The Role Of Nrf2in Radiotherapy Of Human Glioma Cell U251

Background and objective:Glioblastoma multiform (GBM) is the most malignant glioma of astrocytomas, which is of WHO grade Ⅳ. GBM grows fast, and the median survival time is short. Radiotherapy is one of the main treatment of GBM after surgery to kill remaining cancer cells, however, GBM resistance to radiotherapy limits its therapeutic effect. Nrf2is a redox-sensitive response of a transcription factor regulating antioxidant enzymes and the expression of anti-apoptotic protein. Nrf2has the power of protective effect of antioxidant and antiapoptotic. Nrf2-ARE signaling pathway maintains cellular redox balance, and the prevention of environmental pollution and carcinogens stimulate. However, Nrf2also have a protective effect on tumor cells. Nrf2can reduce the sensitivity of cells to chemotherapy drugs has been relatively clearly. Then this study is to see whether Nrf2has something to do with radiotherapy resistance. By inhibition and increasing Nrf2,we will find the impact of10Gy radiotherapy in human glioma cell U251by observing cell proliferation and apoptosis. Therefore, by investigation of the relationship between Nrf2and radiotherapy, we may find new ideas for clinical GBM treatment.The aim of this experiment is:1、observe the change of Nrf2in human glioma cell U251after10Gy radiotherapy;2、after inhibition and increasing Nrf2in human glioma cell U251, we observe cell apoptosis after10Gy radiotherapy;3、do immunohistochemistry to human glioblastoma surgical pathology specimens, to see whether Nrf2changed after radiotherapy.Methods:1. Radiation: select lOGy as radiation dose in human glioma cell U251, unradiatived cells as the negative control group. After radiation, we select3h,6h,12h,24h as time points using real time RT-PCR to determin Nrf2expression;2. Plasmid transfection Divide into four groups:①lipofectamine2000group:U251cells+lipo2000②blank vector group (pEGFP):U251cells+lipo2000+blank vector (pEGFP)③Si-Nrf2group:U251cells+lipo2000+Si-Nrf2 ④pEGFP-Nrf2group:U251cells+lipo2000+pEGFP-Nrf2Measurement methods:48h after transfection using flow cytometry to determin the apoptosis rate;3. X-ray irradiation:divide into four groups, first we do plasmid transfection, then after48h we do lOGy irradiation,:①lipofectamine2000group:U251cells+lipo2000②blank vector group (pEGFP):U251cells+lipo2000+blank vector (pEGFP)③Si-Nrf2group:U251cells+lipo2000+Si-Nrf2④pEGFP-Nrf2group:U251cells+lipo2000+pEGFP-Nrf2Measurement methods:Using flow cytometry, the apoptosis rate is determined8hours after irradiation4. in Jinling Hospital, many cases of glioblastoma patients were selected,we have to take pathological specimens before and after radiotherapy. By immunohistochemistry, the change of Nrf2expression can be observed before and after radiotherapy.Results:1. After10Gy radiotherapy of human glioma cell U251, the mRNA level of Nrf2changed from0.0319to0.0266after3hours,0.0207after6hours, 0.0216after12hours,0.0288after24hours. Compared with the initial value, the mRNA level of6h and12h decreased significantly (p<0.05);2.48hours after plasmid transfection, mesured by flow cytometry. The cell apoptosis rate of Lipofectamine2000group was0.06%, blank vectors group was1.08%, pEGFP-Nrf2group was0.06%, Si-Nrf2group was1.39%. The difference is statistically significant;3.48h after plasmid transfected, and then8h after10Gy radiotherapy, mesured by flow cytometry. The cell apoptosis rate of Lipofectamine2000group was0.386667%, blank vectors group was3.40333%, pEGFP-Nrf2group was1.55%,Si-Nrf2group was4.23667%. The difference is statistically significant;4. In the specimens of glioblastoma, we found that Nrf2expression was significantly increased after radiotherapy, and Nrf2was increased in both cytoplasm and nucleus, especially in cytoplasm.Conclusion:1.When do radiotherapy to human glioma cell U251,we can see Nrf2decrease within24hours;2. Nrf2play a protective role in the radiotherapy of U251glioma cells, downregulate of Nrf2can increase cell apoptosis and upregulate of Nrf2can decrease cell apoptosis;3. The expression of Nrf2of glioblastoma patients who received radiotherapy increased significantly whether in the cytoplasm and nucleus, especially in cytoplasm.