Study On The ATM Gene Silence Mediated By RNAi And The Effects Of Radiosensitivity In Hela Cells

Objective To construct the Hela cell model with ATM gene silenced(Hela^ATM-cell) by transfecting chemically synthesized siRNA. To study the radiosensitivity of Hela cells with ATM gene silenced by using conventional chromosome aberration analysis method and cytokinesis-block micronucleus method. This research provided a cell model for further research on the bionomics of tumor cells with ATM silenced,and present a new idea and experimental data for the research on the radiosensitivity of tumor cells.Methods (1) siRNA was designed and synthesized ,including 4 pairs of siRNA targeting ATM gene,the negative and positive control siRNA and one pair of FAM-marked negative control siRNA. Transfected these siRNA into Hela cells by using liposome. Transfection effect was observed under Fluorescent Microscope. Using RT-PCR to detect ATM gene expression of Hela cells 24h after siRNA transfected. Transfected the more effective siRNA into Hela cells and detected ATM gene expression respectively after 24h,48h,72h and 96h.Constructed the Hela^ATM-cell with ATM gene silenced . (2) Using conventional chromosome aberration analysis method,chromosome aberration frequencies (CAF) of Hela^ATM-cells and Hela cells with 0,1,2,3,4 and 5 Gy exposures to 60Coγ-ray were observed,compared with Hela cells. (3) Using cytokinesis-block micronucleus method,the micronucleus frequencies (MNF) and micronucleus cell frequencies (MNCF) of Hela^ATM-cells and Hela cells with 0,1,2,3,4 and 5 Gy exposures to 60Coγ-ray were observed,compared with Hela cells.Results (1) It showed that FAM-marked negative control siRNA was successfully transfected into Hela cells under Fluorescent Microscope. (2) RT-PCR showed that ATM gene expression was depressed significantly in Hela1057cells which had been transfected by 1057-1075nt siRNA 24h before (P<0.05). Hela^ATM-cell with ATM gene silenced was constructed .(3) RT-PCR showed the 1057-1075nt siRNA worked untill 96 hours after transfected (P<0.05). ATM gene expression was decreased significantly at 24h and 48h after transfected than 96h (P<0.05),no significant difference between 24h and 48h,72h and 96h after transfected (P>0.05);ATM gene expression was decreased significantly at 24h and 48h than 72h and 96h after transfection (P<0.05). (4) After exposed to 0,1,2,3,4 and 5 Gy ⁶⁰Coγ-ray,the main pattern of chromosome aberration was dic and the radiation-induced level of CAF was significantly higher in Hela^ATM- cells than in Hela cells at every expose dose point (P<0.01) . In the two cells,CAF had a positive correlation with dose,and their linear regression equations were Y=a+bD. The slope of CAF linear regression equations of Hela^ATM- cells was larger than that of Hela cells (P<0.05). (5) After exposed to 0,1,2,3,4 and 5Gy 60Coγ-ray, the radiation-induced levels of MNF and MNCF were significantly higher in the Hela^ATM-cells compared with the Hela cells (P<0.01). In the two cells, the MNF and MNCF had a correlation with dose,and their linear regression equations were Y=a+bD+cD².Conclusion (1) Hela^ATM-cell with ATM gene silenced was constructed by transfecting chemically synthesized siRNA. (2) The CAF of Hela^ATM- cells was significantly higher than Hela cells by using conventional chromosome aberration analysis method. (3) The MNF and MNCF of Hela^ATM- cells were significantly higher than Hela cells by using Cytokinesis-Block micronucleus method.