应用cDNA微阵列基因芯片筛选胃低分化腺癌相关基因的研究

背景与目的：胃低分化腺癌癌变的分子机制至今不清楚,关键是未找到与胃低分化腺癌密切相关的基因.本研究拟建立胃低分化腺癌基因表达谱,筛选差异表达基因,进一步分析差异表达基因与胃癌发生、发展关系.方法：用含10 000个已知基因的cDNA微阵列分析胃低分化腺癌和癌旁正常胃黏膜基因表达谱的变化,免疫组化研究差异表达基因与胃癌的关系.结果：二倍以上的差异表达基因212个,其中在胃低分化腺中表达上调169个,表达下调43个.S-P免疫组化结果显示：EMS1蛋白表达定位于胞质,呈黄色至棕黄色;EMS1蛋白在20例正常胃黏膜阳性表达率为20%（4/20）,在146 例胃癌中阳性表达率为89.72% （131/146）;EMS1蛋白在胃癌中的表达高于正常胃黏膜（P＜0.001）.结论：发现EMS1与胃癌有关,为进一步寻找胃癌相关基因提供了重要的研究线索.     

胃癌基因表达谱芯片差异基因的生物信息学分析

目的：筛选胃癌发生发展过程中的关键基因和信号通路,为寻找有价值的胃癌分子标志物提供依据。方法：从GEO数据库下载5个胃癌基因芯片数据集：GSE35809、GSE54129、GSE79973、GSE66229和GSE51105。合并5个数据集中的样本,去除数据集间的批次效应,对合并后的基因表达数据进行标准化,并通过主成分分析监测数据标准化情况。利用R语言中的limma包筛选胃癌组织和正常组织中表达差异的基因。利用DAVID数据库对胃癌发生发展过程中的差异基因进行功能富集分析,并通过STRING数据库和Cytoscape分析差异基因编码蛋白之间的相互作用网络并进行可视化。结果：总共筛选出1 205个差异基因,包括480个上调基因,725个下调基因。差异基因的生物学功能主要富集于细胞-细胞信号传导、炎症反应的调节、细胞粘附、细胞凋亡和离子的跨膜转运。KEGG信号通路分析显示差异基因主要富集于p53信号通路、PI3K-Akt信号通路、NF-κB信号通路。通过构建蛋白质相互作用网络筛选出了CENPE、KIF15、MELK、KIF2C、CENPF、KIF11、NUSAP1、UBE2C、TTK、AURKB、DLGAP5、TOP2A等29个Hub基因。结论：通过合并不同数据集,利用生物信息学方法筛选出胃癌发生发展过程中的关键基因和信号通路,为胃癌的诊疗提供新的候选标志物。     

Prognostic significance of aberrant gene methylation in gastric cancer

Promoter methylation acts as an important alternative to genetic alterations for gene inactivation in gastric carcinogenesis. Although a number of gastric cancer-associated genes have been found to be methylated in gastric cancer, valuable methylation markers for early diagnosis and prognostic evaluation of this cancer remain largely unknown. In the present study, we used methylation-specific PCR (MSP) to analyze promoter methylation of 9 gastric cancer-associated genes, including MLF1, MGMT, p16, RASSF2, hMLH1, HAND1, HRASLS, TM, and FLNc, and their association with clinicopathological characteristics and clinical outcome in a large cohort of gastric cancers. Our data showed that all of these genes were aberrantly methylated in gastric cancer, ranging from 8% to 51%. Moreover, gene methylation was strongly associated with certain clinicopathological characteristics, such as tumor differentiation, lymph node metastasis, and cancer-related death. Of interest, methylation of MGMT, p16, RASSF2, hMLH1, HAND1, and FLNc was closely associated with poor survival in gastric cancer, particularly MGMT, p16, RASSF2 and FLNc. Thus, our findings suggested these epigenetic events may contribute to the initiation and progression of gastric cancer. Importantly, methylation of some genes were closely relevant to poor prognosis in gastric cancer, providing the strong evidences that these hypermethylated genes may be served as valuable biomarkers for prognostic evaluation in this cancer.     

