Isolation and bioinformatics analysis of differentially methylated genomic fragments in human gastric cancer

AIM：To isolate and analyze the DNA sequences which are methylated differentially between gastric cancer and normal gastric mucosa.
METHODS： The differentially methylated DNA sequences between gastric cancer and normal gastric mucosa were isolated by methylation-sensitive representational difference analysis （MS-RDA）. Similarities between the separated fragments and the human genomic DNA were analyzed with Basic Local Alignment Search Tool （BLAST）.
RESULTS： Three differentially methylated DNA sequences were obtained, two of which have been accepted by GenBank. The accession numbers are AY887106 and AY887107. AY887107 was highly similar to the 11th exon of LOC440683 （98%）, 3＇ end of LOC440887 （99%）, and promoter and exon regions of DRD5 （94%）. AY887106 was consistent （98%） with a CpG island in ribosomal RNA isolated from colorectal cancer by Minoru Toyota in 1999.
CONCLUSION： The methylation degree is different between gastric cancer and normal gastric mucosa. The differentially methylated DNA sequences can be isolated effectively by MS-RDA.     

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.     

Two novel gastric cancer-associated genes identified by differential display

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应用MS-RDA筛选胃癌甲基化差异片段

目的:应用甲基化敏感性代表性差异分析法(methylation-sensitive representational difference analysis,MS-RDA)筛选胃癌和正常胃组织间甲基化差异DNA片段．方法:通过MS-RDA筛选胃癌和正常胃组织间DNA甲基化差异片段,经克隆、测序后进行生物信息学分析．结果:获得3个甲基化差异片段,分别为 CRS1308,CRS1309,CRS1310k列,其中 CRS1309和CRS1310已被GenBank收录,登陆亏分别为AY887106和AY887107,CRS1309 序列与LOC440683基因第11外显子、 LOC440887基因的3’端,DRD5基因启动子和外显子区域均有很高的相似性(分别为98％,99％, 94％),CRS1310序列与1999年Minoru Toyota在人类结肠癌中分离出的核糖体RNA上的甲基化差异性CpG岛有很高的相似性(98％)．结论:胃癌和正常胃组织间DNA甲基化存在差异,MS-RDA可有效分析这两种不同组织间甲基化的差异,筛选出有意义的甲基化差异片段.     

人胰腺癌抑制消减cDNA文库的构建

目的应用抑制消减杂交技术(SSH)构建胰腺癌和正常胰腺组织间差异表达的抑制消减cDNA文库。方法分别提取胰腺癌(tester)和癌旁正常胰腺组织(driver)中的总RNA和mRNA．合成双链cDNA，经RsaI酶切后，将胰腺癌双链cDNA分为两组，分别加上不同的接头，再与正常胰腺组织cDNA进行两次消减杂交及两次抑制性PCR，分离出胰腺癌差异表达基因的cDNA片段。将该差异表达片段克隆至T／A载体，并转化大肠杆菌TOP10F’，经蓝白斑筛选后，再用PcR方法筛选阳性克隆，从而构建胰腺癌抑制消减cDNA文库。结果文库扩增后得到257个白色克隆，随机挑取50个阳性克隆进行PCR扩增分析，其中47个克隆有插入片段．克隆阳性率为94％，片段大小主要集中在300～600bp之间。结论成功构建了人胰腺癌抑制消减cDNA文库，为进一步筛选、克隆胰腺癌特异性表达基因奠定了基础。     

用基因表达谱芯片研究人正常胃和胃癌中与发育相关基因的价值

用8464条人cDNA制成表达谱芯片,利用胃和胃癌组织的mRNA,通过逆转录方法将Cy3和Cy5两种荧光分别标记到两组cDNA上,利用这种cDNA探针与表达谱芯片进行杂交后扫描,通过计算机分析判定基因是否在上述组织中存在差异表达,结果示在197条与生长发育相关的基因中,存在差异表达的共10条,上调的8条（0．095％）,下调的2条（0．024％）,应用这种方法识别出的基因对胃癌的诊断和治疗具有重要的潜在价值。     

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.     

应用基因表达谱芯片筛选胃腺癌相关基因

目的:利用基因芯片技术筛选胃腺癌组织和癌旁正常组织间的差异表达基因．方法:分别抽取胃腺癌组织和癌旁正常组织的总RNA．采用逆转录的方法,制成cDNA链, 并以两种荧光Cy5和Cy3标记后作为探针,与含有14 784条人类14KcDNA基因表达谱芯片进行杂交．以Agilent荧光扫描仪扫描芯片上两种荧光信号,并用计算机处理和分析．结果:在14 784条基因中,4例胃腺癌组织和癌旁正常组织共同差异表达基因29条,其中上调基因10条,下调基因19条,上调的基因中有2 条功能信息不明．结论:胃腺癌发生过程中有多基因的参与,胃腺癌与癌旁正常组织共同差异表达的29条基因可能与胃癌的发生、发展有关．     

Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer

AIM： TO screen out the differentially methylated DNA sequences between gastric primary tumor and metastatic lymph nodes, test the methylation difference of gene PTPRG between primary gastric tumor and metastatic lymph nodes, and test the regulatory function of 5-aza2-deoxycytidine which is an agent with suppression on methylation and the level of methylation in gastric cancer cell line. METHODS： Methylated DNA sequences in genome were enriched with methylated CpG islands amplification （MCA） to undergo representational difference analysis （RDA）, with MCA production of metastatic lymph nodes as tester and that of primary tumor as driver. The obtained differentially methylated fragments were cloned and sequenced to acquire the base sequence, which was analyzed with bioinformatics. With methylation-specific PCR （MSP） and RT-PCR, methylation difference of gene PTPRG was detected between primary tumor and metastatic lymph nodes in 36 cases of gastric cancer. Methylation of gene PTPRG and its regulated expression were observed in gastric cancer cell line before and after being treated with methylation-suppressive agent. RESULTS： Nineteen differentially methylated sequences were obtained and located at 5＇ end, exons, introns and 3＇ end, in which KL59 was observed to be located at 9p21 as the first exon of gene p16 and KL22 to be located at promoter region of PRPRG. KL22, as the probes, was hybridized with driver, tester and 3-round RDA products respectively with all positive signalsexcept with the driver. Significant difference was observed in both methylation rate of gene PTPRG and PTPRG mRNA expression rate between primary tumor and metastatic lymph nodes. Demethylation of gene PTPRG, with recovered expression of PTPRG mRNA, was observed after gastric cancer cell line being treated with methylation-suppressive agent. CONCLUSION： Difference exists in DNA methylation between primary tumor and metastatic lymph nodes of gastric cancer, with MCA-RDA as one of the good analytical methods. Significant