The Preliminary Study On The Roles Of GAS41in The Glioma Cell

Objective:By RNAi interference GAS41, study the early apoptosis of glioblastoma cell line A172. It could initial us to understand the roles of GAS41in the glioma and provide the experimental evidence for the diagnosis and treatment of glioma.Method:1. Discussion the optimum multiplicity of infection (MOI):we cultured the A172cells in vitro and took the cells in the logarithmic growth phase for experiments. Designing transfection complex gradient was:5,10,15. Lentivirus-mediated the GAS41-siRNA transfected A172cells, treated for8h. Then we calculated the number of cells, that emited the green fluorescence, and the total cells in the white vision of the same area.2. Analysis of GAS41, p53and p21expression:we have cultured A172cells in vitro, and taken the cells in the logarithmic phase for experiment. At the same time, we have diveded the the test gropes for the blank group and the experimental groups (GAS41-siRNA, dsRNA, transfection reagents). With the different regents, we treated the cells for8h. RT-PCR to detect the expression of a GAS41on the RNA level, Western blot detection the protein expression of GAS41, p53and p21.3. Counted the number of apoptotic cells by the flow cytometry:we have cultured A172cells in vitro, and taken the cells in the logarithmic phase for experiment. Then diveded the the test gropes for the blank group and the experimental groups (GAS41-siRNA, dsRNA, transfection reagents) and treated them with different regents. Each group repeated three times. After the Anexin V/FITC and propidium iodide staining process, counted the cells by the machine.Results:1. With the transfection complex increasing, the number of cells decreased significantly, and the cell morphology was severely deformed. When the MOI was up to the ten, the cells showed the stable prolifiration and the intensity fluorescence. By calcurated the cells and χ2test, MOI=10that we wanted was the optimum MOI (P<0.05).2. The expression of GAS41, p53and p21:RT-PCR trails indicated that, the expression of the GAS41group was frankly the lowest in all groups. On the protein level, Western blot results confimed that the GAS41-siRNA group was also the lowest. The same trend of the p53and the p21was verified that the experimental groups were higher than the control group, and the highest expression levels of GAS41-siRNA interference group.3. Counted the number of apoptotic cells by flow cytometry:we compared the proportion of early apoptotic cells, the fourth quadrant of each groups. The apoptosis rate of the GAS41-siRNA group, the dsRNA-siRNA group, the reagent group and the blank group were19.58±2.52%,4.87±0.38%,5.45±1.03%,1.15±0.29%respectively. The apoptosis rate of the GAS41-siRNA group was significantly higher than the other groups (p<0.05), the dsRNA-siRNA transfection reagent was no significant difference (p>0.05), while the experimental group and the control group were significant differences (p<0.05)Conclusion:1. The interference of GAS41promoted the apoptosis of the cells.2. The interference of the GAS41resulted of the up-regulation of p53and p21, which suggested that the interference of GAS41promoted the apoptosis of the cells could come true through the p53signaling.