Dual Effects Of3-methyladenine On Cells Autophagy And The Preliminary Observation On The Effects Of NBA1in The Cellular Response To Radiation

ObjectiveTo investigate the cell autophagy effect after treat with PI3K inhibitors3-MA, DNA-PKcs knockdown and irradiation with fractionated dose.To generate a number of cell models with different NBA1status, including knockout, knockdown and overexpression, then preliminary observe the DNA damage response of NBA1on above cellular, in order to provid the ideas and experimental basis for the further mechanistic investigations of ionizing radiation-induced autophagy.MethodCell Counting Kit-8(CCK-8) assay and Annexin V-FITC/PI double staining were used respectively to detect the viability rate and observe the apoptosis rate in order to make a clear understanding about the cell proliferation toxic-effect of3-Methyladenine (3-MA) and IR. The GFP-LC3puncta were examined under a confocal microscope after transfect GFP-LC3by lipofectamin2000. HeLa cells with a depressed DNA-PKcs were treated with3-MA and fractionated doses (2Gy×2) or a single dose of4Gy y-ray radiation. The expression of autophagy-related proteins were detected by Western blot. TALEN technique was used to create the plasmid for NBA1knockout, and then identify its activity and efficiency by SSA and digestion. The cell line of NBA1knockdown and overexpression has also been achieved by small interference RNA(siRNA), western blotting and qRT-PCR were used to identified its effect. Colony formation assay were used to detect the sensitivity of NBA1knockdown cells to y-ray irradiation. Flow cytometry were performed to measure the changes of cell cycle distribution. Results1.3-MA can promote the IR-induced cell apoptosis. Although3-MA alone at the concentration of5mM was demonstrated to promote the autophagy and apoptosis of HeLa cells, however, it was shown to inhibit the IR-induced cell autophagy; The depression of DNA-PKcs increased cell autophagy induced by4Gy IR; As compared to fractionated doses (2Gy×2), a single dose of4Gy irradiation with was more effective than induction of cell autophagy.2. Three pairs of TALENs (T12、T34and T56) for specifically recognizing and binding to the target DNA sequence are successfully constructed. T56has a higher efficiency of mutation and can be used for NBA1knockout after identification by SSA and digestion.3. The results of western blotting and qRT-PCR showed that the stable cell lines of NBA1knockdown or overexpression were established successfully. The colony formation assay showed that NBA1knockdown increased the sensitivity of HeLa cells to y-ray irradiation. The NBA1knockdown resulted in the abnormal of G2/M arrest induced by y-ray irradiation.Conclusion1. The3-Methyladenine (3-MA), an inhibitor of phosphoinositide3-kinase(PI3K), can influence the cell proliferation in the form of concentration and time dependent, and also can influence the cell survive by suppress or promoting autophagy. Both DNA-PKcs expression and the way of radiation can influence the autophagy activity.2. It is more effectively to build the TALEN targeting sequence for NBA1knockout by the way of Unit assembly.3. NBA1knockdown increased the sensitivity of HeLa cells to y-ray irradiation, and resulted in the abnormal of G2/M arrest induced by y-ray irradiation.