Cloning And Function Analysis Of Human Survivin Gene In Gastric Cancer

Aims: The anti-apoptotic gene survivin(SW) has drawn much attention since it was identified in 1997. Many researches revealed the relationship of SW and human neoplasms, meanwhile, SW was acknowledged as reliable molecular marker for tumors. But, the biological function of SW in the process of human malignancy has not been identified, nor was there illustration of cloning SW from human gastric cancer. Now, we planned to clone SW gene from human gastric cancer cell line SGC7901, and to analyze its biological function in the carcinogenesis and progress of human gastric cancer.Methods:1. Applying RT-PCR, clone Survivin gene from human gastric cancer cell line, SGC7901.2. The obtained bands were cloned into pGEM T-Easy vector, and the positive clone were confirmed by EcoR I enzyme digestion. Then, the sequences of inserted fragments were analyzed. These sequences were compared with GeneBank database through BLASTN and BLASTP program for homologue analysis, in order to identify that the obtained fragment is identical to the known SW gene.3. After introduced the sites of restrictive endonuclease enzyme NdeI and BamH I, the identified SW band were cloned into prokarytoic expression vector pRSET, to fulfill its efficient expression in E Coli BL2KDE3).4. After designing primers according to the SW sequence, the sense and anti-sense fragment of SW were obtained by PCR, they are SW-a and SW-b correspondingly. They were cloned into eukaryotic expression vector pEGFP-Nl after introduced the sites of restrictive endonuclease enzyme EcoR I and BamH I.5. Transient transfecting the human gastric cancer cell SGC7901 with the positive recon contained the sense SW fragment, applying confocal microscopy to observe the location of transfected SW in gastric cancer cell.6. Stable transfecting the human gastric cancer cell SGC7901, MKN45 and mice NIH3T3 through G418 screening . To observe the cell growth curve and cell cycle of above-mentioned cells after transfected with pEGFP-Nl vector only, pEGFP-Nl-a and pEGFP-Nl-b seperately. Meanwhile, to detect the effects to the cell apoptosis of above-mentioned three transfection conditions using TUNEL method, to examine the change of expression level of PCNA using immunohistochemistry, to check the sensitivity of gastric cancer cells to the chemotherapy drug 5~FU and VP16 by MIT.7. With the specifically designed SW primers, RT-PCR was used to explore its expression among 5 gastric adenocarcinoma patients, 1hepatic carcinoma patient, colon cancer cell HT29 and leukemia cell HL60.8. By using In Situ Hybridization, expression of S^ and SiB were analyzed in gastric cancer tissues, colon cancer tissues, lung cancer tissues and prostate cancer tissues. Specimens used in this part were paraffin-embedded tissues including 61 stomach ademocarcinoma patients, 2 colon cancer patients, 2 lung cancer and 2 prostate cancer patients. Image analysis software was used to quantitate the expression of S,|A and SiB, and mean OD was used as the quantitative index.Results1. Obtained the full-length cDNA of human SW gene- S^ from gastric cancer cell.2. Through sequencing and BLAST analysis, the obtained band S4A was proved to be identical to the known SW. At the same time, a new band, SiB, was gained, which is 119bp shorter than S4A. Blast analysis revealed that SiB lost the third exon of SW, and it encodes 137aa which is highly homological to SW protein, thus, we called S3 variant of SW. This is the first time for this variant of SW to be obtained from gastric cancer.3. Fulfill the efficient expression of SW in E Coli BL21(DE3).4. Though sequencing, sense SW and anti-sense SW were proved to be successfully cloned into eukaryotic expression vector pEGFP-Nl, we called them pEGFP-Nl-SW-a and pEGFP-Nl-SW-b correspondingly.5. Transient transfecting the human gastric cancer cell SGC7901 with the positive recombinant contained the sense SW fragment, pEGFP-Nl-SW-a, By using confocal technique, we identified that SW was expressioned in the cytoplasm of gas...