Preliminary Study Of Mutation Of Dna Polβ Promoter In The Esophageal Carcinoma Tissue

Backgroud and ObjectiveDNA repair enzymes play an important role in maintaining the stability of the cells genome and biological heredity.The defect or abnormity in DNA repair system might be associated with certain steps in carcinogenesis,which is given close attention and became a hot research of the etiology of cancer.DNA polymerase beta (polβ) is considered to be an important part in the DNA repair machinery in mammalian cells.It mainly participate in base exicision repair(BER),repair to cut base,and spanning lesion repair to repair the damaged and muted gene.The alteration of the structure or expression of polβwas induced by all kinds of hamful factors from endogenous and exogenous sources may lead to development of tumor.The studies showed that the overexpression of polβin the cells will affect the stability of chromosomes,which participated in the development of tumor.In recent research,there are also overexpression of polβmRNA in esophageal carcinoma in the early stage.This is relevant with the development of esophageal carcinoma:The core region of a gene promoter usually can regulate the gene expression.The mutation of base in the core region of promoter will affect the activity of the promoter,thus changes the activity and content of gene expression products,there is no more papers to be shown whether or not the overexpression of polβgene concerned with the abnormal regulation of polβgene promoter.So we plan to construct the luciferase reporter expression vector which contained human wild-type and mutant DNA polβgene promoter,and compare the difference of the expression activity in the cell between of them to explore the regulation of the gene expression by base mutation in the core region of polβgene promoter.This may provide the foundation to study the roles of polβgene promoter in the development of esophageal carcinoma and the target gene therapy.Materials and Methods1.Specimen source:The specimen of normal mucous membrane of esophagus, a distant from the carcinoma place,and esophageal carcinoma tissue was obtained from the Tumor Hospital of Linzhou city.They were confirmed as the squamous cell infiltrating carcinoma by pathological examination.These patients didn't get any chemotherapy or radiotherapy before surgery.2.Amplificating the DNA fragments of human polβgene promoter and screening mutational site:The primer pair amplificating the DNA fragments of human polβgene promoter was designed according to the GenBank and literature about the sequence of polβgene promoter.The DNA fragments of wild-typed and mutant of human polβpromoter were obtained with PCR,and then were ligated to pGEM-T Easy with T4 DNA ligase and transfered them into the DH5αcompetent cells.The recombinant clones,pGEM-T-polβ/promoter,were selected and identified throughα-complementation test and PCR with the primers T7/SP6.The pros and cons two-way sequencing was made.The blast was performed to the sequence of polβgene promoter with GenBank in the intemet by DNASIS,OMIGA software,and analyse the mutational site of mutant.3.Construction and identification of luciferase reporter gene expression vector containing human polβgene promoter:The positive pGEM-T-polβ/promoter recombinant which were extracted and sequenced,and pGL3-neo-enhancer luciferase reporter vector,both of them were cut down by Xholâ… and Hindâ…¢restriction enzyme respectively.Then,the target fragment of polβgene promoter which was obtained by restriction enzyme was subcloned into pGL3-neo-enhancer luciferase reporter vector after purified with gel extraction kit. After identifing the positive clones with Xholâ… and Hindâ…¢restriction enzyme and PCR,and then Pros and cons two-way sequencing confirmed the positive wild-type and mutant recombinants again,and confirmed the mutation site is also right.The luciferase reporter gene expression vector,pGL3(W/M)polβ/promoter containing human DNA polβgene promoter was constructed successfully.4.Cells culture and transfection:The cells were divided to 4 groups.(1) blank group:not to transfer any plasmid into Eca 9706,(2)control group:to transfer pGL3-neo-enhancer blank plasmid into Eca 9706,(3)wild-type group:to transfer pGL3Wpolβ/promoter containing wild-type DNA fragment of human polβgene promoter and(4)mutant group:to transfer pGL3Mpolβ/promoter(-37 site Câ†'A mutation) containing mutational site DNA fragment of human polβgene promoter. Group(2),(3) and(4),were introduced into Eca9706 cells by complexing with Lipofectamine 2000 respectively,and screened positive clones with G418 selective medium.The experiment was carried out at least three times repeatedly in each group.5.Luciferase activity measure:The luciferase activity was determined with fluorescence detector,and got average value in every group.6.Statistical analysis:Data were expressed as mean±standard deviation(SD). Means of groups were compared using one-way ANOV method used SPSS14.0 Statistical software.Probability values less than 0.05 were considered significant.Results1.AmpLIficating the DNA fragments of human polβgene promoter and screening mutational site:The core sequence of DNA polβgene promoter which was amplified from human genome DNA by PCR was 392bp,which corresponded with its sequence provided by GenBank.Pros and cons two-way sequencing,blasting the sequence in GenBank in the interact,it was confirmed that the wild-type sequence of polβgene promoter had the same sequence with GenBank and literature reference, the polβgene promoter containing mutation form of Câ†'A at -37nt was confirmed mutant.2.Construction and identification of wild-typed and mutant pGL3(W/M) polβ/promoter recombinant plasmid:The wild-typed and mutant polβgene promoters were inserted into multiple cloning sites of luciferase reporter gene vector, identifing the positive clones with Xholâ… and Hindâ…¢restriction enzyme and PCR,it was shown that the length of the core sequence of DNA polβgene promoters was 392bp,which corresponded with its sequence provided by GenBank.Then pros and cons two-way sequencing confirmed that the wild-typed segment was fight,and the mutation site of the mutant is also right.The outcome indicated that the luciferase reporter gene expression vector,pGL3(W/M) polβ/promoter containing human DNA polβgene promoter was constructed successfully.3.Luciferase activity assay:The results of comparison among the blank group(400),control group,wild-type group and mutant group showed that the differences were statistically significant.The luciferase activity of the mutant of polβpromoter containing mutation form of Câ†'A at -37nt,pGL3M polβ/promoter (7882559±201824.9) was higher than its of the wild-typed pGL3W polβ/promoter (1112976β82900.2),P<0.001.The wild-type group and mutant group were compared with the control group respectively,the differences of the results were significant.Conclusions1.The luciferase reporter gene expression vector containing human DNA pol gene promoter,pGL3(W/M) polβ/promote,was constructed successfully.2.The polβgene promoter of pGL3(W/M) polβ/promoter was capable of promoting luciferase gene expression strongly,the polβgene promoter containing mutation form of Câ†'A at -37nt could enhance the luciferase gene expression very strongly.The results of this study provide the foundation for deeply studying the roles of polβgene promoter in the development of esophageal carcinoma and target gene therapy.