Effect On Gadd45α Gene Expression In Human Esophageal Cancer Cells Ec9706 After 6mv-x Ray Irradiation

Background and ObjectiveGadd45αgene is a growth arrest gene and DNA damage gene and it was also the first to be detected downstream of the p53 gene. Gadd45αgene was induced to express increasingly through the two channels of the p53-dependent and non-dependent and it plays a key role in monitoring the cell cycle, apoptosis , DNA damage repair, in the maintenance of genome stability, as well as other important signal transduction regulation of cell life. It involved in the development and suppression of tumor. Many studies found that Gadd45αgene is sensitive to ionizing radiation and ionizing radiation can be produced a strong reaction to its high expression changes. 6MV-X ray is used commonly in clinical radiotherapy .We used it to irradiate the human esophageal cancer cells EC9706 to induce Gadd45αgene expression , then, to determine the relative Gadd45αgene expression with RT-PCR method and observe the changes of Gadd45αexpression in the cells of EC9706 .We explored the effect on Gadd45αgene expression in EC9706 after 6MV- X ray irradiation by comparison and statistical treatment, which maybe give a certain experimental evidence and a valuable clinical reference on the comprehensive radio-chemotherapy for esophageal cancer in the future.Materials and MethodsIn vitro, we cultured human esophageal cancer cells EC9706 , respectively irradiating by 6MV-X ray. In accordance with different doses into 3 groups, 0Gy (control group), 4Gy (experimental group) and 6Gy (experimental group), each shot in accordance with the time-dose point 1h, 6h, 24h, respectively, three parallel samples for a total of 27 samples. After irradiation, we placed them into the culturing-box with 37℃, 5%CO₂ and 100% relative humidity for continuously culturing at once. At every different time point, we extracted the total RNA respectively, then ,reverse transcription for cDNA. We chose the special primer for Gadd45αgene from literature and verified it on the Genbank, then, amplified the purpose gene in the tubes. After that, we used RT-PCR technology, considering 18srRNA as control gene, to measure the relative Gadd45αgene expression .Each test has 27 independences studies, and repeat 3 times. The means±standard error were used as results (relative expression value). The data in each time intervals of each dose were compared with 0Gy .We used T-test to analyze them and to find the relation between gene expression and doses at different time points and at the same time point between control group and experimental group. Then, we observed whether there are statistic difference (P<0.05) .ResultDatum of control group (0Gy) between samples had no statistic difference with each other at1h, 6h and24h. Gadd45αgene expression is stable at different time and had no statistic difference (P>0.05). After irradiation at the dose point of 4Gy and 6Gy, the tendency of Gadd45αgene expression change were same: At the these two dose points, the increase of Gadd45αgene expression were time-dependent, they increased after irradiation and reach peak after 6h exposure. After that they decreased, but 24h after a downward trend, they were not reduced to the initial level. After irradiation, Gadd45αgene expression of 4Gy and 6Gy increased and had statistic difference (P<0.05). At every time point, the increasing of gene expression were dose-dependent.Conclusion:1.Using internal relative quantitative/semi-quantitative RT-PCR technology to measure the relative amount of Gadd45αgene expression comparing with control gene, the method is of high sensitivity, specificity, simple, fast, and good repeatability. 2.Gadd45αgene expresses stably in human esophageal cancer cells EC9706. 3.After 6MV-X ray irradiation, Gadd45αgene expression is increased dose-dependently and time-dependently. 4.So, we could predict that Gadd45αgene be as a target gene for the comprehensive radio-chemotherapy of esophageal cancer in the future.