| BACKGROUND & OBJECTIVE: The gastric cancer is first cause of death induced by carcinoma in our country. It was thought that gastric cancer was developed from premaligment lesion(atrophic gastritis, intestinal metaplasia and dysplasia). mRNA differential display(DD) was a method of isolating and cloning genes in study of carcinoma.The search for differentially expressed genes in gastric cancer and its premalignant lesions by DD may be helpful to define molecular alterations in the gastric mucosa that may precede the development of gastric cancer.It was more important and practical as compared with research of genes differentially expressed between gastric cancer and normal mucosa.METHODS: The differentially expressed cDNA bands beween gastric cancer,premalignant lesion and normal mucosa were isolated and identified by fluorescent differential display (FDD) and reamplified by PCR.After being cloned by T vector ,all cDNA fragments were sequenced. Through BLASTN software of GenBank database, the cDNA bands sequenced were compared with GenBank database for homology analysis.Northern blot analysis was performed for determining the express of cDNA fragments in 3 groups of tissues (gastric cancer,premalignant lesion and normal mucosa) . Expression of Genes was measured by semi-quantitative RT-PCR.The rapid amplification of 5' cDNA ends (5 ' -RACE) was used to amplify and expland one of new cDNAfragments for full length of cDNA.The open reading frame(ORF) and location of gene on chromosome were determined by bioinformatic programs on Internet, such as ORF finder, Bioedit and Map View.The distribution of of expression of the gene in the multiple tissues was detected by RT-PCR and series analysis of gene expression (SAGE) .The charateristic of protein sequence was analysed by bioinformatic programs on Internet, such as composition of amino acid, the prediction of functional domains , the subcellular location of protein,the homology of protein sequence, transmembrane segments , blocks and secondary structure.RESULTS: 30 differentially expressed cDNA bands were cut from polyacrylamide gel. Twelve of them were reamplified ,cloned and sequenced. The four of cDNA bands were identificated new express sequence tag(EST) because they were not found homologue to the known sequence in DNA sequence database and given accession number of EST by GenBank database.The eight of cDNA bands were identificated the known sequences because they were found homologue to the known sequence in the database.3 of them were homologue to SPTAN1 , CD9,ribosomal protein S24, fespectively and the other five cDNA bands were found homologue to genes whose function were unknown until now.Northern blot analysis of 4 new EST revealed that their expression was consistent to the result of DD. The SPTAN1 was over-expressed in gastric cancer,but the level of its expression in premalignant lesions and the normal gastric mucosa was decreased.The CD9 was expressed in normal gastric mucosa, but the level of its expression in premalignant lesions and gastric cancer was decreased. A full length of 849 bp cDNA named GCR and containing an entire ORF of 501 bp was obtained by 5 ' RACE,cloning and sequencing, which ecodes a polypeptide containing 166 amino acid and is located in human chromosome 3p21.GCR is expressed in colonic mucosa, esophogus mucosa anddown-expressed in colonic cancer,esophogus cancer,normal hepatic tissue and cancer of liver. Expression of GCR was increased in medulloblastoma, ependymoma brain ependymoma, normal breast stroma.The analysis of bioinformatic showed that main amino acid of the protein sequence was serine and arginine.The protein is located in nuclear and mitochondrial of cell.The transmembranes segments of GCR was not found. GCR was found homologue to glyceraldehyde-3-phosphate dehydrogenase (GDPD) (identity was 31%) . Second structure of GCR showd that the protein was classed as the mixed clas... |
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