大鼠大肠癌模型建立及其基因表达谱初步研究

目的建立实验性大肠癌动物模型,利用基因芯片技术筛选大肠癌发病进程中差异表达基因,探讨大肠癌发生发展过程中可能的信号通路。方法利用二甲肼诱发大鼠大肠癌,收集4例大鼠中的息肉、腺瘤、癌组织及对应正常组织,提取总RNA,应用大鼠全基因组表达谱芯片检测疾病及对应正常组织的基因表达情况,筛选不同组织之间的差异表达基因,以实时荧光定量PCR技术对基因芯片检测结果进行验证,最后利用生物信息技术对差异表达基因进行通路及功能归类。结果获得成功建模大鼠4只。基因芯片结果显示,息肉组织、腺瘤组织及大肠癌组织与正常大肠黏膜组织相比,差异表达基因分别为493、3 472及3 060个。大肠癌序列相关基因在3种病理组织中的表达均发生不同程度的变化。通路分析结果显示,在息肉组织中,细胞因子-细胞因子受体相互作用通路相关基因变化最为显著,P=5.62E-08;在腺瘤组织中,嘌呤代谢通路相关基因变化最为显著,P=7.73E-30;而在大肠癌组织中,核糖体通路相关基因变化最为显著,P=1.40E-27。功能分析结果显示,生物过程功能类归属基因表达变化最多,分子功能类基因其次,而细胞组分类基因最少。RT-PCR验证结果与芯片结果完全相符。结论在成功建立大肠癌动物模型的基础上,通过芯片技术筛选和鉴定了大肠癌疾病发生及发展相关基因,建立了肠息肉、腺瘤及大肠癌组织基因表达谱,为探寻大肠癌早期发病机制及基因靶向治疗提供了分子依据。     

Primarily screening and analyzing ESTs differentially expressed in rats’ primary liver cancer

Objective: To screen and analyze key express sequence tags (ESTs) which were differentially displayed in every period of SD rats’ primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. Methods: Using diethylnitrosamine (DENA) as a cancerigenic agent, animal models with different phases of primary hepatic cancer were constructed in SD rats. Rats were respectively sacrificed at d 14, d 28, d 56, d 77, d 105 and d 112 after the rats received DENA by gavage, then the livers were harvested. One part of the livers was classified according to their pathological changes, while the other was reserved for molecular mechanism studies on hepatocarcinogenesis. The differentially expressed genes were isolated from both normal and morbid tissues by mRNA differential display technique (DDRT-PCR). After the fragments were sequenced, bioinformatics were used to analyze the results. Results: Twelve differentially expressed cDNA fragments were obtained. Nine fragments had the homology with known cDNA clones, especially EST-7 was similar to BN/SsNHsdMCW mitochondrion gene and the identity was 100% which suggested EST-7 may be the part of BN/SsNHsdMCW mitochondrion gene. In contrast, other three fragments (EST-1, EST-3 and EST-5) had extremely low identity to any genes registered in GENBANK databases. Conclusions: BN/SsNHsdMCW mitochondrion gene was expressed in different periods of hepatocarcinogenesis. Moreover, EST-1, EST-3 and EST-5 were suggested to contribute to the development of rat hepatocarcinogenesis, and thus may be candidates of new targets of oncogenes or cancer suppressor genes.     

胃癌及癌前病变差异表达基因的初步研究

目的:寻找胃癌、癌前病变中差异表达的基因,以确定胃癌发生前胃粘膜的分子变化方法:应用荧光mRNA差异显示技术分析胃癌(3例)、癌前病变(3例)和正常胃粘膜(3例),鉴别并分离差异表达的基因片段,进行PCR再扩增将扩增的cDNA片段克隆后测序,测序结果提交GenBank,经BLASTN软件检索以进行同源性分析.差异条带经Northern印迹验证.结果:发现2个差异表达的cDNA片段,1个在正常组织和癌前病变中高表达的cDNA片段在GenBank数据库中未找到同源序列,获得GenBank登陆号CB833297,并由UNIGENE基因库收录,为新基因片段.另1个在正常胃粘膜组织中高表达的cDNA片段,在GenBank数据库中与RP11-315A19克隆高度同源,但其功能目前尚不清楚.结论:本研究发现的2个在胃癌、癌前病变及正常胃粘膜组织中差异表达的基因,它们可能参与了胃癌的发病过程.     

