Establishment Of Radiotherapy Resistance Cell Line In Hypopharyngeal Carcinoma And Exploration Of The Resistant Mechanism

ObjectiveHypopharyngeal carcinoma is one of the malignant head and neck tumors threatening human health with poor prognosis.Radiotherapy is one of the main treatments for hypopharyngeal carcinoma,which has obvious advantages in preserving organ function.With the development in radiotherapy technology in recent years,many efforts have been made to improve the curative effect.However,the outcome of hypopharyngeal carcinoma is not satisfied.One reason for treatment failure is local recurrence or regional lymph node recurrence.The reason for the relapse may be the constant mutation during radiation of some tumor cells,inducing gradually resistant to radiation of cancer.Therefore,radiation resistance is one of the major obstacles to the treatment of hypopharyngeal carcinoma.It is of great significance to explore the mechanism of radiotherapy resistance in hypopharyngeal carcinoma and find the molecular markers for predicting the outcomes of hypopharyngeal carcinoma,as well as the molecular target of radiotherapy sensitization,to improve the quality of life of patients with hypopharyngeal carcinoma.Methods1)The radiological resistance of hypopharyngeal cancer was established and its radiological resistance was detected.FaDu-RR cells were established after repeated and intermittent X-ray irradiation.The radioresistant characteristics of FaDu-RR were detected by cloning assay,cell proliferation and cell cycle distribution analysis.2)Microarray was used to construct the mRNA differential expression profiles of radioresistance in hypopharyngeal carcinoma.The function of different expressed mRNA was analyzed.3)The DNA damage and repaire capacity of FaDu and FaDu-RR cells was detected by comet assay and immunofluorescence.QRT-PCR,western blot and immunofluorescence were used to detect the expressions of RPA1,Rad51,BRCA1 and BRCA2 in the two cells before and after iron irradiation.The outcomes were verified in nude mice xenograft model.Results1)Successfully established the radioresistant hypopharyngeal carcinoma cell line FaDu-RR.a.The cloning assay showed that the clones of FaDu-RR cell were more and larger than that of FaDu cells,indicating FaDu-RR cell was more resistant to IR than FaDu cells and radiation resistance was heritable.b.Cell proliferation assay showed that FaDu cells proliferate slightly faster than the FaDu-RR cells before irradiation.After 10 Gy radiation,FaDu-RR cells proliferated faster than FaDu cells.c.Cell cycle distribution analysis showed that the percentage of FaDu-RR cells in G1 phase was significantly lower than FaDu cells,but significantly higher in G2 phase before irradiation(P<0.05).After 10 Gy radiation,the percentage of FaDu-RR cells in G1 phase decreased significantly,and the proportion of S phase and G2/M cells increased significantly.2)The difference expression profile of radioresistance for hypopharyngeal carcinoma was successfully established.a.A total of 4,751 differentially expressed mRNA were selected by microarrays.Among them,2649 mRNAs were up-regulated,and 2102 mRNAs were down-regulated in FaDu-RR cells.b.The GO analysis of the differentially expressed gene suggested that the biological processes related with DNA function played a crucial role in the radiological resistance of the hypopharyngeal carcinoma.c.QRT-PCR was performed to verify the expression of four genes associated with DNA damage repair.The trend was consistent with the results of microarray,confirming the reliability of the microarrays.3)The different expressions of homologous recombinant repair proteins in FaDu cells and FaDu-RR cells.a.Detection of DNA damage and repaire capacity: the detection of?-H2 AX by immunofluorescence showed the DSBs in FaDu-RR cells decreased faster than in FaDu cells after irradiation.Comet assay suggested that the tail moment of the FaDu-RR cells was significant longer than that of FaDu cells before irradiated(P<0.01).However,it shortened significantly faster in FaDu-RR cells than in FaDu cells after irradiation,suggesting FaDu-RR cells were more capable in repairing DSBs than FaDu cells.b.QRT-PCR,western blot and immunofluorescence showed that the expressions of RPA1,Rad51,BRCA1 and BRCA2 in FaDu-RR cells were significantly higher than in FaDu cells.After irradiation,these four genes were continuously increased in FaDu-RR cells,and the expressions were significantly higher than those in FaDu cells at 30 min and 12 h after irradiation,suggesting that the FaDu-RR cells were more capable in HR repair.c.The resistant nude mice xenograft models of FaDu and FaDu-RR cells were established to verify the HR genes expressions.After intermittent irradiation,the tumor volume of FaDu cells reduced faster than the FaDu-RR cells.At 28 days after radiotherapy,the tumor volume and weight of FaDu cells decreased significantly,while the tumor volume and weight of FaDu-RR cells had no significant reductions.These suggest that FaDu-RR cells were more resistant to irradiation in the nude mice xenograft models.Immunohistochemical results showed that the expressions of Rad51,RPA1,BRCA1 and BRCA2 were significantly higher in FaDu-RR cell tumor,indicating the HR repair pathway plays an important role in the radiation resistance of FaDu-RR cell,which were consistent with the cell experimental results.Conclusions1)The radioresistant hypopharyngeal carcinoma cell line FaDu-RR was successfully established.2)The mRNA different expression profiles of hypopharyngeal carcinoma radioresistance were successfully established by microarrays.GO analysis suggests that the biological processes involved in DNA functions play a crucial role in the radioresistance of hypopharyngeal carcinoma cells.3)FaDu-RR cells were more capable in repairing DNA damage.4)The expression of HR proteins in FaDu-RR cells was significantly higher than in FaDu cells,suggesting that the HR played an important role in the radioresistance in hypopharyngeal carcinoma.