| BackgroundCervical cancer is the fourth most common type of cancer in women in the world and in some low-income countries it is the most common cancer in women. Globally, there are 528,000 new cases of cervical cancer per year, 266,000 women die of cervical cancer, 85% of which occur in low-income countries; highlighting the significant inequities that exist in global health. Persistent infection with human papillomavirus(HPV) is a necessary, although not sufficient, factor in the development of this cancer. cervical cancers caused by HPV genotypes 16 and 18 account for about 80% of cervical cancers worldwide.The current management for cervical cancer consists primarily of surgery, radiation and chemotherapy. Radiation critical to treat cervical cancer, mainly for the advanced, recurrence cases and patients who can't accept operation, or joint application with surgery. Ionizing radiation(IR) is a potent inducer of double-strand breaks(DSB), which are considered the most serious form of DNA damage. Complex systems have evolved to rapidly detect and repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to IR and other DNA damaging agents. The DNA repair ability of tumor cells also prevents the accumulation of lethal DNA damage from cytotoxic agents and ionizing radiation(IR), leading to tumor resistance. Homologous recombination(HR) and nonhomologous end-joining(NHEJ) represent the two major DSB repair pathways in cells. NHEJ is believed to play a more important role then HR in mammalian cells. The catalytic subunit of DNA-dependent protein kinase(DNA-PKcs) is an essential component of the NHEJ which is the major DNA DSB repair pathway in mammalian cells. ATM plays a key role in detecting DNA DSBs and HR DNA repair.Clinical studies have revealed that malignancies with overexpressed of DNA-PKcs and ATM are associated with disease progression, poor clinical outcomes and radiationresistance. Cells experiment have demonstrate that suppress the expression of DNA-PKcs or ATM by shRNA can enhance the radiosensitivity and malignant biological behavior of carcinoma cell lines. But there was no research investigate the effect of suppressing the expression of DNA-PKcs and ATM by siRNA at the same time on biological behavior, radiosensitivity of HeLa cell. ObjectiveTo investigated the effect of suppressing the expression of DNA-PKcs and ATM by RNAi separately and both on the biological behavior and radiosensitivity of HeLa cell. Materials and Methods1. Construction of plasmid with shRNA to knockdown DNA-PKcs and ATM: Endogenous DNA-PKcs and ATM was knocked down using the plasmid vector containing short hairpin RNA(shRNA) against DNA-PKcs and ATM.2. Construction of stably transfected cell lines: The cells were transfected with plasmid vector contain shRNA against DNA-PKcs(DNA-PKcs group), ATM(ATM group), both DNA-PKcs and ATM(ATM+DNA-PKcs group). Mixed resistant clones were collected and cultured in medium containing puromycin. QRT-PCR and Western Blot was used to detect the expression of DNA-PKcs and ATM in all of the cell lines described above.3. The effect of knocking down DNA-PKcs, ATM on HeLa cells on cell survival was detected by CCK8 assay.4. The effect on proportion of apoptotic by knocking down DNA-PKcs, ATM on HeLa cells was detected by flow cytometry.5. The effect on invasion by knocking down DNA-PKcs, ATM on HeLa cells was detected by Transwell cell invasion assay.6. The effect on radiosensitivity by knocking down DNA-PKcs, ATM on HeLa cells was detected by clonogenic assay.7. Statistical analysis : SPSS17.0 statistical package is used to analyze and process the datas. Measurement data are normality, homogeneity of variance test results meet the conditions of normality are mean ± standard deviation( x ± S) said that among the groups were compared using ANOVA or non-parametric test. Pairwise comparison between groups with SNK(Student-Newman-Keuls, SNK) method. All statistical tests were two-sided probability test, P <0.05 was considered statistically significant. Results:1. Stably transfected HeLa cells was screened by Puromycin and the efficiency of knockdown DNA-PKcs, ATM was tested by Western Blot and Quantitative Real-time PCR:The Screening concentration of Puromycin on HeLa cell was 0.8ug/ml after 2 weeks of culture with culture medium contained different concentration of Puromycin. Stably transfected HeLa cells was screened by screening culture medium. Stably transfected HeLa cells had significantly decreased levels of DNA-PKcs, ATM compared to the control groups at both the mRNA level and the protein level by tested by Western Blot and Quantitative Real-time PCR, and the difference was statistically significant(P <0.05).2. The cell proliferation assay showed that knockdown of DNA-PKcs+ATM could reduce the proliferation of HeLa cells at 24 h, 48 h, 72 h, 96 h than control group(P <0.05). Knockdown of DNA-PKcs +ATM could reduce the proliferation of HeLa cells at 48 h, 72 h, 96 h than ATM group(P <0.05). The proliferation of DNA-PKcs group was lower than control group at 48 h, 72 h, 96h(P <0.05). The proliferation of ATM+DNA-PKcs group was lower than DNA-PKcs group and the proliferation of DNA-PKcs group was lower than control group at 72 h, 96h(P <0.05). The proliferation of DNA-PKcs group was lower than ATM group at 96h(P <0.05). The rest difference of different group with no statistical significance(P> 0.05).3. The apoptosis assay showed that the apoptosis rate of ATM+DNA-PKcs group was higher than DNA-PKcs group, ATM group and control group(P <0.05). The apoptosis rate of DNA-PKcs group was lower than ATM group with significant difference(P <0.05). The rest of different group with no statistical significance(P> 0.05).4. The Transwell test showed that the number of invaded cell was lower in DNA-PKcs group, ATM group and ATM+DNA-PKcs group than control group, the difference was statistically significant(P <0.05). The number of invaded cell was lower in ATM+DNA-PKcs group than DNA-PKcs group and ATM group, the difference was statistically significant(P <0.05). The number of invaded cells in ATM+DNA-PKcs group was minimum. The rest of different group with no statistical significance(P> 0.05).5. Radiosensitivity experiments showed that the cloning efficiency of DNA-PKcs group, ATM group and ATM+DNA-PKcs group was lower than control group and the cloning efficiency of ATM+DNA-PKcs group was lower than DNA-PKcs group and ATM group in the radiation dose of 2,4,6 Gy. The cloning efficiency of DNA-PKcs group was lower than ATM group in the radiation dose of 6 Gy. The cloning efficiency of ATM+DNA-PKcs group was the lowest. The rest of different group with no statistical significance(P> 0.05).Conclusion:1. DNA-PKcs plays a more important role in cell proliferation, apoptosis and radiosensitivity than ATM in HeLa cell.2. Knockdown of DNA-PKcs and ATM at the same time can more efficiently reduce the proliferation, invasion and especially increase the radiosensitivity of HeLa cells. |
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