Influence Of Dna Polymerase β Promoter Mutation To Its Transcriptional Activity In Esophageal Carcinoma Cell Ec-1

Backgroud and ObjectiveDNA repair enzymes play an important role in the maintenance of genomic stability of cells. After studing the genetics of esophageal cancer ,some schoars found that DNA repair capacity in the peripheral blood of patients with esophageal cancer after DNA damage significantly decreases, and DNA brittleness increases, and cancer cells have many significant changes in the chromosome. For this reason,they suggested that the of development of esophageal cancer accompanied with the process of repairing DNA damage. Human DNA polymeraseβ(DNA polymerase beta, polβ) is one of the human repair enzymes, which mainly involved in base excision repair (base exicision repair, BER) and the inter-repair of damaged or mutated gene. A variety of endogenous and exogenous harmful factors polβgene structure or expression,which may eventually contribute to influeace and development of cancer. Some studies have show that polβover expression and mutation occur early in carcinogenesis. It indicates that polβgene mutation and over-expression may be associated with the development of esophageal cancer. Gene transcription is regulated by the promoter, enhancer and the responsive element, such as cis-acting element; and affected by transcription regulatory factor,incuding basic transcription factors, upstream transcription factor,and other trans-acting factor . Therefore,the changes in gene transcription activity may be not only on the abnormal base pairs of promoter and,but also on trans-acting factors that act on the promoter. Previous study show that polβpromoter mutation may exist in many different forms of mutations and lead to its change of transcriptional activity in esophageal carcinoma. Based on this, the promoter of DNA polβgene in the EC-1 of human esophageal cancer cells and normal cells has been studied. First of all, human wild-type and mutant polβgene promoter luciferase expression vector were built,and wild-type DNA polβpromoter the cone of DNA polβmutant promoter and trasfered into tumor cells (EC-1 and Eca9706 cells), and normal immortalized cells. Then strong or weak promoter activity were investigated. and the results show that the promoter of polβin tumor cells increases promoter activity.Materials and Methods1. PCR Amplification of the DNA fragments of EC-1 and 293T polβgene promoter and screening of mutational site: The primers for amplificating the DNA fragments of EC-1 and 293T polβgene promoter was designed according to the GenBank and literature. The DNA fragments of wild-typed and mutant of EC-1 and 293T polβpromoter were sythesed by PCR. Then they respectivety were ligated to pGEM-T Easy with T4 DNA ligase,and transfered them into the DH5αcells. The recombinant clones, pGEM- T-polβ/ promoter, were selected and identified throughα-complementation test and PCR , and sequenced,compaired with the sequence of polβgene promoter in GenBank by DNASIS,OMIGA software, and the mutational site was analyzed.2. Construction and identification of luciferase reporter gene expression vector containing EC-1 and 293T polβgene promoter: The positive pGEM-T-polβ/promoter recombinant were extracted and sequenced. and PGL3-neo-enhancer luciferase reporter vector. were cut by Xho I and Hind III restriction enzyme respectively. Then, the target fragment of polβgene promoter was subcloned into PGL3-neo-enhancer luciferase reporter vector After identifing the positive clones with Xho I and HindIII by restriction enzyme and PCR, they were sequenced for confirming the positive wild-type and mutant recombinants again, and the result show that the mutation site is right. Like the luciferase reporter gene expression vector, pGL3 (W/M) polβ/promoter.It contains EC-1 and 293T DNA polβgene promoter.3. Cells culture and transfection: The cells were divided to 4 groups. (1) blank group: not to transfer any plasmid into Eca9706,EC-1 and 293T; (2)control group: to transfer PGL3-neo-enhancer blank plasmid into Eca9706, EC-1 and 293T; (3)wild -type group: to transfer PGL3-neo-293 containing wild-type DNA fragment of polβ gene promoter;(4)mutant group: to transfer PGL3-neo-EC-1 (-137 site and -164 site Gâ†'A mutation),containing mutational site DNA fragment of polβgene promoter.(2),(3) and (4)were introduced into Eca9706 cells,EC-1 cells and 293T cells by mixing up with Lipofectamine 2000 respectively, and positive clones were screened with G418 selective medium. The experiment was repeated three times.5. Luciferase activity measuring: The luciferase activity was detected with fluorescence detector,and average value in every group was acculated.6. Statistical analysis: Data were expressed as mean±standard deviation(SD). Means between groups were compared by using one-way ANOV method and SPSS14.0 Statistical software. Probability values less than 0.05 were considered to be significant.Results1. Amplificating the DNA fragments of EC-1 and 293T polβgene promoter and screening mutational site: The core sequence of DNA polβgene promoter was amplified from EC-1 and 293T genome DNA by PCR, which was 499bp and corresponded with its sequence provided by GenBank. Sequencing, it was confirmed that the wild-type sequence of polβgene promoter had the same sequence with GenBank and literatures. But the polβgene promoter mutation contains mutations of Gâ†'A at -137 and -166 site.2. Construction and identification of wild-typed and mutant pGL3 (W/M) polβ/promoter recombinant plasmid: Polβgene promoters were inserted into multiple cloning sites of luciferase reporter gene vector in the 233T cells and EC-1 cells ,and the positive clones with reaction sites of Xhol I and HindIII restriction enzyme were indentified. The length of the core sequence of DNA polβgene promoters was 392bp,which corresponded with its sequence provided by GenBank. By sequencing. It had been confirmed that the wild-typed sequence was right,and the mutation site of the mutant is also right. The outcome indicated that the luciferase reporter gene expression vector,PGL3-neo-EC-1 and PGL3-neol-293containing EC-1 and 293T DNA polβgene promoter were constructed successfully.3. Luciferase activity assay: Three different plasmid pGL3-neo (control group), pGL3-neo-293T-polβ/promoter(wild type)and pGL3-neo-EC-1-polβ/promoter (mutant) were transfected into EC-1 cells,and separately transfected into Eca9706 and 293T cells. By compairing each group,wild group and the control group (pGL3-neo), the difference are significant(P <0.001).Compairing wild and mutant group,the differences were statistically significant(P<0.001).pGL3-neo(control group)pGL3-neo-293T-polβ/promoter plasmid or re-combinant plasmid (wild type) were transfected into three different cells, luciferase activity did not statistical significance(P> 0.001); and pGL3-neo-EC-1-polβ/promoter were transfected into three different cells, luciferase activity was no difference.Conclusions1. It has been confirmed in EC-1 cells that exists mutation site -137 and -166 Gâ†'A; DNA polβgene promoter in 293T cell.2. The luciferase reporter gene expression vector containing mutatued DNA polβgene promoter,and the recombinations plasmid of DNA polβgene promoter (PGL3- neo-Ec-1 and PGL3-neo-293T)has been was constructed success in Ec-1 cells fully.3. The activity of mutation type of DNA polB promoter is stronger than that of 293T cell.4. The promoter activity of wild type, mutation type of DNA polβpromoter reporter gene in EC-1 cell is much stronger than one in normal cells. It indicates that there is some factors to stengthen the activity of DNA polβpromo- ter in EC-1 cells.