Preliminary Exploration On The Effect Of Autophagy On The Radiation Sensitivity Of Human Lung Adencarcinoma A549Cell

Background： Radiation therapy is one of the most important treatmentmethods of lung cancer, yet it is easily to be recurrence and residue because ofradiation resistance.A major determinant of tumor response to radiation is tumor cellradiosensitivity which dependent on its capability of repairing DNA damage. Recentresearch has indicated that radiation induced tumor cell autophagy happen.Autophagyis chosely related with the process of DNA damage repair. Whether it can increase theradiation sensitivity of lung cancer through regulation of autophagy to affect DNAdamage repairing is still unclear.Objective：The aim of this research is to study the effects of autophagy activatorRapamycin on the radiosensitivity of human adenocarcinoma A549cells and itsmolecular mechanism.Methods：Human adenocarcinoma A549cell line was chosen to be experimentalsubject, then divided into three group:control group（N）,radiation group（R）,rapamycin and radiation group（R+RAPA）.①MTT assay method was used todetermine the proliferation of A549cell which was treated with rapamycin, the tenpercent inhibitory concentration(IC10).②Group R was treated with ray from0Gy to12Gy，the cell survival was determined by colony formation assay.③Group R wastreated with ray(4Gy)，group R+RAPA was pretreated with IC10rapamycin beforeradiation，Real-time PCR was used to detect Rad51mRNA、Ku70/Ku80mRNA，γ-H2AX protein、Rad51protein、Ku70/Ku80protein、p62protein、LC3protein wasdetected by western blot；autophagy body was observed by transmission electronmicroscope,SF4was determined by colony formation assay.Results：①The effect of different concentration of rapamycin onA549cell.In the concentration range of rapamycin (10nmol/L-120nmol/L),the inhibitionratio of A549cell was gradually increased in a dose dependent manner, and the IC10was18.4nmol/L after rapamycin action for24h.②The influence of rapamycin regulate of autophagy on radiosensitivity. In the dose range of0-12Gy,radiation can kill tumor cell, and the IC50=4.194Gy. The SF4of Group R was62.11±3.56%,the SF4of Group R+RAPA was32.44±2.18%.Electon microscopy detected that radiation and rapamycin can increaseautophagosome formation of A549cell，contrast with group R and Group RAPA，theautophagosome formation of A549cell was increased.Western blot detected thatcontrast with Group N, LC3I was decreased, LC3II was increased, the ratio ofLC3II/I was increased, p62was decreased in Group R and Group R+RAPA; contrastwith Group R, LC3I was decreased, LC3II was increased, the ratio of LC3II/LC3Iwas increased, p62was decreased(p＜0.05）.It was indicated that it can increaseradiosensitivity of A549cell by autophagy.③The mechanis of the effect of rapamycin on radiosensitivity throughautophagy.Contrast with Group N,1h after radiation,γ-H2AX protein was increased ingroup R and group R+RAPA（p＜0.05),and there was no significant change betweenthe two groups；contrast with1h after radiation,γ-H2AX protein was decreased in24hin the two groups（p＜0.05);24h after radiation,contrast with group R, γ-H2AXprotein was increased （p＜0.05）.It was indicated that radiation can resultDSBs,upregulate autophagy by rapamycin can cause DSBs persisted.Contrast with group N, the expression of Rad51protein was increased（p＜0.05）,and the expression of Ku70protein and Ku80protein expression has no significantchange, the expression of Rad51protein and Ku80protein was decreased（p＜0.05）,and the expression of Ku70protein expression has no significant change in groupR+RAPA. It was indicated that upregulate the level ofautophagy in A549cell byrapamycin, can decreased DSBs repair by inhibit Rad51protein and Ku80proteinexpression.After the radiation of4Gy, the expression of Rad51mRNA was increased（p＜0.05），and the expression of Ku70mRNA and Ku80mRNA expression has nosignificant change, there was no significant difference between the two groups.Contrast with group R, the expression of Rad51mRNA was increasd in2h and4h in group R,Ku70mRNA and Ku80has no significant change;contrast with group N,the expression was increasd in2h and4h in group R+RAPA（p＜0.05), Ku70mRNA ans Ku80mRNA has no significant change; the expression of Rad51mRNA、Ku70mRNA、Ku80mRNA has no significant difference between groupR andgroupR+PAPA.It was indicated that radiation can increase the expression of Rad51mRNA,but has no significant influence of Ku70mRNA and Ku80mRNA, upregulatethe level of autophagy in A549cell didn’t change the mRNA expression of DSBsmolecular.Conclusions：①Rapamycin can inhibit the survival of A549cells in a dose-dependent manner.②Autophagy can incease radiation sensitivity of A549cell.③Autophagy can inhibit the process of DSBs repairto increase radiationsensitivity of A549cell.