胃癌患者外周血与癌组织基因差异表达比较

[目的]利用基因芯片技术筛选与早期胃癌癌变相关的基因.[方法]用U133A基因芯片分别检测胃癌组(T)、癌旁上皮组(P)和切缘正常胃黏膜上皮组(C)及胃癌患者外周血(WB)和正常对照组外周血(NB)的基因表达谱,对荧光强度进行扫描并数字化,用专业软件对检测结果进行分析.[结果]胃癌、癌旁和正常胃黏膜上皮相比,有327个基因共同表达上调,211个基因共同表达下调.胃癌和癌旁上皮同时有差异表达的基因中,在外周血也有差异表达,其中同时表达上调有39个,同时表达下调有4个.[结论]虽然在癌旁上皮病理组织图像上尚未见异常,但在基因表达水平上已经显示有多个与胃癌相关基因表达.尤其在患者外周血有核细胞的基因表达中,也发现某些基因与胃癌及癌旁基因差异表达正相关.提示这些基因可能与早期胃癌的启动和演化有关,通过外周血有核细胞基因芯片检查可能早期发现胃癌患者,提高患者的治愈率.     

基于加权基因共表达网络分析构建胃癌转移预测模型

背景与目的：通过对高通量功能基因组数据库（Gene Expression Omnibus,GEO）中一组含有转移和非转移性胃癌以及癌旁组织的基因芯片进行加权基因共表达网络分析（weighted gene co-expression network analysis,WGCNA）,筛选出与胃癌发生和转移显著相关的分子,为胃癌的治疗和生存期延长的研究提供参考。方法：采用WGCNA方法对19例胃癌患者基因表达进行差异分析;结合临床数据,选取与临床信息高度相关的基因模块构建网络。结果：利用WGCNA我们筛选出了Lightsteelblue模块与胃癌转移明确相关,同时对模块中的基因进一步进行分析,筛选出4个基因：C5AR1、AP3M2、TYMP、ANXA2P1作为核心靶基因。通过表达分析和受试者工作特征（receiver operating characteristic,ROC）曲线分析验证上述基因与胃癌发生、转移明确相关。同时,通过外部ONCOMINE和Kaplan-Meier plot数据库验证上述基因在胃癌中高表达,高表达这些基因的患者有着更差的预后。并利用GSE14210数据集构建基于这些基因的预测患者预后和疾病进展模型。结果提示我们所筛选的4个基因具有成为潜在胃癌转移和治疗生物标志物的可能。结论：鉴定筛选出与胃癌发生和转移相关的4个基因,可为胃癌发生、转移和治疗的研究提供参考。     

Identification and characterization of genes involved in the carcinogenesis of human squamous cell cervical carcinoma

We utilized RT-PCR differential display and cDNA microarrays to identify cellular genes involved in the multi-step carcinogenesis of squamous cell cervical carcinoma. Thirty-eight cervical cancer patients in various stages of the disease and 5 non-cervical cancer patients were studied. Twenty-five cDNA clones were identified and these were subsequently demonstrated to be consistently over-expressed in squamous cell cervical carcinoma biopsies of various FIGO stages. To further evaluate the possible role that these genes may play in the progression of disease, we performed Northern blot analysis and RNA-RNA in situ hybridization studies using cervical cancer biopsies of various FIGO stages. Of particular interest are the 2 clones G32C4B and G30CC that have been identified to be the NADH dehydrogenase 4 gene and the gene that encodes ribosomal protein S12 respectively when compared to sequences available in the GenBank database. Increased expression of these 2 genes were detected in the matched normal tissues collected together with the late FIGO stages of cervical cancer biopsies. In comparison, upregulation of these 2 genes was not detected in cervical squamous epithelium collected from patients admitted for surgery for non-malignant conditions, suggesting that expression of these 2 genes may have altered in the adjacent histopathologically "normal" cervical squamous epithelial tissue from cervical cancer patients. The ribosomal protein S12 and the NADH dehydrogenase 4 genes may therefore be potentially useful as early pre-transformation diagnostic markers for human cervical cancer.     

核糖体蛋白S24基因在胃癌及其癌前病变中差异表达的研究

背景与目的:将胃癌组织、正常胃黏膜及胃癌前病变的基因表达进行比较,找出与人胃癌密切相关的基因片段,为进一步探讨胃癌的发生机制、胃癌的早期诊断和治疗方案提供重要的理论根据。材料与方法:应用荧光mRNA差异显示技术(FluorescentmRNAdifferentialdisplay,FDD)分析3例胃癌、3例正常胃粘膜、3例胃癌前病变组织,分析基因差异表达情况。分离差异表达基因片段,进行PCR扩增。将cDNA片段测序,测序结果提交Genbank,经BLAST软件检索与同源性分析。选取其中差异表达的核糖体蛋白S24(RibosomalproteinS24,RPS24)基因,应用Northern杂交进行验证,并进一步对RPS24基因进行基因表达系列分析和组织表达谱分析。结果:mRNA差异显示发现RPS24基因在胃癌组织中的表达明显高于癌前病变和正常胃黏膜组织,并且Northern印迹验证阳性。基因表达系列分析和组织表达谱分析发现RPS24基因在多种肿瘤组织中表达增高以及分布广泛。结论:与非癌组织相比,RPS24基因在胃癌中表达明显增高,RPS24的高表达可能与胃癌的发生、恶性进展有关。     

