Effect Of RPA1 On The Radiosensitivity Of Nasopharyngeal Carcinoma And Its Mechanism

Nasopharyngeal carcinoma(NPC)is a common malignant tumor in the Southerneast Asia and the Southerneast coastal areas of China.Thetreatment technology of NPC had got great progress,but the local recurrence and metastasis after treatment was still a leading cause of failure.It was closely associated with the radiosensitivity of NPC cells.Today,radiotherapy was the prefer choice for NPC and the treatment protocol was formulated according to tumor-nodal-metastasis stage(TNM stage)of the patient.But the tumor cells in the same patient had variety radiosensitivity owing to their intrinsic heterogeneity.With the advent of the era of precise therapy,the biomarkers for predicting therapeutic outcome and prognosis of malignant tumor have been exploring and are wished to facilitate the formulation of individual treatment protocol for the patients with malignant tumor.Therefore,it is urgent to find a biomarker for predicting the radiosensitivity of NPC and guiding individual treatment.Ionizing radiation(IR)and/or cytotoxic drug induced DNA double strand break(DSB)was a lethal damage,which was intensely related to the radiosensitivity.Replication protein A(RPA),a major element of homologous recombination(HR),bound with single strand DNA(ssDNA)and protected ssDNA from formation of second structure.Studies had found that the expression of RPA was related to esophageal cancer,gastric carcinoma,hepatoma carcinoma and colon carcinoma,and the higher the expression,the worse the prognosis.Among the complex,RPA1,one of a subunit of RPA complex,was viewed as the most important due to its high affinity with ssDNA.Two other studies had developed that knockdown of RPA1 of esophageal cancer TE-1 cells enhanced radiosensitivity.It is suggested that RPA1 may be a predictor for the radiosensitivity of the tumor.At present,it was unclear the expression of RPA 1 in nasopharyngeal carcinoma and whether it can be used as a predictor of the radiosensitivity in NPC.Object:To investigate the effect of RPA1 on radiosensitivity of nasopharyngeal carcinoma and its mechanism.Methods:(1)The data of clinicopathological features of NPC was collected.All the patients were divided into two groups-complete response(CR)and non-complete response(nCR)-according to the imaging data before and 1 month after radiotherapy and response evaluation criteria for solid tumor(RECST).The expression of RPA1 mRNA in 24 and protein in 182 patients with NPC were detected with the use of RT-PCR and immunohistochemistry(IHC),respectively.The difference of RPA1 in the two groups was analyzed and the relationship was also analyzed between the expression of RPA1 and clinicopathological features.(2)The RPA1 low expression of radiation resistant NPC stable cell lines were established using the lentivirus mediated shRNA interference technology.After lOGy-X ionizing radiation(IR),proliferation,radiosensitivity,DNA damage repair,cell cycle and apoptosis were tested with the use of cell viability assay,clonogenic assay,alkaline comet assay andflow cytometry,repectively.In addition,the effects of RPA1 combined with radiation on tumor growth were studied using a tumor bearing nude mouse model.(3)Western blotting weas used to test the proteins expression of RPA 1,RAD51,y-H2ax and IF was used to test RAD51,y-H2ax foci formation.Result:(1)RPA1 mainly expressed in the nuclear,nCR group with a high level and CR group with a low level.In CR group,in the patients with low expression of RPA1,64 cases obtained complete remission(68.09%),which was obviously higher than nCR group(?2=15.860,P=0.000).The level of RPA1 expression was merely related to the radionsensitivity of NPC.Further,the result of multivariate analysis showed that,besides EBV,RPA1 was another predictor of radiosensitivity in NPC(HR3.755,95%CI 1.990-7.085).(2)The CNE-2R cells were transfected by three lentivirus mediated shRNA,RPA1-RNA1,RPA1-shRNA2 and RPA1-shRNA3,respectively.After selected with purinomycin,RPA1-shRNA1 had the best inhibition effect among them.The result of clonogenic assay showed that the colon number in RPA1-shRNA group was obviously less than that in the control group(p<0.05),and the radiosensitization ratio(SER)was 1.21.The result of CCK8 assay showed that proliferation of the cells in RPA1-shRNA group declined compared with that in control group.After lOGy-IR,the change was more obvious(p<0.05).The result of Alkaline comet assay displayed that the comet tail in the two groups was very obvious and the tail length is almost equal at 30min after IR.Six hours later,the comet tail in the control group disappeared,while the comet tail in the RPA1-shRNA group was still visible(p<0.05).The results of flow cytometry showed that the G2/M phase cells in the RPA1-shRNA group were significantly more than those in the control group after 10Gy-IR(P<0.05).And the number of apoptotic cells in RPA1-shRNA group increased significantly compared with NC-shRNA group(p<0.05).The result of tumor bearing nude mouse model displayed that the tumor volume in the RPA1-shRNA was significantly smaller than that in control group(p<0.05).After 6Gy-IR,the cells growth in the both groups retarded,and the volume of tumor was smaller than that in the untreated groups.And the tumor volume in RPA1-shRNA group was obviously smaller than that in the control group(p<0.05).The same trend was observed in the weight of tumor in the two groups.(3)The result of WB showed that the expression of RAD51 in the RPA1-shRNA group was appreciably lower than that in the control group at 1hour after 10Gy-IR(p<0.05),while the expression of ?-H2ax had no significant difference between the two groups(P>0.05).After 6 hours,the expression of RAD51 were attenuated in the two groups(P>0.05),while?-H2ax expression in RPA1-shRNA group was significantly higher than that in the control group(p<0.05).The result of IF displayed that the number of RAD51 foci in RPA1-shRNA group was significantly lower than that in the control group at 1 hour after 10Gy-IR.Six hours later,RAD51 foci in RPA1-shRNA group disappeared,while it was still visible in the control group.Twelve hours after IR,the ?-H2ax foci in the RPA1-shRNA group was still clear,while it almost disappeared in the control group.Conclusions:1.RPA1 highly expressed in NPC patients with radioresistance.2.RPA1 can be used as a predictor of radiosensitivity in NPC.3.The mechanism that RPA1 affects the radiosensitivity of CNE-2R cells may be that the downregulation of RPA1 reduces the recruitment of RAD51 at the site of DNA lesion and inhibits the repair of DSBs.