Two novel gastric cancer-associated genes identified by differential display

AIM To clone novel gastric cancer-associated genes and investigate their roles in gastric cancer occurrence.METHODS A method called differential display was used which allows the identification of differentially expressed genes by using PAGE to display PCR-amplified cDNA fragments between gastric cancer cells and normal gastric mucosa cells. These fragments were cloned into plasmid vector pUC18. Homology analysis was made after sequencing these fragments.RESULTS Two novel genes were identified compared with sequences from GenBank. One was registered with the AD number AF 051783. In situ hybridization showed that these two novel genes expressed specifically in gastric cancer tissues.CONCLUSION The two novel genes obtained by differential display were confirmed to be gastric cancer-associated genes using in situ hybridization.     

Differential display of vincristine-resistance-related genes in gastric cancer SGC7901 cell

AIM: To isolate and clone the vincristine-resistance-related genes in gastric cancer SGC7901 cell line and to clarify the multidrug-resistant molecular mechanism of gastric cancer cells. METHODS: The modified differential-display polymerase chain reaction (DD-PCR) was used to examine differences in the mRNA composition of Vincristine-resistant gastric cancer SGC 7901 cells (SGC7901/VCR), induced by vincristine sulfate versus SGC7901cells. The differentially expressed cDNA fragments were confirmed by reverse Northern analysis, sequencing, BLAST analysis and Northern bolt analysis. RESULTS: The DD-PCR identified that 54 cDNA fragments were preferentially expressed in SGC 7901/VCR cells. When these cDNA fragments were analyzed by reverse Northern blot, twenty were reproducibly expressed at high level in SGC7901/VCR. Sequencing and BLAST analysis revealed that seven of the genes were known genes:ADP-ribosylation factor 4, Cytochrome oxidase subunit II, Ss-A/Ro ribonucleoprtein autoantigen 60kd subunit,ribosomal protein S13, galaectin-8 gene, oligophrenin 1 mRNA, ribosomal protein L23 mRNA; thirteen of the genes were unknown genes. The length and abundance of the four unknown genes were further confirmed by Northern blot analysis. CONCLUSION: The twenty differential known and unknown genes may be related to the vincristine-resistant mechanism in human gastric cancer SGC7901 cell line.     

采用单引物AP-PCR技术对胃癌相关基因片段的分离分析

目的 分离与胃癌发生相关的基因片段并予以鉴定。方法     

Intracellular and interstitial expression of Helicobacter pylori virulence genes in gastric precancerous intestinal metaplasia and adenocarcinoma

Gastric intestinal metaplasia (IM) and gastric cancer are associated with Helicobacter pylori, but the bacterium often is undetectable in these lesions. To unravel this apparent paradox, IM, H. pylori presence, and the expression of H. pylori virulence genes were quantified concurrently using histologic testing, in situ hybridization, and immunohistochemistry. H. pylori was detected inside metaplastic, dysplastic, and neoplastic epithelial cells, and cagA and babA2 expression was colocalized. Importantly, expression of cagA was significantly higher in patients with IM and adenocarcinoma than in control subjects. The preneoplastic "acidic" MUC2 mucin was detected only in the presence of H. pylori, and MUC2 expression was higher in patients with IM, dysplasia, and cancer. These novel findings are compatible with the hypothesis that all stages of gastric carcinogenesis are fostered by persistent intracellular expression of H. pylori virulence genes, especially cagA inside MUC2-producing precancerous gastric cells and pleomorphic cancer cells.     

肿瘤相关基因NAG6、NAG-7、BRD7在胃癌中的表达

目的　NAG6、NAG 7、BRD7为最近克隆的肿瘤相关新基因 ,对它们在胃癌及癌旁组织中的表达进行检测 ,探讨这些基因在胃癌发生发展中的作用。方法　采用RT PCR、Northern杂交、点杂交方法检测 34例胃癌和癌旁组织中这些基因的表达。结果　胃癌组织中 ,有 61 .8% (2 1 / 34)NAG6基因表达缺失 ,NAG6在胃癌组织中的表达较癌旁组织显著下调 (P <0 .0 1 ) ,但NAG6表达下调与胃癌淋巴结或远处转移无明显关系 (P >0 .0 5)。NAG 7基因在胃癌和癌旁组织中表达率分别为 88.2 % (30 / 34)和 82 .3 % (2 8/ 34)。BRD7基因在胃癌及癌旁组织中均有表达。RT PCR、Northern杂交、点杂交结果均显示NAG 7、BRD7基因在胃癌与癌旁组织之间的表达差异无显著性。同时 ,点杂交也证实NAG6在胃癌组织中表达下调。结论　NAG6基因在胃癌组织中表达显著下调 ,这可能在胃癌的发生发展过程中起重要作用 ,但可能与淋巴结或远处转移无关。BRD7、NAG 7基因在胃癌中未发现表达水平改变 ,初步提示这两种基因可能在胃癌的发生发展中不起作用     

Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues

Background and Aim:  MicroRNAs (miRNAs) play important roles in carcinogenesis. The global miRNA expression profile of gastric cancer has not been reported. The purpose of the present study was to determine the miRNA expression profile of gastric cancer.
Methods:  Total RNA were first extracted from primary gastric cancer tissues and adjacent non-tumorous tissues and then small isolated RNAs (< 300 nt) were 3'-extended with a poly(A) tail. Hybridization was carried out on a μParaflo™ microfluidic chip (LC Sciences, Houston, TX, USA). After hybridization detection by fluorescence labeling using tag-specific Cy3 and Cy5 dyes, hybridization images were collected using a laser scanner and digitized using Array-Pro image analysis software (Media Cybernetics, Silver Spring, MD, USA). To validate the results and investigate the biological meaning of differential expressed miRNAs, immunohistochemistry was used to detect the differential expression of target genes.
Results:  The most highly expressed miRNAs in non-tumorous tissues were miR-768-3p, miR-139-5p, miR-378, miR-31, miR-195, miR-497 and miR-133b. Three of them, miR-139-5p, miR-497 and miR-768-3p, were first found in non-tumorous tissues. The most highly expressed miRNAs in gastric cancer tissues were miR-20b, miR-20a, miR-17, miR-106a, miR-18a, miR-21, miR-106b, miR-18b, miR-421, miR-340*, miR-19a and miR-658. Among them, miR-340*, miR-421 and miR-658 were first found highly expressed in cancer cells. The expression of some target genes (such as Rb and PTEN ) in cancer tissues was found to be decreased.
Conclusion:  To our knowledge, this is the first report about these miRNAs associated with gastric cancer. This new information may suggest the potential roles of these miRNAs in the diagnosis of gastric cancer.     

Differential display of vincristine-resistance-related genes in gastric cancer SGC7901 cells

AIM: To isolate and clone the vincristine-resistine-relatedgenes in gastric cancer SGC7901 cell line and to clarify themultidrug-resistant molecular mechanism of gastric cancercells.METHODS: The modified differential-display polymerasechain reaction (DD-PCR) was used to examine thedifferences in the mRNA composition of Vincristine-resistantgastric cancer SGC 7901 cells (SGC7901/VCR), induced byvincristine sulfate versus SGC7901cells. The differentiallyexpressed cDNA fragments were confirmed byreverseNorthern analysis, sequencing, BLAST analysis andNorthern bolt analysis.RESULTS: DD-PCR identified that 54 cDNA fragments werepreferentially expressed in SGC 7901/VCR cells. When thesecDNA fragments were analyzed by reverseNorthern blot, 20were reproducibly expressed at a high level in SGC7901/VCR. Sequencing and BLAST analysis revealed that sevenof the genes were known genes: ADP-ribosylation factor 4,cytochrorne oxidase subunit Ⅱ, Ss-A/Ro ribonucleoprteinautoantigen 60kd subunit, ribosomal protein S13, galaectin-8 gene, oligophrenin 1 mRNA, and ribosomal protein L23mRNA; and thirteen of the genes were unknown genes. Thelength and abundance of the four unknown genes mRNAwere further confirmed by Northern blot analysis.CONCLUSION: The twenty differential known and unknowngenes may be related to the vincristine-resistant mechanismin human gastric cancer SGC7901 cell line.     

Identification of mouse genes with highly specific expression patterns in differentiated intestinal epithelium

BACKGROUND & AIMS: Few genes that regulate intestinal epithelium development, homeostasis, or function are known. We reasoned that potential candidate regulators of these processes could be identified based on their activation during intestinal epithelium development and their subsequent specific and restricted expression. METHODS: Genes were identified by differential display and microarray analyses, further selected according to sequence and UniGene expression profiles, and analyzed by RNA in situ hybridization of mouse fetal and adult intestines and in intestinal polyp tissue. RESULTS: Five genes with unknown physiological function predominantly or exclusively expressed in the intestinal epithelium were identified. Their expression is activated at distinct times during intestinal development and maturation and is maintained in highly specific, spatially distinct patterns in the adult intestinal epithelium. Two of the genes were up-regulated in intestinal tumors, 1 was down-regulated, and 2 were apparently unaltered. CONCLUSIONS: Based on sequence and expression, the identified genes represent good candidates for regulators of intestinal epithelium integrity or function. Their expression patterns suggest a morphologically not obvious molecular regionalization of the intestinal epithelium along the crypt villus axis. This approach should be an efficient means to identify novel genes required for intestinal epithelium homeostasis and function.     

