The Effects Of RAD51AP1 On Radiotherapy Resistance Of The Nasopharyngeal Carcinoma

【Background】The nasopharyngeal carcinoma is a high incidence in southern China region and is also one of the most common types of head and neck cancer. The main treatment is radiotherapy while chemotherapy and surgery is complementary comprehensive treatment guidelines.Because the lesion location of nasopharyngeal carcinoma is relatively hidden and there are no obvious specificity symptoms, the first examination in most patients is associated with lymph node invasion or back to the nasal discharge with the blood which indicates these patients have developed a terminal-stage cancer. In the stage of treatment, radiotherapy resistance becomes one of the major factors that lead to the treatment failure. The problems how to figure out the reason why nasopharyngeal carcinoma generates radiotherapy resistance and how to increase the sensitivity of the radiotherapy needs to be solve urgently.Tumor cells under the ionizing radiation will emerge DNA double-strand breaks(DSBs).Through genetic self-repair mechanism which includes homologous recombination and non-homologous end joining,the tumor cell can maintain the integrity of the double-strand DNA breaks. By the interaction of homologous recombination and non-homologous end joining, double-stranded DNA can be kept relatively stable.RAD51AP1, called DNA repair relevant protein 51-associated protein 1,is a homologous recombination repair molecule found in yeast, which, combined with molecular RAD51,can increase the repair capacity of DNA chain for double-strand DNA breaks(DSBs).This study aims at observing the effects of the homologous recombination repair molecular RAD51AP1 on nasopharyngeal carcinoma radiotherapy resistance in order to provide a new therapeutic targets for the treatment of nasopharyngeal carcinoma(NPC).【Objective】The paper illuminates the influence of the expression change of the target gene RAD51AP1 on the sensitivity change of the radiotherapy for the nasopharyngeal carcinoma cell CNE1, aiming at deeply uncovering the molecular mechanisms about how the radiotherapy resistance of the nasopharyngeal carcinoma is generated and providing theoretical basis for the search of the target gene which is used for the sensitization therapy for the nasopharyngeal carcinoma radiotherapy. All this work achieves a valuable exploration of how to effectively improve the clinically therapeutic effect on the nasopharyngeal carcinoma.【Method】This experiment mainly includes the following five parts:1 Expression change of the target gene RAD51AP1 in Real-time fluorescent quantitative PCR after irradiationCNE1 can be divided into two groups. One group is exposed into irradiation with 6 Gy dose while the other group is not exposed to irradiation,then the cells are placed into an incubator for 6 hours and in the end we will extract the RNA from the two groups. Through the compare of the real-time fluorescent quantitative PCR between two groups, expression quantity change of RAD51AP1 gene after irradiation can be observed.2 Construction of cell knock down the gene RAD51AP1This part applies Liposome transfection method to introduce RAD51AP1 interference fragment into CNE1 cell to silence target gene RAD51AP1, and builds stable cell knock down RAD51AP1 through pressurizing antibiotics. RT-PCR and Western blotting methods will be used to test if the target gene RAD51AP1 is silenced. If it is success, the CNE1 cell which obtain the stable and silenced target gene RAD51AP1 will be marked sh-RAD51AP1 as experimental group, the CNE1 cell which transfect empty plasmid vector will be marked sh-Cont as the control group.3 Detection of the cell proliferation ability in MTT methodIn this part,MTT method is applied to test OD of the sh-RAD51AP1 cell in the experiment group and the sh-Cont cell in the control group after incubation with 6 h, 24 h,48 h, 72 h, 96 h, 120 h, 144 h, and the cells proliferation ability of two groups is compared and their growth curves are drawn.4 Detection of the cells’ radiosensitivity change in clone formation assayIn this part, the sh-RAD51AP1 cell in the experiment group and the sh-Cont cell in the control group will be incubated for 14 days after radiation exposure in different doses(0Gy,2 Gy,4 Gy,6 Gy), then the cells are fixed, in order to count the cloned cell mass.Survival rate of the cells from two groups with the same dose is used to compare changes in cellular sensitivity to radiation.5 Detection of cell apoptosis in flow cytometry method1 After the sh-RAD51AP1 cells in the experiment group and the sh-Cont cells in the control group are exposed to irradiation with the same dose(2 Gy, 6 Gy) and then incubated with different incubation time(7 h, 24 h, 48 h)..2 Flow cytometry method is applied to detect the cycle change of cell sh- RAD51AP1 and cell sh – Cont after irradiation exposure with different doses(0 Gy, 2 Gy, 4 Gy, 6 Gy)and with 7 hour incubation. And the ratio of the cells blocked in G2 / M to the whole cells will be calculated, and the G2 / M phase of the cells from two groups under different radiation dose will be compared.【Results】1 After the test of the real-time fluorescent quantitative PCR, comparing the mRNA extracted from the CNE1 under no irradiation with the m RNA extracted from the CNE1 under irradiation of 6 Gy dose, we find that the expression of the target gene RAD51AP1 increases to 1.563 times of the original.2 Through lipofection transfection method, the target gene RAD51AP1 is knocked down in nasopharyngeal carcinoma cell CNE1. Applying PCR and western blot testing method,the m RNA level and protein level of the target gene RAD51AP1 in CNE1 both markedly decrease, especially the inhibition ratio reaches 71.2% in RT-PCR’s resμlt, which indicates that RAD51AP1 is stable and has low expression in the cell model we obtain.3 Testing the cell proliferation condition of the sh-RAD51AP1 cell in the experiment group and the sh-Cont cell in the control group by MTT method, we find that there is no markedly difference between the two groups(P≥5%).Through the clone formation assay, it is found that there is no markedly difference between the two groups(P≥5%)with four dosage gradients(0 Gy,2 Gy,4 Gy,6Gy).4 The resμlt of the flow cytometry method shows, compared with incubation for 24 hours and 48 hours, the sh-RAD51AP1 cell in the experiment group and the sh-Cont cell in the control group after irradiation exposure with the same dose(2 Gy, 6Gy) have the most obvious function and the cells are mainly blocked in G2 / M phase when incubated for 7hours. There is no markedly difference in G2 / M phase between the two groups(P≥5%)with four dosage gradients(0 Gy, 2 Gy, 4 Gy, 6Gy).【Conclusion】1 It successfμlly builds stable cell knock down RAD51AP1.2 Irradiation results in a phenomenon that the nasopharyngeal carcinoma cell CNE1 is blocked in G2/M stage, which is more obvious especially after 7 hours’ irradiation.3 The radiotherapy sensitization enhancement effect of the single silent target gene RAD51AP1 on the nasopharyngeal carcinoma cell CNE1 is not obvious.