Location Of Gastric Cancer Related Gene GCRG213 In Eukaryotic Cells And Effect To Cell MKN45

A differentially expressed new gene, GCRG213, was screened directly by fluorescent differential display reverse transcription polymerase chain reaction (DDRT-PCR) analysis from human intestinal type gastric cancer, paratumor and non-tumor tissues in our laboratory. GCRG213 consists of 1094 base pairs with an open reading frame (ORF) which encodes 142 amino acids . The results of In Situ Hybridization and Northern blot showed that its expression was much higher in tumor and paratumor samples than in their normal counterparts. Through conserved domain database search in GenBank, a putative conserved domain, Human apurinic (apyrimidinic) endonucleas/redox-factor 1 (APE/Ref-1), was detected in the deduced amino acid sequence of GCRG213 — ORF, it shared 61.0% alignment with the C-terminal region of APE/Ref-1 conserved domain. APE/Ref-1 is involved in the repair of DNA damage as well as in the transcriptional regulation of genes. APE/Ref-1 levels have been found to be elevated in a number of cancers such as ovarian, cervical, prostate, colorectal and germ cell tumors. It is likely that GCRG213-ORF was a new member of the APE family. As such, to clarity the function of GCRG213 may play an important role in the diagnosis and therapy of gastric cancer.Aims: To study the location of gastric cancer related gene GCRG213 in eukaryotic cells and investigate the effect of gene GCRG213 transfection(sense, antisense and siRNA)to gastric cancer cell MKN45.Methods1 .GCRG213's DNA fragment with complete open reading frame were amplified by PCR from plasmid pGEM-T, and then were cloned into eukaryotic expression vector pEGFP-N1 after introduced the sites of restrictive endonucleaseenzyme Pst I and BamH I.2. Transient transfecting the human gastric cancer cell MKN45 with the recombinant plasmid pEGFP-Nl-GCRG213 , applying confocal microscopy to observe the location of transfected GCRG213 in gastric cancer cell.3. The sense and anti-sense fragment of GCRG213 were obtained by PCR, they are GCRG213-a and GCRG213-b respectively. They were cloned into eukaryotic expression vector pcDNA3.1(+) after introduced the sites of restrictive endonuclease enzyme Kpnl, BamH I and EcoRI, BamH I.4.Two pairs of DNA sequences containing small hairpin structure to GCRG213 were designed and synthesized. The complement form was obtained by annealing and inserted into RNAi expression vector IMG-800. they are IMG-800-land IMG-800-2 correspondingly.5.The recombinant plasmid pcDNA3.1-a, pcDNA3.1-b, IMG-800-1, IMG-800-2 and the vector pcDNA3.1,IMG-800 were transfected separately into MKN45 cells conducted by lipofectamine? 2000. After G418 selection, the cells were transfected steadily. Expression of GCRG213 was detected with semi-quantitative RT-PCR and Western Blot.6. The growth graph was protracted by the methods of cell counting to six steady transfected cells. The clone formation rate in plate and in nude mouse was tested to investigate the oncogenesis character of six steady transfected cells in vitro and in vivo. FACS detected the cell cycle and Annexin V FITC/PI two parameter detect the effects to the cell apoptosis of above-mentioned cells. MTT has applied to check the sensitivity of gastric cancer cells to the chemotherapy drug 5-FU, VP16, MMC, PTX and Oxaliplatin. Furthermore, ultra-microscopic structure was observed by transmission electric microscopy,Resultsl.DNA sequence analysis showed that the GCRG213 sequence was right in the recombinant plasmid pEGFP-Nl-GCRG213.2.Transient transfecting the eukaryotic cell COS-7 withpEGFP-Nl-GCRG213. By using confocal technique, we identified that GCRG213 was expressioned in the cytoplasm and nuclei of COS-7 cells.3. Though sequencing, sense GCRG213 and anti-sense GCRG213 were proved to be successfully cloned into eukaryotic expression vector pcDNA3.1, we called them pcDNA3.1-a and pcDNA3.1-b correspondingly. And Two pairs of DNA sequences containing small hairpin structure to GCRG213 were proved to be successfully cloned into siRNA expression vector IMG-800, we called them IMG-800-1 and IMG-800-2 correspondingly.4. The recombinant plasmid pcDNA3.1-a, pcDNA3.1-b, IMG-800-1, IMG-800-2 and the vector pcDNA3.1,IMG-800 were transfected separately into MKN45 cells conducted by lipofectamine? 2000. After G418 selecting, the cells were transfected steadily.5. Sense vector(pcDNA3.1-a) transfection into the MKN45 significantly increased the expression of GCRG213 ,both in mRNA level and protein level. Anti-sense vector(pcDNA3.1-b),siRNA vector(IMG-800-l, IMG-800-2 ) transfection into the MKN45 significantly decreased the expression of GCRG213 ,both at mRNA level and protein level.6. The growth graph was protracted by the methods of cell counting showed that the growth of pcDNA3.1-a transfected cells was faster than that of vector transfected cells. But pcDNA3.1-b, IMG-800-1 and IMG-800-2 transfected cells were slower than that of vector transfected cells.7. Cell cycle examination showed that the proportion of cells in G2/M and/or S period increased in the cells transfected with pcDNA3.1-a compared with those transfected with vector, while the proportion of cells in G2/M and/or S period decreased in the cells transfected with pcDNA3.1-b, IMG-800-1 and IMG-800-2.8. Annexin V FITC/PI two parameter showed the cell apoptosis decreased in the cells transfected with pcDNA3.1-a compared with those transfected with vector, while the cell apoptosis increased in the cells transfected withpcDNA3.1-b, IMG-800-1 and IMG-800-2.9.1n vitro, the average clone formation rate increased in the cells transfected with pcDNA3.1-a compared with those transfected with vector, while the average clone formation rate decreased in the cells transfected with pcDNA3.1-b, IMG-800-1 and IMG-800-2.lO.In vivo, the time of oncogenesis of pcDNA3.1-a transducted cells in nude mouse is shorter and the tumor node is bigger. The time of oncogenesis of pcDNA3.1-b, IMG-800-1 and IMG-800-2 transducted cells in nude mouse is prolonged and the tumor node is smaller.ll.Furthermore, in transmission electric microscopy, there were more microvilli on the surface of pcDNA3.1-a transducted cells, and there were fewer microvilli on the surface of pcDNA3.1-b, IMG-800-1 and IMG-800-2 transducted cells.12.MTT cytoxicity assay indicated pcDNA3.1-a transducted cells were less sensitive to 5-FU and Oxaliplatin ,but IMG-800-1 and IMG-800-2 transducted cells were more sensitive to 5-FU and Oxaliplatin. The cells' sensitivity to VP16, MMC and PTX did not change.Conclusion1.Transient transfection showed that GCRG213 was located in cytoplasm and nuclei of COS-7 cells.2.Stable transfection showed that GCRG213 promoted cell growth and proliferation, inhibited the cells into apoptosis, induced the cells' malignancy in vitro and in vivo. GCRG213 might be a new promoter to tumor.3.SiRNA and anti-sense transduction could inhibited cell growth and proliferation, promoted the cells into apoptosis, inhibited the cells' malignancy in vitro and in vivo.4. Transmission electric microscopy showed that GCRG213 induced the cells' malignancy and metastasis, siRNA and anti-sense transduction could inhibit the cells' malignancy and metastasis.