The Effect Of Oncogene BMI-1on The U87MG Glioblastoma Cells Exposed To Radiation And The Mechanism Of Cellular Senescence

Purpose:Glioma is the most common tumors in central nervous system, which was classified into WHO grade Ⅰ-Ⅳ. The proportion of grade III and grade IV glioma can be77.5%of all gliomas. The median survival time of WHO grade III glioma is usually2-3years and the median survival time of glioblastoma (WHO grade IV) is only one year. Prognosis of gliomas depends on many factors and treatment measures. Though the treatment of gliomas is comprehensive treatment include surgery, radiotherapy, chemotherapy and other treatments at present, there are still quite a lot of patients with recurrence in1year. Due to specific parts of glioma growth, it made that the surgical radical resection is considerable difficult. Because of the complications after surgery, the quality of patients’life usually decreased. Although new chemotherapy drugs for example temozolomide improved the curative effect of glioma, the effect of chemotherapy was extremely limited because of the presence of the blood-brain barrier. Radiotherapy plays an important role in the treatment of gliomas. Postoperative radiotherapy can reduce the recurrence chance of gliomas and eliminate or inhibit the residual tumor cells result in the improvement of cure. For these glioma patients who was unable to accept surgery, radiotherapy can be used to control tumor growth for prolonging the survival period and improving the quality of life. But, the gliomas are characterized by anti-apoptotic and radioresistance, as a result that the therapy of gliomas often fails in the radiotherapy and recurrent at last. Recently the many researches of focus on how to improve the radiosensitivity of glioma. In recent years, it was reported that certain glioma cells would not apoptosis but senescence after exposure to radiation. Senescence is the irreversible cell cycle arrest in some phase (generally in G0/G1phase).The senescence cells have still some metabolism activity but it can’t proliferate any more. It is an important mechanism to suppress tumor. Escape from senescence may has important position in the radioresistance of glioma. Bmi-1is usually highly expressed in glioma which participates cellular senescence through various signaling pathways. Bmi-1may be one of the key genes involved in escape from senescence, anti-apoptotic effect and chemoradiotherapy resistance. There is extrodinary biological and clinical significance of studing on the relationship between Bmi-1and radioresistance of glioma especially highly grade ones. MicroRNA-128a is abundant in normal brain tissue. But the expression level of microRNA-128a is significantly lower in the glioma tissue samples which is associated with the ability of glioma proliferation and glioma stem cell self-renewal. MicroRNA-128a can inhibit the expression of Bmi-1therefore affect its biological functions including oncogene epigenetic regulation, promoting cell proliferation and maintaining stem cell self-renewal. In our reseach, we selected human glioblastoma cell line U87MG to be used for studies. We determinated the radioresistance and anti-apoptosis of glioma, cellular senescence after exposure to X-ray and the effect of radiation on expression of Bmi-1or MicroRNA-128a. We explored the possibility of changing glioma radiosesenitivity though regulating MicroRNA-128a expression to target the expression of Bmi-1. We expect to show the possibility for MicroRNA-128a and Bmi-1as a new target of improving radiosesentivity of glioma.Materials and methods:1. Human glioblastoma cell line U87MG was purchased from cell bank of Chinese academy of sciences. The method involving cell culture, passage, frozing and recovery were conventional.2. The U87MG glioma cells were cultured for experiments. Linear accelerator was used for radiation treatment by SSD technique. The parameters about radiation dose and fraction methods was reported to experienced radiation therapy physicist who designed radiation planning and carried the accurate verification for the radiation dose before experiments.3. After exposure to different doses X-ray, U87MG cells were double staining by PI and Annexin V-FITC. The flow cytometry was used to detect cell apoptosis.4. Different doses X-rays were used to treat U87MG cells. Senescence cells usually get large and express the P-galactose glucoside enzyme which has highly activity at pH6.0. So the β-galactose glucoside enzyme staining was used to detect cell senescence ratio.5. After exposure to different doses X-ray, the total protein was extracted. The concentration of protein was measured by BCA method. Western blot method was be used to detect the protein expression.6. After treatment of different dose X-ray, the total RNA of U87MG cells was extracted and purified to detect the quantity and purity. The real-time PCR method was be used to detect expression of Bmi-1mRNA and MicroRNA-128a.7. After exposure to different doses X-ray, the fluorescent probe DCFH-DA was loaded in U87MG cells. The fluorescence of2’,7’-dichloride