| Background: Nasopharyngeal carcinoma(NPC)is a regional high-risk head and neck malignant tumor,which is common in southern China,including Guangdong and Guangxi provinces.Chemoradiotherapy is still the main treatment for NPC.Chemotherapy is a systemic treatment,and its individualized differential drug delivery regimen may result in primary and secondary chemotherapy resistance,which is related to NPC recurrence and metastasis,making clinical treatment a great challenge.With the development of pharmacogenetics,individualized treatment under gene guidance has become the only way to improve the therapeutic effect of cancer and reduce ineffective treatment.Relevant genetic markers that predict differences in treament efficacy,such as single nucleotide polymorphism(SNP),can predict the patient's therapeutic efficacy and adverse reactions,and provide a basis for individualized treatment of cancer,thereby guiding clinical treatment and improving efficacy.Studies have found that the resistance of many cancers to clinical treatment may be related to the increased ability of DNA oxidative damage repair.There are many mechanisms to repair DNA.Some studies have shown that human 8-oxoguanine DNA glycosylase 1(hOGG1)is involved in the mechanism of treatment resistance in the base excision repair(BER)pathway.Our previous study in screening the genetic polymorphisms of DNA from peripheral blood samples of NPC patients and healthy controls in a Guangxi population,and we found that the hOGG1 rs1052133 GG genotype was a functional SNP and its distribution was significantly different between the two groups.Objective: To evaluate the effect of genotype of rs1052133 SNP in hOGG1 on the prognosis of patients with NPC,and to establish cell models harboring hOGG1 with rs1052133 SNP GG or CC genotypes and study their respective function.Comprehensive studies were undertaken to analyze the differences of DNA damage repair function between hOGG1 with GG genotype and that with CC,the corresponding mechanisms of these SNPs affecting NPC prognosis is also to be investigated.Method: 1.NPC patients,who were selected for hOGG1 rs1052133 genotype in our preliminary study,were chosen as candidates to explore the effect of rs1052133 SNP on NPC prognosis.2.We constructed vectors overexpressing different rs1052133 SNPs in hOGG1,which were packed in lentivirus and then were used to infect a NPC cell line,HK1,to establish cell models stably overexpressing hOGG1 with rs1052133 CC or GG SNP.Fluorescence microscopy,RT-qPCR and WB were utilized to evaluate transfection efficiency of the target genes,which were subsequently sequenced.3.Using MTT assay,real-time observation by live cell workstation,flow cytometry,RT-qPCR,WB,immunofluorescence,etc.,the functions of hOGG1 with rs1052133 GG and CC genotype were investigated,and possible proteins that may mutually interact with hOGG1 were also investigated by immunoprecipitation and mass spectrometry.Result: 1.The relation between hOGG1 rs1052133 GG or CC genotypes and the NPC prognosis: we selected eligible 352 cases from 488 of those with NPC,who were genotyped for hOGG1 rs1052133 SNP in our preliminary investigation.Our univariate analysis showed that CC genotype(p=0.018),T classification(p=0.020)was significantly associated with overall survival(OS),and smoking(p=0.055)was related to OS with a marginal significance.Our univariate analysis also indicated that T classification(p=0.009)was significantly related to tumorfree survival(TFS),and CC genotype(p=0.066)had a trend to be associated with TFS.The multivariate analysis showed that CC genotype(HR 2.133,95%CI 1.217-3.738,p=0.008),T classification(HR 2.113,95%CI 1.021-4.374,p=0.044)were independent factor for OS,and CC genotype(HR 1.789,95%CI 1.046-3.057,p=0.034),T classification(HR 2.105,95%CI 1.090-4.066,p=0.027)were independent indicators for TFS.Three hundred and twenty-three NPC cases with late stage(? and ? stage)were also involved in our univariate analysis,and the results suggested that CC genotype(p=0.008 and 0.039),T classification(p=0.049 and 0.031)were significantly associated with OS and TFS,respectively.Multivariate