Preliminary Study of Mutation of DNA polβ Promoter in the Esophageal Carcinoma Tissue
|Course||Biochemistry and Molecular Biology|
|Keywords||DNA polymerase beta Promoter Luciferase reporter vector Esophageal cancer Mutation|
Background and purpose of DNA repair enzymes play an important role in the maintenance of cell genome stability and biological and genetic stability, DNA repair system defects or abnormal tumor has also been a concern, has become one of the cancer etiology hot spots. DNA polymerase β (DNA polymerase beta, polβ) is one of the main polymerase involved in DNA repair, mainly involved in base excision repair the (base exicisionrepair BER) as well as cross-damage repair, from the repair of damaged or mutated gene. A variety of endogenous or exogenous harmful factors polβ gene structure or expression changes are likely to eventually prompted the occurrence and development of the tumor. Chang at overexpression in the cell will affect the stability of the chromosome involved in tumor development. The results show that in recent years, there are also high polβ gene mRNA expression in esophageal carcinoma and occurred in the early stages of cancer. Over prompt polβ gene expression may be related to the occurrence and development of esophageal cancer. Gene expression usually be made subject to the regulation of the core promoter region. The core gene promoter region nucleotide mutation may affect its promoter activity causing gene product activity and changes in the content. High expression is related to abnormal control of with polβ gene promoter region has not been reported in the literature. Therefore, this project intends to build human wild-type and mutant polβ gene promoter luciferase expression vector, compares the activity of luciferase expression in cells, to explore polβ gene core promoter region base mutation the regulation of gene expression, gene promoter for more depth study polβ, occurrence and development of esophageal cancer and provide the basis for targeted gene therapy. Materials and Methods 1. Specimen source: esophageal normal mucosa and esophageal cancer tissue samples from the the Linzhou Cancer Hospital, pathologically proven squamous cell invasive cervical cancer, the preoperative patients did not receive any chemotherapy or radiotherapy. 2 amplification the people polβ gene promoter region DNA fragments and mutation screening: reference polβ gene promoter sequence references, designed to amplify DNA of pol gene promoter primer, human wild-type and mutant polβ gene was amplified by PCR core promoter fragment, connect the vector pGEM-T Easy and transformed into competent bacteria DH-5 alpha, alpha complementary and using universal primers T7/SP6-line PCR identification the recombinant pGEM-T-polβ/promoter target fragment is transferred to positive after the anti bidirectional sequencing using DNASIS the OMIGA software will the sequencing results with GenBank the found DNA polβ promoter sequence homology comparison, analysis determines wild type polβ start promoter sequence and mutant polβ of promoter mutation sites. (3) Construction and identification with people polβ gene promoter luciferase reporter gene expression vector: extract the correct sequencing of recombinant T vector (pGEM-T-poβ/promoter) plasmid and pGL3-Neo-enhancer fluorescent luciferase reporter vector, respectively Xho1 I and Hind III double digestion, purified the double stick polβ promoter target fragment was subcloned into the double-stick linear pGL3-neo-enhancer luciferase reporter vector, the recombinant plasmid pGL3-neo-of pol / promoter by Xho1 Ⅰ and Hind Ⅲ, double digestion and PCR amplification, the pros and cons of bi-directional sequencing to identify the construct containing the human wild-type and correctness of the the mutant polβ gene sub-luciferase reporter gene expression vector, and confirmed that the mutation was present sites still found no other differences exist. The construct containing human wild-type and mutant polβ gene promoter luciferase reporter gene expression vector (pGL3 (W / M) of pol / promoter). (4) Cell culture and plasmid transfection: cells were divided into four groups: (1) blank control group: No transfection any plasmid Eca9706 of cells; (2) control group: the empty vector transfected with pGL3-neo-enhancer; (3) Wild Group: transfected with a luciferase enzyme reporter gene expression vector containing the gene promoter sequence in human wild type polβ of pGL3Wpolβ/promoter '; (4) mutation group: Transfection -37 point C → A mutation-containing polβ promoter sequence firefly luciferase reporter gene the expression vector pGL3Mpolβ/promoter. Liposomes Lipofectamine2000 (2), (3) and (4) group transfer transfection Eca9706 cells, G418 screening positive cell clones. Each set of experiment was repeated at least three times. 5 detection of luciferase activity: Luciferase fluorescence detector for the determination of luciferase activity of each group, each group of measured values ??were averaged. 6 Statistical analysis: detection of luciferase activity data analysis and processing (?) ± s said, the one-way ANOVA using SPSS 14.0 statistical software analysis, P <0.01 was considered significant. Results 1.PCR amplification containing the person polβ promoter region of the DNA fragments and mutation Filter: extracted from human genomic DNA as a template, amplified by PCR polβ gene promoter sequence, electrophoresis shows a single band at 392bp, the theoretical value of the fragment, the PCR amplification product of the positive and negative bidirectional sequencing and homology comparison and analysis, to determine the gene promoter sequence with GenBank and reported in the literature polβ the normal sequence of the wild type, containing -37 C → A point mutation mutant. PCR amplification of the wild-type and mutant the pGL3 (W / M) of pol / promoter recombinant plasmid and identification: wild and mutation polβ gene promoter sequence into the luciferase reporter vector cloning sites line increase and Xho I and Hind III double digestion positive clones were developed at the 392bp visible electrophoretic bands, consistent with the theoretical value of the fragment. DNh positive and negative bidirectional sequencing inserted into the wild-type sequence is normal and mutant mutation sites correctly unchanged, to show that pGL3Wpolβ/promoter and pGL3Mpolβ/promoter expression vector was constructed successfully. 3 luciferase activity analysis: blank group (value 400), between the control group, wild group and mutation group, the difference was statistically significant. -37 Point C → A mutation (7882559 ± 201824.9) was significantly higher than the mutant pGL3Mpolβ/promoter luciferase activity the wild type pGL3Wpolβ/promoter (1,112,976 ± 82900.2), P <0.001. The wild group and the mutation group compared with the control group (2743.50 ± 227.3), the differences were statistically significant (P <0.001). Conclusion 1. Successfully constructed containing human wild-type and mutant DNA of pol gene promoter luciferase reporter gene expression vector pGL3 (W / M) of pol / promoter. 2.pGL3 (W / M) of pol / promoter polβ the promoter has a strong promoter activity of luciferase gene expression and polβ start the promoter region of -37 C → A base mutation can significantly enhance the luciferase the expression. For more in-depth research laid the foundation for polβ gene promoter in esophageal cancer development and targeted gene therapy.