Expression of somatostatin mRNA in various differentiated types of gastric carcinoma

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Gene expression profiling reveals sequential changes in gastric tubular adenoma and carcinoma in situ

AIM: To analyze the expression profiles of premalignant and/or preclinical lesions of gastric cancers. METHODS: We analyzed the expression profiles of normal gastric pit, tubular adenoma and carcinoma in situ using microdissected cells from routine gastric biopsies. For the DNA microarray analysis of formalin-fixed samples, we developed a simple and reproducible RNA extraction and linear amplification procedure applying two polymerasebinding sites. The amplification procedure took only 8 h and yielded comparable DNA microarray data between formalin-fixed tissues and unfixed controls. RESULTS: In comparison with normal pit, adenoma/ carcinoma showed 504 up-regulated and 29 down-regulated genes at the expected false significance rate 0.15%. The differential expression between adenoma and carcinoma in situ was subtle: 50 and 22 genes were up-, and downregulated in carcinomas at the expected false significance rate of 0.61%, respectively. Differentially expressed genes were grouped according to patterns of the sequential changes for the 'tendency analysis' in the gastric mucosa-adenoma-carcinoma sequence. CONCLUSION: Groups of genes are shown to reflect the sequential expression changes in the early carcinogenic steps of stomach cancer. It is suggested that molecular carcinogenic pathways could be analyzed using routinely processed biopsies.     

Laser-mediated microdissection facilitates analysis of area-specific gene expression in rheumatoid synovium

OBJECTIVE: Current approaches to analyzing gene expression in rheumatoid arthritis (RA) synovium are based on RNA isolated either from cultured synovial cells or from synovial biopsy specimens. This strategy does not, in general, allow distinction of specific gene expression between cells originating from different synovial areas, due to potential mixture of expression profiles. Therefore, we established the combination of laser-mediated microdissection (LMM) and differential display to analyze profiles of gene expression in histologically defined areas of rheumatoid synovium. The present study was undertaken to establish parameters for this technique and assess its usefulness for gene expression analysis. METHODS: Cryosections derived from RA synovial tissues were used to obtain cell samples from synovial lining versus sublining, using a microbeam laser microscope. RNA was isolated and analyzed by nested RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) for differential display fingerprinting. Differentially expressed bands were cut out, and PCR products were eluted, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry analysis. RESULTS: Microdissected sections of RA synovial tissue containing approximately 600 cells yielded enough RNA to produce a reproducible RNA fingerprint pattern. Several genes could be identified as being expressed differentially between the synovial lining and the sublining, and their expression could be confirmed at the messenger RNA and protein levels. CONCLUSION: The combination of LMM and RAP-PCR presents a valuable tool to obtain novel insights into the area-dependent differential regulation of gene expression in RA synovium. Both known and previously unknown genes were revealed with this technique. This study is the first to demonstrate the potential of this analytic strategy in the investigation of a nonmalignant, multifactorial, inflammatory disease.     

DNA methylation in gastric cancer,related to Helicobacter pylori and Epstein-Barr virus

Gastric cancer is a leading cause of cancer death worldwide,and significant effort has been focused on clarifying the pathology of gastric cancer.In particular,the development of genome-wide analysis tools has enabled the detection of genetic and epigenetic alterations in gastric cancer;for example,aberrant DNA methylation in gene promoter regions is thought to play a crucial role in gastric carcinogenesis.The etiological viewpoint is also essential for the study of gastric cancers,and two distinct pathogens,Helicobacter pylori(H.pylori)and Epstein-Barr virus(EBV),are known to participate in gastric carcinogenesis.Chronic inflammation of the gastric epithelium due to H.pylori infection induces aberrant polyclonal methylation that may lead to an increased risk of gastric cancer.In addition,EBV infection is known to cause extensive methylation,and EBV-positive gastric cancers display a high methylation epigenotype,in which aberrant methylation extends to not only Polycomb repressive complex(PRC)-target genes in embryonic stem cells but also non-PRC-target genes.Here,we review aberrant DNA methylation in gastric cancer and the association between methylation and infection with H.pylori and EBV.     

mRNA差异显示技术分离猬迭宫绦虫幼虫特异表达基因

目的　查找猬迭宫绦虫幼虫裂头蚴阶段特异性表达基因。　方法　裂头蚴和成虫组织用异硫氢酸胍一步法提取总 RNA,用 DNA酶去除总 RNA中污染的 DNA。使用 T1 2 MA、T1 2 MC、T1 2 MG和 T1 2 MT4种锚定引物反转录合成 c DNA,再用 1种随机引物与上述 4种锚定引物在含同位素的反应液中进行 PCR反应。将 PCR产物用变性聚丙烯酰胺凝胶电泳分离 ,经放射自显影后 ,从凝胶上选出裂头蚴与成虫不同的差异带 ,PCR扩增后经杂交试验鉴定出不同种类的基因片段 ;以筛选出的差异带作探针 ,分别与裂头蚴和成虫 RNA进行 Northern杂交证实裂头蚴阶段表达基因 ;将差异带测序后与 Gen Bank中的序列进行同源性比较。　结果　从凝胶中共选出 11条差异带。将回收的 11条带再经PCR扩增和杂交试验 ,从中选出 3种不同的基因片段。经 Northern杂交证实片段 1和片段 2为裂头蚴特异表达基因 ,而片段 3为裂头蚴和成虫共同表达的基因。3个基因片段测序后与 Gen Bank中的基因进行同源性分析 ,基因片断 1和 2无同源序列 ;基因片段 3与多种生物的 2 8S r RNA同源。　结论　通过 m RNA差异显示技术查找出 2个裂头蚴阶段特异性表达基因片段