fluorescent yellow was detected by flow cytometry to evaluate the level of cellular ROS.Results:1. As cells growth curve shown, all group cells showed a trend of logarithmic proliferation. X-ray could not decreased the number of cells. The trend proliferation of1,2Gy groups had no significant difference to control group. After24h exposed to radiation, the trend proliferation of1,2Gy groups increased compared to control. The deference has statistical significance (P<0.05). After72h exposed to radiation, the trend proliferation of4,6and8Gy groups decreased apparently and has the statistically significant difference (P<0.05). It was shown that the U87MG cells has the certain radioresistance.2. The inhibition ratio curve had shown the proliferation of U87MG glioma cells was increased in1,2Gy groups within the5days after exposure to radiation. The results are statistically significant (P<0.05). The inhibition ratio on the7th day after exposure to radiation was about20%. In6,8Gy groups, the inhibition effect on U87MG cells could be obviously observed in a dose-dependent manner and increased over time. The results have statistical significance. The significant inhibition effect was shown in all groups. The mean inhibition ratio was as follows:1Gy group was20.35%,2Gy group was20.35%,4Gy group was48.8%,6Gy group was68.51%,8Gy group was68.51%. The effect intensity was related to radiation dose. It was shown that X-ray has inhibitory effect on proliferation of U87MG cells.3. After72h exposed to different dose of X-ray radiation, the cell of each group did not appear obvious apoptosis. It was shown that X-ray could not induce U87MG glioma cell apoptosis. Its inhibition effect had nothing to do with cell apoptosis. The other inhibition mechanism should exist.4. After exposed to different doses of radiation, the cell morphology was observed under phase contrast microscope. It can be observed that senescence cells showed significantly larger volume, flattened. The nucleus became lager and stained more dark. Granular inclusions could be seen in the nucleus. β-galactose glucoside enzyme staining was used to detect the proportion of senescence cells which was positively related to the intensity of radiation. The difference between each radiation group and control was statistically significant (P<0.05). It showed that the X-ray inhibition effect on U87MG glioma cells is implemented by promoting senescence.5. After24h exposed to X-ray radiation, the S phase cells increased in1,2Gy groups compared to control. It suggested DNA synthesis increased under the stimulus of low doses radiation. G0/G1phase cells in each radiation group increased in a dose-dependent manner. It partly suggested that senescence was involved in the inhibition effect on U87MG glioma cells of X-ray. After72h exposed to radiation, the S phase cells in all radiation groups increased compared to the control group. It might be because that radiation leads to redistribution of the cell cycle.The late S phase cells was not sensitive to radiation, as a result, S phase cell percentage increased. G2/M phase cells proportion increased in a dose-dependent manner. In the6,8Gy groups, G0/G1phase cells decreased significantly and G2/M phase cells increased. There may be certain mechanism to make the U87MG cells escape from senescence. It was the reason that U87MG cells was characterized by radioresistance.6. After72hours exposed to radiation, the expression of Bmi-1protein and mRNA of U87MG glioma cells increased significantly in6,8Gy groups. It suggested that Bmi-1gene was involved in the escape from senescence.7. After72h exposed to radiation, the expression of microRNA-128a in8Gy group decreased significantly. The result had statistical significance (P<0.05). The expression of microRNA-128a in1,2Gy groups increased significantly,. The result was statistically significant (P<0.05).8. After2h exposed to radiation, the ROS level in glioma cells in2Gy group increased compared to other group. After12h exposed to radiation, the ROS level in glioma cells in all radiation groups increased compared to control. The NAC was added in U87MG glioma cells before exposed to8Gy dose X-ray. As a result, the inhibition effect on U87MG glioma cells of X-ray was decreased.Conclusions:1U87MG glioma cells have the characteristics of the radioresistance and anti apoptosis.2. X-ray can inhibit the growth of U87MG glioma cells in a dose-dependent manner. The mechanism is the promoting effect on cell senescence.3The escape from senescence may happen in U87MG glioma cells after exposure to radiation which is one of the radioresistance mechanism.4. ROS is one of the key molecules for the inhibition effect of radiation on U87MG glioma. Down-regulation of intracellular ROS generation could increase radioresistance of U87MG glioma cells.5. Bmi-1may be involved in the escape from senescence after U87MG cells were exposed to X-ray and play a key role.6. The microRNA-128a expression of U87MG glioma was related to the dose intensity after exposure to X-ray. It is likely involved in regulation of Bmi-1expression.