MAD2和p27在大肠癌组织中的表达及意义

目的探讨MAD2和p27表达与大肠癌发生、发展的关系。方法采用实时荧光定量PCR和免疫组织化学方法,检测大肠癌、腺瘤及正常大肠黏膜组织中MAD2表达,并对大肠癌组织中MAD2扩增产物进行测序。同时应用免疫组织化学方法检测大肠癌和正常组织中p27表达情况。结果大肠癌组织中MAD2阳性表达率明显高于腺瘤和正常大肠黏膜组织（66．7％VS39．6％VS22．9％）,三者间比较差异均有统计学意义（P〈0．01）。MAD2表达与大肠癌肿瘤分化和患者无瘤生存时间有关（P〈0．05）。大肠癌组织中未见MAD2基因突变。正常大肠黏膜组织中p27阳性表达率为81．3％,大肠癌中其阳性表达率为41．7％,两者比较差异有统计学意义（P〈0．05）。结论MAD2和p27基因表达与大肠癌的发生及发展有关。MAD2可能是大肠癌预后的1个重要指标。     

Down-regulation of FHIT inhibits apoptosis of colorectal cancer: mechanism and clinical implication

Fragile histidine triad (FHIT) gene, a candidate tumor suppressor gene located at 3p14.2, has been shown to be involved in carcinogenesis of many human tissues, including digestive tract tissues. However, the expression and role of FHIT in the initiation and the development of the colorectal cancer (CRC) are poorly understood. In our present study, we have demonstrated that the FHIT gene exhibits significantly decreased expression in human CRC compared to colorectal adenoma and normal colorectal tissue by tissue microarray (TMA). The positive of FHIT gene expression in normal colorectal tissue, adenoma and adenocarcinoma were 93.75%, 68.75% and 46.25%, respectively. We showed that decreased FHIT expression was significantly correlated with the progression of colorectal carcinoma (P<0.05) as well as differentiation and lymph node metastasis (P<0.05). Two somatic mutations in the FHIT gene were also detected in human CRC. The presence of these mutations correlated significantly with decreased FHIT expression in the human CRC. In addition, we identified decreased FHIT expression resulting in apoptosis inhibition and decreasing apoptosis associated with abnormal levels of some pro- and anti-apoptotic proteins (Bax, Bcl-2 and Survivin) by TUNEL and TMA. Our results demonstrated that the mutation in the FHIT gene significantly reduced FHIT expression in human CRC. Both TUNEL and TMA experiments demonstrated significantly inhibited apoptosis by down-regulation of Bax and up-regulation of Survivin and Bcl-2. Collectively, these studies identify the mechanism by which an important tumor suppressor gene, FHIT, inactivated specifically in human CRC, and contributes to our understanding of the mechanism of colorectal carcinogenesis.     

TSA对人胃癌细胞SGC-7901基因表达的影响

目的：研究乙酰化转移酶抑制剂（TSA）对人胃癌细胞SGC-7901基因表达的影响,初步探讨TSA对胃癌细胞抑制作用的机理.方法：用含有22575个样点,覆盖21073个人类基因的Agilent Human 1A基因表达谱芯片来研究TSA对胃癌细胞基因表达的影响.分离纯化未作任何处理的胃癌细胞和用TSA处理过的胃癌细胞mRNA,制备荧光标记的cRNA靶物（Cy3或Cy5标记）.靶物与基因芯片杂交,用Axon 4000B型扫描仪进行芯片荧光信号扫描.利用计算机分析用TSA处理过的胃癌细胞和未处理的胃癌细胞基因表达的差异.同时利用real time PCR验证部分基因的改变.结果：在21073个基因中,有显著差异的基因共4710个基因,其中TSA处理组相对于未处理组基因上调2倍以上的有905个,下调0.5倍以下的基因3805个.结论：TSA通过多基因共同作用来抑制胃癌细胞的生长.这些基因在胃癌的发病中可能起着非常重要的作用.     

