Study Of Effect Of ATM Gene Silencing On The Radiosensitivity Of Colorectal Cancer Cells

OBJECTIVE Using RNA interference technique to investigate the effect of ATM gene silencing on the radiosensitivity of colorectal cancer HT29 cells in vitro.METHODS Small interfering RNA(siRNA) of ATM gene, eukaryotic expression plasmid called pSilencer2.1-ATM, was designed and constructioned to transfect into human colorectal cancer HT29 cells as positive group. RNAi oligonucleotide sequence of ATM gene was rearranged randomly to construct pSilencer2.1-nonspecific plasmid as negative group. Control group was not transfected. The transcription of ATM gene mRNA in each group was determined with Reverse Transcription-Polymerase Chain Reaction(RT-PCR). Protein expression of ATM gene mRNA in each group was detected by Western blot(WB). After 8 Gy X-ray irradiation, each group was divided into two halves. One was cultured continuously for 24h and the other for 48h. Then at different points of time(24h,48h), flow cytometry was used to detect group of positive, negative and control of cell cycle distribution and apoptosis. Finally, SPSS20.0 software was used for statistically analyzing the results.RESULTS ATM gene siRNA eukaryotic expression plasmid was constructed and transfected into colorectal cancer HT29 cells successfully. RT-PCR test result confirmed that mRNA content of ATM gene was significantly decreased in the positive group. Western blot results confirmed that expression protein of ATM gene was significantly decreased in the positive group.24h after X-ray irradiation, proportions of cells in G1/G0 phase and G2/M phase of positive group was lower than those in control group(t=-11.59, P<0.001; t=-4.30, P<0.001). But negative group had no significant difference comparing with control group(t=0.02,.P=0.98; t=-0.03, P=0.97).48h after X-ray irradiation, like 24h after X-ray irradiation, proportions of cells in G1/G0 phase and G2/M phase of positive group was lower than those in control group(t=-9.91, P< 0.001; t=-4.94, P< 0.001). There was also no statistical difference between negative group and the control group(t=0.08, P=0.94; t=0.09, P=0.93).24h and 48h after irradiation, apoptosis rate of positive group was both higher than those of control group(t=21.15, P< 0.001;t=32.94, P< 0.001) and there showed no statistical difference between negative group and the control group(f=1.56, P=0.12;t=1.86,P=0.06).CONCLUSION 1. By constructing the eukaryotic expression plasmid of ATM gene siRNA and transfection into human colorectal cancer HT29 cells, the transcription and expression of ATM gene was effectively inhibited.2. The radiosensitivity of colorectal cancer HT29 cells was enhanced after ATM gene was silenced. Its mechanism probably was ralated to the inhibition of DNA damage repair, cell cycle checkpoint inactivation in Gi/Go phase and G2/M phase and the increasing of apoptosis rate.