| mRNA differential expression was detected between gastric cancer and counterpart normal gastric mucous membrane of five patients using cDNA Microarray.The 18000 genes on used cDNA Microarray membrane come from liver library, hepatocellular carcinoma library,adrenal gland library,pirutary library, hypothalmus and DC library.Another 1200 genes on used cDNA Microarray membrane are rumor related genes.mRNA expression signals on hybridization membrane were quantified with computer software.Over 3 times down-regulated genes in gastric cancer were screened. 67 genes were confirmed down-regulated genes in gastric cancer including tumor suppressor genes, apoptosis related genes,DNA replication and transcription or translation related genes, cell cycle related genes, cell migration related genes,immunity related genes ,NGF and Trk receptors families genes etc. They are involved in gastric tumorigenicity and metastasis of gastric cancer.cDNA suppression subtractive library was established by counterpart normalgastric mucous membrane mRNA (Tester) subtracting gastric cancer tissues mRNA(Driver) of five patients. After subtractive efficiency identification of the subtractive library in PCR and reverse hybridization , positive clones chosen randomly were sequenced , and down-regulated novel genes in gastric cancer were screened .The results demonstrate that the library was high specific ,low false positive and sensitive to clone low abundance mRNA. Two low abundance expressing novel gene fragments named GDDZ(AP494508) ,GDDR(AF494509) were cloned respectively. They were confirmed down-regulation in gastric cancer by semi-quantity RT-PCR. 802bp GDDZ and 751bp full-length of GDDR genes were obtained by Race. GDDR gene encodes protein homolog to CA11, a candidate of tumor suppressor gene. GDDZ and GDDR may play a important role in gastric tumorigenicity and metastasis of gastric cancer.CA11 was screened in subtractive library.CAll was just found , and 600bp ORF encodes 199 amino acids. It's property and funtion is unknown. Down-regulated gene CA11 in gastric cancer was confirmed by Northern blot and RT-PCR. Functional forecast on net shows CA11 encoding protein is a secrete peptide. CA11 mRNA was only expressed in stomach among 13 varieties of tissues and organs. CA11 mRNA was located in situation mRNA hybridization, and result demonstrated CA11 mRNA was located in gastric mucous epithelial cells.CAll cloned into vector pcDNA3.1/Myc-His(-)A was transfected into COS-7 cells by Lipofectamin. The loading sample of SDS-PAGE electrophoresis came from culture medium of transiently transfected COS-7 cells that stayed in Log growth period when Western blot was undergone with primary anti-Myc tag antibody. Western blot can not prove existence of CAll-Myc fusion protein in culture medium of COS-7 cells. COS-7 cells were transiently transfected by CA11, and primary anti-Myc tag antibody added bound to FITC -labelled secondantibody, its expressing fusion protein in cytoplasma was observed under fluorescent microscope.At last, by immunohistochemistry in primary anti-Myc tag antibody, whole secrete passage of CAll-Myc fusion protein was grasped from cytoplasma to trans-membrane,and to outside cell in gastric cancer cell lines 7901 by steadily transfecting with CA11. First CA11 encoding protein was proven to be a secrete peptide.CAll was steadily transfected into gastric cancer cell lines 7901 by lipofectamin. RT-PCR,Western blot and immunohistochemistry stain confirm that CAll was transfected into gastric cancer cell lines 7901, and CAll-Myc fusion protein was expressed. Gastric cancer cell lines 7901 by steadily transfecting with CAll, showed a marked decrease in growth rate by growth curve or MTT test results (72h, 0.379?.019 A vs 0.467?.021 A,P<0.01),and growth inhibition in nude mice. Irregular shapes , less ribosomes or cilia on cell membrane were observed in gastric cell lines 7901 steadily transfected by CAll. GI peroid cells increased, and S period cells decreased in gastric cell lines 7901 steadily transfected b...
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