analysis showed that CC genotype(HR 2.158,95%CI 1.236-3.768,p=0.007),T classfication(HR 2.236,95%CI 1.107-4.514,p=0.043)were independent prognostic factors for worse OS.And for TFS,CC genotype(HR 1.783,95%CI 1.050-3.028,p=0.032),T classfication(HR 2.043,95%CI 1.077-3.875,p=0.031)were independent prognostic factor for the decline in TFS.2.Cell models stably overexpressing hOGG1 with rs1052133 GG or CC were successfully established by infection of lentivirus:(1)morphology study showed that no obvious morpholgoical changes in all of the cell lines infected;contrast detection of fluorescence and luminescence in overlaping field showed that lentivirous infection efficiencies of all the cell lines were extremely high.(2)The expression levels of hOGG1 mRNA in hOGG1-GG-HK1 and hOGGl-CCHK1 were about as 2.28-fold and 2.26-fold as than that of N-HK1,respectively.(3)WB results showed that the expression levels of hOGG1 of hOGG1-GG-HK1 and hOGG1-CC-HK1 cells were slightly higher than those of N-HK1 cells.(4)Sequencing results showed that the two sequences of hOGG1 in hOGG1-GGHK1 and hOGG1-CC-HK1 had GG/CC mutation only at the rs1052133 site,with the other sequences unchanged.3.Functional exploration of hOGG1 rs1052133 SNP GG/CC:(1)The cell biological behavior of the hOGG1-GG-HK1 and hOGG1-CC-HK1 under no emergency stress showed no significant difference of growth rate between each cell line.After DDP or DPI administration,stress damage repair ability in both hOGG1-GG-HK1 and hOGG1-CC-HK1 were stronger than that of control group N-HK1.The results also indicated that hOGG1-GG-HK1 cells have weaker resistance to low dose DDP or DPI than hOGG1-CC-HK1 cells.(2)The real-time observation of the low-dose DDP effect on cell proliferation showed that hOGG1-CC-HK1 cells had the highest proliferation ability after DDP administration,followed by that of hOGG1-GG-HK1 cells,with N-HK1 cells having the weakest ability.(3)Flow cytometry was used to detect the effect of DDP and DPI on the cell cycle.The results showed that N-HK1 cells were arrested in G2/S phase in the most degree,followed by that hOGG1-GG-HK1 cells,with that of hOGG1-CC-HK1 cells being the least after DDP administration.(4)The detection of hOGG1 mRNA levels before and after lowdose DDP or DPI treatment showed a hOGG1 mRNA up-regulation in hOGG1-GG-HK1 cells,but in a lower degree than that of hOGG1-CC-HK1 cells.(5)Immunofluorescence results showed a highest hOGG1 protein expression level in hOGG1-CC-HK1 cells following DDP treatment.(6)The WB results showed that the hOGG1 protein level of hOGG1-CC-HK1 cells was up-regulated in a dose-dependent manner with the concentration of the DDP or DPI.The hOGG1 protein level in hOGG1-GG-HK1 cells was slightly negatively associated with the DDP or DPI concentration.(7)Before DDP adminstration,the DUSP6 expression level in hOGG1-GG-HK1 cells was the highest,compared with those of hOGG1-CC-HK1 and N-HK1.After DDP administration,DUSP6 levels in both hOGG1-GG-HK1 and hOGG1-CC-HK1 declined in different degree,with that in the latter doing more obviously.But when DDP concentration continue to increase to a certain extent,DUSP6 was resumed to be up-regulated again.(8)The results of co-immunoprecipitation showed that hOGG1 and DUSP6 did not interact directly.Conclusion: 1.hOGG1 rs1052133 CC genotype may be an independent risk factor predicting the prognosis of NPC.2.NPC cell models,hOGG1-GG-HK1 and hOGG1-CC-HK1 stably transfected with hOGG1 rs1052133 CC or GG SNP were successfully established.3.Damage repair function of hOGG1 with rs1052133 CC genotype is stronger than that of hOGG1 with rs1052133 GG genotype.4.hOGG1 with rs1052133 CC genotype inhibited DUSP6 expression more significantly than that with rs1052133 GG genotype in NPC cells.5.hOGG1 may not bind DUSP6 directly to perform its function,and the detail mechanism of their mutual interaction remain to be further studied.6.For those NPC patients hOGG1 rs1052133 CC genotype,the need of increasing the treatment agents concentration to improve the therapeutic effect and the prognosis deserve further investigation. |
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