CD147和Ki-67在甲状腺乳头状癌中的表达及意义

目的:探讨甲状腺乳头状癌组织中CD147和Ki-67表达情况及临床意义。方法:应用免疫组化法检测甲状腺乳头状癌、滤泡性腺瘤和正常甲状腺组织（各50例）中CD147和Ki-67的表达。结果:甲状腺乳头状癌组织中CD147阳性表达率明显高于滤泡性腺瘤和正常甲状腺组织（68％ vs 20% vs 0）,三者比较差异有统计学意义（P<0.001）。CD147表达与淋巴结转移和TNM分期有关（P<0.05）。正常甲状腺组织中Ki-67未见阳性表达,甲状腺乳头状癌、滤泡性腺瘤中阳性表达率为（36％ vs 18%）,三者比较差异有统计学意义（P<0.05）。结论:CD147和Ki-67表达异常是甲状腺乳头状癌产生的机制之一,与甲状腺乳头状癌的发生及发展有关。CD147可能是甲状腺乳头状癌预后的一项重要预测指标。     

Comparison of gene-expression profiles between diffuse- and intestinal-type gastric cancers using a genome-wide cDNA microarray

Gastric cancer is the fourth leading cause of cancer-related death in the world. Two histologically distinct types of gastric carcinoma, 'intestinal' and 'diffuse', have different epidemiological and pathophysiological features that suggest different mechanisms of carcinogenesis. A number of studies have investigated intestinal-type gastric cancers at the molecular level, but little is known about mechanisms involved in the diffuse type, which has a more invasive phenotype and poorer prognosis. To clarify the mechanisms that underlie its development and/or progression, we compared the expression profiles of 20 laser-microbeam-microdissected diffuse-type gastric-cancer tissues with corresponding noncancerous mucosae by means of a cDNA microarray containing 23,040 genes. We identified 153 genes that were commonly upregulated and more than 1500 that were commonly downregulated in the tumors. We also identified a number of genes related to tumor progression. Furthermore, comparison of the expression profiles of diffuse-type with those of intestinal-type gastric cancers identified 46 genes that may represent distinct molecular signatures of each histological type. The putative signature of diffuse-type cancer exhibited altered expression of genes related to cell-matrix interaction and extracellular-matrix (ECM) components, whereas that of intestinal-type cancer represented enhancement of cell growth. These data provide insight into different mechanisms underlying gastric carcinogenesis and may also serve as a starting point for identifying novel diagnostic markers and/or therapeutic targets for diffuse-type gastric cancers.     

Klotho is silenced through promoter hypermethylation in gastric cancer

As one of major epigenetic changes to inactivate tumor suppressor genes in human carcinogenesis, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes and predict the prognosis of cancer patients. In the present study, we found KL (klotho) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. KL expression was downregulated in primary gastric carcinoma tissues (n=22, p<0.05) and all of gastric cancer cells lines examined. Ectopic expression of KL inhibited the growth of gastric cancer cells partially through the induction of apoptosis, demonstrating a tumor suppressive role of KL in gastric cancer. Demethylation with 5-aza-2'-deoxycytidine (Aza) increased KL expression and KL promoter was hypermethylated in gastric cancer cell lines as well as some of primary gastric carcinoma tissues (47/99) but none of normal gastric tissues. Importantly, promoter methylation of KL was significantly associated with the poor outcome of gastric cancer patients (p=0.025, Log-rank test), highlighting the relevance of epigenetic inactivation of KL in gastric carcinogenesis. As a summary, we found that KL is a novel tumor suppressor gene epigenetically inactivated in gastric cancer and promoter methylation of KL could be used to predict the prognosis of gastric cancer patients.     

人胃癌腹膜高转移潜能细胞系的基因表达分析

目的:比较人胃癌细胞系与其腹膜高转移潜能细胞系的基因表达谱,寻找与胃癌腹膜转移相关的基因表达改变。方法:采用基因芯片技术,比较遗传背景相同但转移力有明显差异的两个细胞系GC9811和GC9811-P,分析其基因表达谱的差异。选取部分基因行RT-PCR进一步验证基因芯片的准确性。结果:在11901个候选基因中,筛选出248个(2.1%)差异表达基因。GC9811-P与GC9811相比,表达上调的基因有218个,下调的有30个,包括DNA合成和错配修复基因如H3F3A、细胞增生基因、蛋白合成与修饰基因、信号传导基因和离子通道与运输蛋白相关基因等。半定量RT-PCR对PTEN、S100A4和ZNRD1基因检测结果验证了基因芯片数据的可靠性。结论:胃癌腹膜转移是多基因作用的综合结果,筛选的基因对预测胃癌腹膜转移和抗转移干预措施可能有指导意义。