ABSTRACT
It is important to identify the differentially expressed gene in gastric cancer for elucidating the molecular mechanisms of tumorigenesis of stomach. Here, 38 genes differentially expressed genes between gastric cancer and normal gastric mucosa by in silico approaches. A potassium channel protein KCNE2, identified as a down-regulated gene in gastric cancer, was chosen for further study. We investigated the expression of KCNE2 in gastric cancer tissues and cell lines and examined the effect of KCNE2 on proliferation of gastric cancer. The expression of KCNE2 was markedly down-regulated in gastric cancer tissues and cell lines. Forced overexpression of KCNE2 suppressed the growth of SGC7901 cells and cell cycle progression significantly, which might be related to the down-regulation of Cyclin D1. KCNE2 also inhibited SGC7901 cell growth in soft agar and its tumorigenicity in nude mice. Taken together, our work showed that in silico analysis approaches could be used to identify cancer-related genes effectively. KCNE2, as a novel down-regulated gene in gastric cancer, suppressed cell proliferation and tumorigenesis of stomach.
Subject(s)
Cell Proliferation , Potassium Channels, Voltage-Gated/metabolism , Stomach Neoplasms/pathology , Animals , Blotting, Western , Cell Cycle/genetics , Cell Line, Transformed , Cell Line, Tumor , Computational Biology/methods , Cyclin B/genetics , Cyclin B/metabolism , Cyclin B1 , Cyclin D1/genetics , Cyclin D1/metabolism , Down-Regulation , Flow Cytometry , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Mice , Mice, Nude , Potassium Channels, Voltage-Gated/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Transfection , Xenograft Model Antitumor Assays/methodsABSTRACT
OBJECTIVE: To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti-Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer. METHODS: Balb/c mice were immunized i.p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA. RESULTS: The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed beta or gamma type anti-Id ScFv. CONCLUSION: The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.
Subject(s)
Antibodies, Anti-Idiotypic/biosynthesis , Antibodies, Monoclonal/immunology , Immunoglobulin Fragments/biosynthesis , Stomach Neoplasms/immunology , Animals , Antibodies, Anti-Idiotypic/genetics , Antibodies, Monoclonal/genetics , Bacteriophages/genetics , Cloning, Molecular , Immunoglobulin Fragments/genetics , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Mice , Mice, Inbred BALB C , RNA, Messenger/genetics , Vaccines, DNA/genetics , Vaccines, DNA/immunologyABSTRACT
<p><b>OBJECTIVE</b>To analyze the distribution and significance of occludin mRNA expression in human gastric cancer, as well as its relationship with gastric cancer pathology and multidrug resistance (MDR) in vivo.</p><p><b>METHODS</b>In situ hybridization (ISH) technique was used to evaluate the expression of occludin mRNA in 42 gastric carcinoma specimens obtained by surgery and 23 relatively normal gastric mucosa obtained by gastric endoscopy. All specimens had been stored in cryostatic section.</p><p><b>RESULTS</b>Occludin mRNA was found positive in the cytoplasm of gastric glandulous epithelia as blue particles with intensive stain in 14 of 42 gastric carcinomas (33.3%), 23 of 42 paracancerous gastric tissues (54.8%), 14 of 23 relatively normal gastric tissues (60.9%), 9 of 16 well differentiated carcinomas (56.3%), 4 of 14 moderately differentiated carcinomas (28.6%), 1 of 10 poorly differentiated carcinomas (10.0%) and none of 2 mucosal carcinomas. There were significant differences in occludin mRNA positive rate between relatively normal gastric tissue and gastric cancer as well as between paracancerous gastric tissue and gastric cancer. The expression of occludin mRNA in moderately and poorly differentiated groups was gradually reduced when compared with well differentiated group, which suggests that there be a significant correlation between tumor differentiation and the expression of occludin mRNA. Furthermore, the positive signals of occludin mRNA distributed extensively in the cytoplasm of SGC7901/VCR cell, being vincristine resistant, derived from parental gastric cell line SGC7901. The positive signals of SGC7901/VCR were stronger than those of SGC7901 cells.</p><p><b>CONCLUSION</b>Occludin mRNA, being mainly located in epithelial cells and its expression correlated with tumor differentiation, may be involved in the development of multi-drug resistance in gastric cancer.</p>
Subject(s)
Humans , Drug Resistance, Multiple , Physiology , Drug Resistance, Neoplasm , Physiology , In Situ Hybridization , Membrane Proteins , Genetics , Metabolism , Occludin , RNA, Messenger , Metabolism , Stomach Neoplasms , Metabolism , Tight Junctions , MetabolismABSTRACT
Objective:To generate phage displayed anti idiotypic antibody single chain variable fragments(anti Id ScFv)to monoclonal antibody MC3 directed against colorectal carcinoma.Methods:Balb/c mice were immunized i.p. with MC3(McAb against colorectal Carcinoma) conjugated with KLH,and mRNA was isolated from the spleens of the immunized mice.VH and VL DNAs of the antibody were amplified separately and assembled into ScFv DNAs with a linker DNA by PCR.The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield phage antibody ScFv library.After four rounds of panning to the library with MC3,the MC3 positive clones were selected by ELISA from the preselected phages.Results:The VH,VL and ScFv DNAs were about 340,320 and 750 bp respectively.After four rounds of panning to the antibody library,15 MC3 positive phage clones displayed anti Id ScFv were selected from 50 enriched phage clones.Conclusion:The phage displayed anti Id ScFv to MC3 were successfully selected by phage antibody library technology,which might provide new putative candidate molecules for developing recombinant anti Id vaccine of colorectal carcinoma.
