步的研究以明确。已有报道,与胃远端腺癌相比,贲门腺癌不仅位置独特,发病机制也存在不同。目的：筛选胃远端腺癌中差异表达的miRNAs,观察其与临床病理因素的关系,并就其在胃癌发生、发展中的作用及其机制进行初步的研究。方法：1.取我科实验室液氮冻存的胃远端腺癌及配对正常组织进行HE染色,确认病理情况；采用Agilent人miRNA寡核苷酸基因芯片,对3对胃远端(体、窦)腺癌组织及配对正常组织进行检测,筛选胃远端腺癌差异表达的miRNAs.2.选择特定miRNAs,在20例胃远端腺癌及配对正常组织中用实时荧光定量PCR进行验证,并观察其与患者年龄、肿瘤大小、分期、淋巴结转移等临床病理因素的关系；在得到确认差异表达的miRNAs后,同法观察其在17例贲门腺癌与胃远端腺癌之间表达是否存在差异、胃腺癌患者血清中的表达状况、人胃粘膜细胞系与人胃癌细胞系的表达状况。3.选择特定miRNA转染人胃癌细胞系,运用MTS法、流式细胞术观察其对胃癌细胞增殖、凋亡的影响。4.运用TargetScan等生物信息学方法,预测特定miRNA可能的靶基因。结果：1.通过对液氮冻存胃远端腺癌及配对正常组织的HE染色,确认了各自的病理状况;miRNAs芯片检测,发现共47条miRNAs在胃远端腺癌组织和配对正常组织中表达差异显著,上调的24条,下调的23条；在这些差异表达的miRNAs中,miR-204. miR-196b、miR-101、miR-107等27条首次报道于胃远端腺癌中;2.选择3个高表达miRNAs:miR-18b、miR-93、miR-196b和2个低表达的miRNAs:miR-204、miR-375进行实时荧光定量PCR分析,显示在胃远端腺癌患者样本中,miR-196b、miR-204、miR-375的表达情况与芯片一致,但其与患者年龄、癌栓有无、肿瘤直径、分化程度、分期、淋巴结转移情况均没有关联；同时,miR-204水平在胃腺癌患者血清中显著升高,ROC曲线分析显示,血清miR-204水平对胃腺癌患者和健康对照的鉴别效能为0.844(95%CI：0.579-0.973,p=0.002),敏感性和特异性分别为100%和75%,最佳cut-off值为0.0343(2-△ct,相对于U6 snRNA);在贲门腺癌和胃远端腺癌中,miR-375表达均下调,而胃远端腺癌下调的更明显,两者有显著性差异。3.转染成熟miR-204 mimics于人胃癌细胞系MGC-803、BGC-823.SGC-7901,发现不论是不同转染浓度,抑或是不同时间点,对肿瘤细胞的增殖、凋亡没有明显的影响。4.生物信息学显示BCL-2, NR3C1, SOCS6等可能为miR-204的靶基因。结论：1.胃远端腺癌有其独特的miRNA表达谱；2.验证的差异表达miRNAs与胃远端腺癌临床病理情况无明显相关；贲门腺癌与远端胃腺癌存在miRNA的表达差异；血清miR-204可能作为胃腺癌潜在的分子标志物。3.过表达miR-204对胃癌细胞系的增殖和凋亡没有明显的影响。4.预测BCL-2, NR3C1和SOCS6等可能为miR-204的靶基因。更多还原

【Abstract】 MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 21-25 nt that are found in diverse organisms including plants and animals and involve in negatively post-transcriptional gene expression regulation, mainly through partial base-pairing to the complementary sites within the 3’-UTR (3’-untranslated region,3’-UTR) of their target messenger RNAs (mRNAs). Through years of researches, it was found that miRNAs play a very important role in controlling various cellular processes such as proliferation, differentiation, apoptosis and tumorigenesis. Recently, some miRNAs were observed to function as oncogene or tumor suppressor in the initiation and development of gastric cancer, which provides a novel tool for the diagnosis, treatment and prognostic evaluation of gastric cancer. However, the biological functions of most miRNAs are still largely illusive until now and their functional mechanisms need to be further investigated. Additionally, Several stydies have reported that cardia adenocarcinoma has its specific pathogenesis.To screen and identify the differentially expressed miRNAs in distal (corpus and antrum) gastric adenocarcinoma and then evaluate the relationship of miRNAs’ expression and clinicopathological factors in patients with distal gastric adenocarcinoma. Furthermore, the functional mechanism of corresponding miRNAs in the course of development and progression of gastric carcinoma were preliminarily investigated.1. Three distal gastric adenocarcinoma tissues and their paired normal tissues conserved in liquid nitrogen were selected from the tissues archives in the Labs of our department and their pathological features were confirmed through HE staining.2. Agilent human oligonucleotide miRNA microarray were used to profile miRNAs expression in aforementioned matched tissues. Furthermore, five miRNAs with differential expressions in distal gastric adenocarcinoma were selected and validated in an independent set of 20 distal gastric adenocarcinoma and paired normal tissues by using real-time RT-PCR. Then, the relationships between miRNAs expression levels and age, tumor size, staging, grading and lymphonode metastasis were investigated. Additionally, the differential expression levels between 17 cardia adenocarcinoma and distal gastric adenocarcinoma, the expression conditions in serum and several gastric tumor cell lines were observed by using the same method.3. MTS method and flow cytometry were employed to observe the proliferation and apoptosis of tumor cells that transfected with specific miRNA.4、To predict the possible targets of miRNA through bioinformatics.1. Pathological conditions are validated through HE staining for distal gastric adenocarcinoma and paired normal tissues. By using miRNAs microarray,47 miRNAs were found to be differentially expressed between tumor tissues and normal tissues, with 24 miRNAs up-regulated while 23 down-regulated respectively. Among these miRNAs, miR-204, miR-196b, miR-101, miR-107 and other 23 miRNAs are reported here in distal gastric adenocarcinoma for the first time.2. Differential expression levels of miR-196b, miR-204 and miR-375 found with miRNA microarray are validated by using real time RT-PCR. However, no relationship between the expression levels of these three miRNAs and clinopathological factors is observed. In addition, level of miR-204 is found to increased in serum of gastric adenocarcinoma comparing normal controls. It yields an AUC (the area under the ROC curve) of 0.844 (95% CI:0.579-0.973; p=0.002) with 100% sensitivity and 75% specificity in discriminating gastric adenocarcinoma from healthy controls, using the cut-off value 0.0343 (normalized). The expressions of miR-375 in distal gastric adenocarcinma and cardia adenocarcinoma are both decreased, while the latter is more significant.3. There are no significant impact on the proliferation and apoptosis of gastric tumor cell lines after transfecting mature miR-204 mimics, independent of the transfectional concentration or time points.4. Bioinformatics indicates that BCL-2, NR3C1 and SOCS6 may be the targets of miR-204.1.There is a specific miRNA profile in distal gastric adenocarcinoma.2.There are no significant correlation between levels of specific miRNAs and clinopathological factors in distal gastric adenocarcinoma. The levels of specific miRNAs in distal gastric adenocarcinoma and cardia adenocarcinoma are different. The abundance of miR-204 is significant higher in patients with gastric adenocarcinoma comparing healthy controls. And it may be a potential biomarker for gastric adenocarcinoma.3. There are no significant impact on the proliferation and apoptosis of gastric tumor cell lines after over expression of mature miR-204.4. BCL-2, NR3C1 and SOCS6 may be targets of miR-204.更多还原