Effect Of RNA Interference Mediated C-jun Gene Downregulation On The Radiosensitivity Of Radioresistant Human Nasopharyngeal Carcinoma Cell Line CNE-2R

[Background]Nasopharyngeal carcinoma is a common malignant tumor in Southeast Asia and southern China. Local recurrence and metastasis are the main causes that restrict the efficacy and prognosis after radiotherapy. Weaken the radiation resistance to increase the radiation sensitivity of tumor cells may be a promising way to improve 5-year survival of patients with NPC. Some studies showed that radioresistance was closely related to radiation induced gene expression and gene regulation. Therefore, looking to a biomarker of radiosensitivity has important significance on molecular treatment of nasopharyngeal carcinoma therapy.Our research group pre used Gene ontology、Pathway analysis and Protein interaction network to carry on the comprehensive analysis, which obtain multiple key markers associated with radiation resistance in the network.According to the previous experiment, we selected c-jun gene for further experiment, which was located at the molecular network node and participate in many bioinformatics pathway. To clear the correlation between c-jun gene and nasopharyngeal carcinoma radioresistant, useing RNA interference mediated c-jun gene downregulation expression on the radioresistant human nasopharyngeal carcinoma cell line CNE-2R, and then explores the impact of radiosensitivity of CNE-2R cell. Whether c-jun gene can become the new target of molecular treatment of nasopharyngeal carcinoma is discussed initially.ResearchPart I C-jun shRNA lentiviral vector’s constructed and established CNE-2R stable transfected cell line[Objective]Based on the previous experiment, we found c-jun gene was highly expressed in the radioresistant cell line CNE-2R and involved in several pathways, which was also one of the key nodes in the genetic interation network. To clear the relationship between c-jun gene and radioresistant hunman nasopharyngeal carcinoma, in this part, c-jun gene downregulation expression in CNE-2R by using RNA interference, and then established CNE-2R stable transfected cell line to lay the foundation for the research of the influence on radiation sensitivity of CNE-2R cells.[Method]1. The c-jun gene’s expression was confirmed by RT-PCR and western blot.2.3 groups of siRNA sequences specific for c-jun gene were designed, and lentvirus vector with c-jun shRNA was constructed and infected of CNE-2R.3. The stably silenced expression of c-jun cell was selected by flow cytometry and the gene silencing efficiency of these recombinants was confirmed by RT-PCR and Western blot.[Results]1. The result of RT-PCR and Western blot showed that the change of c-jun gene expression was consistent with previous result of the chip.2. Three of LV-shRNA-c-jun lentiviral vector was successfully constructed, and packed with high virus of 8×108 TU/ml, observed under inverted fluorescence microscopy, the transduction rate was approximately 80%, the lentiviral shRNA was successfully constructed and transduced into the CNE-2R cells.3. The best interference target was identified by RT-PCR and Western blot, the sequence was CAAACCTCAGCAACTTCAA.[Conclusion ]RNA interference can inhibit c-jun expression effectively on radioresistant human nasopharyngeal carcinoma cell line CNE-2R, which laid a foundation for the subsequent experiment.Part Ⅱ Effect of RNA interference mediated c-jun gene downregulation on the behavior and the radiosensitivity of radioresistant human nasopharyngeal carcinoma cell line CNE-2R[Objective]In order to clear the relationship between c-jun gene and radioresistant hunman nasopharyngeal carcinoma, in this part, c-jun gene downregulation expression in CNE-2R by using RNA interference, and then observed cell proliferation, radiosensitivity, cell cycle and apoptosis of CNE-2R cells which were silenced by c-jun, in order to get better understand the relationship between c-jun gene and radiosensitivity of human nasopharyngeal carcinoma.[Method]1. The effects on cell survival of the c-jun shRNA-transfected were examined by CCK-8 assay, to analysis the effect on cell survival rate of these cells after silencing the c-jun gene.2. Colony formation assay was used to evaluate the radiosensitivity of CNE-2R cells after silenced the c-jun gene.3. The cell cycle and apoptosis of each group were exposed respectively to 0 Gy and 2 Gy radiation were measured by FCM, which was used to study the effect on cell cycle and apoptosis of these cells after silenced c-jun gene of CNE-2R cells.[Results]1. OD value of 6 MV X-rays combined with c-jun-RNAi-LV2 in 0,2,4,6, 8 Gy group of spectrophotometery was significantly lower than the other two groups (F=42.70-200.67, P< 0.05)2. The cloning formation capacity of c-jun-RNAi-LV2 group was significantly lower than non-transfected group and NC-transfected group. The values of SF2, Do and Dq were decreased and the sensitizing enhance rate (SER) of Do was 1.41.3. The G2/M-phase cell population of 0,2 Gy X-ray radiation combined with the control group, NC group and c-jun-shRNA group was (18.27±5.28)%, (19.93±4.99)%; (17.13±3.35)%, (17.63±3.01)%; (27.33±1.58)%and (28.57±2.9)% (P<0.05) espectively.4. The apoptosis rate of 0,2 Gy X-ray radiation combined with c-jun-RNAi-LV2 group, non-transfected group and NC-transfected group were respectively (20.93±1.99)%, (38.17±0.83)%; (10.97±0.70)%, (20.43±0.25)%; (10.80±1.25)% and (19.53±1.50)% (F=50.54,330.14, P<0.05)[Conclusion]RNA interference can inhibit c-jun expression enhanced the radiation sensitivity, inhibit cell proliferation and promote cell apoptosis. Our findings implied that the overexpress of c-jun could serve as a potential target to enhance the radiation sensitivity for NPC therapy.