The Establishment Of Cell Experiment Platform For Radioresistance In NSCLC And The Preliminary Study Of Its Mechanism

Objectives:1. To test the change of the damage repair ability of DNA in non-small cell lung cancer conventional fractionated and hypofractionated radioresistant cell lines in mRNA and protein level.2. To test the cell cycle distribution and level of apoptosis of radioresistant cell and analyze the mechanism of radioresistance.Methods:1. A549, A549-2GyR and A549-4GyR were cultured in vitro. All cells underwent radiation exposure. Colony formation tested the ability of cell proliferation.2. According to mRNA micro-array, we selected out several differentially expressed genes which are related to DNA damage repair. A549, A549-2GyR and A549-4GyR were cultured in vitro. We acquired target genetic templates by reversible transcription PCR (RT-PCR), then analyzed the differential expression by real-time quantitative PCR.3. To test the related protein expression, such as Ku70、Ku80、DNA-PKcs、LIG4、 Rad50、XRCC4、Mrell and p53、p21、CylinDl in the radioresistant cell under basal conditions by Western Blot experiment.4. Detect the cell cycle distribution following PI staining and apoptosis with Annexin V/PI double staining by flow cytometry.Results:1. The ability of cell proliferation in A549, A549-2GyR and A549-4GyR became stronger in turn.2. By mRNA micro-array screening, we chose Ku70、Ku80、DNA-PKcs、LIG4、 Rad50、XRCC4、Mrell as target gene. We found XRCC4、Ku80、DNA-PKcs、 Rad50’s transcription reduced in A549, A549-2GyR and A549-4GyR in turn and Ku70and LIG4transcribed least in A549-4GyR cells.3. Western Blot’s result showed that DNA-PKcs、XRCC4、LIG4、Ku80、Ku70、 Rad50、Mrell、NBN、XLF expressed less and less in A549, A549-2GyR and A549-4GyR; cell cycle-related proteins p53、Phospho-p53(Ser20). Phospho-p53(Ser37) expressed also less and less, while p21’s expression increased gradually.4. In the flow cytometry result, we found that in basal conditions,62.63%of A549cell in G1phase,10.04%in G2phase,27.33%in S phase;64.45%of A549-2GyR cell in G1phase,11.77%in G2phase,23.79%in S phase;72.37%of A549-4GyR cell in G1phase,8.7%in G2phase,18.93%in S phase. Apoptosis cell in A549accounted for0.7%,1.7%in A549-2GyR cell and0.3%in A549-2GyR cell.Conclusions:1. The A549radioresistant cell lines could be acquired by repeated exposure to radiation.2. Compared with control and conventional fractionated radioresistant cell, the transcription of DNA damage repair related gene Ku70、Ku80、DNA-PKcs、 LIG4、Rad50、XRCC4in hypofractionated radioresistant cell reduced.3. In protein expression level, these protein expressed less and less in A549, A549-2GyR, A549-4GyR in accordance with transcription in mRNA level.4. In basal conditions, there was no significant differences in apoptosis among the A549, A549-2GyR and A549-4GyR cells.5. There was more cells arrested in Gl/S phase in hypofractionated radioresistant cell than that in control and conventional fractionated radioresistant cell. In consideration of the decreased expression of DNA damage repair protein, the arrest of cell cycle maybe the main reason of radioresistance.