The Detection Of Survivin MRNA Expression In Peripheral Of Colorectal Cancer By RT-PCR-ELISA

ObjectiveColorectal cancer has a high incidence in China. The early diagnosis of colorectal cancer is of the great importance and the early detecting minimal tumor cells dissemination could give us a valuable warrant to predict the current and prognosis. The method of RT - PCR - ELISA used to detect tumor specific genes is so sensitive and convenient. It can effectively detect the minimal tumor cells in the blood of colorectal cancer. Survivin is a new member of inhibitor of apoptosis proteins (IAP). Survivin gene lies in the 17q25 of human chromosome , coding a 16.5 kd protein, survivin protein contains only one BIR( baculo-virus IAP repeat ) domain and does not have the zinc — binding fold terminated with carboxyl. Furthermore under normal circumstances survivin is expressed in embryonic tissues, but completely down regulated in normal adult tissues. Interestingly this protein is found to be re - expressed in a variety of human malignant tumorous tissues, and could be used as a tumor marker of cancer. Our experiment detected the survivin mRNA expression in the peripheral blood of colorectal cancer patients and the healthy controls by RT - PCR - ELISA, evaluated the relationship between survivin mRNA and the clinical significance and the sensitivity and the specificity of survivin mRNA as tumor marker of colorectal cancer.Material and MethodsBlood sample were obtained from 30 colorectal cancer patients attending the department of oncology at the first affiliated hospital of China Medical Universityand the blood sample of 25 healthy people were used as controls.1 ml peripheral blood were collected in a tube containing EDTA, The fraction of blood cells were isolated by centrifugation, Total RNA was extracted u-sing TRIZOL reagent. Then RT - PCR was performed for Survivin mRNA, Added all RT - PCR products to the ELISA strips coated by survivin mRNA probe to perform molecular hybridization. At last perform ELISA reaction, Read color density on a microplate reader at 450/630 nm then plotted standard curve and calculate Survivin mRNA concentrations of the sample. The ^2test was used to compare the difference expression of Survivin mRNA between in colorectal patients and the controls, and the difference of pathobiological characteristics.ResultsIn 30 patients of colorectal cancer 17 (56. 7% ) were Survivn mRNA positive expression, In the 25 healthy people 2(8%) were Survivin mRNA positive expression. Survivin mRNA in the peripheral blood has a significant difference between colorectal cancer patients and the healthy controls.Survivin mRNA positive rate was 42. 9% (6/14) at a size <5cm, 68. 8% (11/16) at a size 5=5cm;100% (2/2) at early colorectal cancer, 40% (4/10) at protrude type, 61.1% (11/18) at ulcerative type;54. 5% (12/22) at well differentiation, 62.5% (5/8) at poorly differentiation;31.3% (5/16) at lymph node non -metastasis, 85.7% (12/14) at lymph node metastasis, 31.3% (5/ 16) at Dukes A and Dukes B stage, 85.7% (12/14) at Dukes C stage. Survivin mRNA positive expression correlated significantly with lymph node metastasis and Dukes stage and it has no correlation with the other pathobiological characteristics.As a tumor marker of colorectal cancer, the sensitively of Survivin mRNA is 56.7% , its specificity is 92% , and its accuracy is 72.9%.Conclusion1. Survivin mRNA has good specificity in detecting minimal cancer cells incirculation of colorectal cancer patients by RT - PCR - ELISA, and could be look as a reliable marker to detect the minimal cancer cells in circulation of colorectal cancer patients.2. Survivin mRNA might show that minimal cancer cells are existing in circulation. It could be looked as a high risk factor of blood and lymph node metastasis to predict the recurrence and prognosis of colorectal cancer.3. Blood dissemination of colorectal cancer might happen early.