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Initial Studies on Regulating the Expression of DNA Polymeraseβ in Esophageal Carcinoma Cell Line(EC9706)

Author: WangTao
Tutor: DongZiMing；ZhaoGuoQiang
School: Zhengzhou University
Course: Pathology and Pathophysiology
Keywords: DNA polymerase beta Esophageal cancer RNA interference siRNA expression vectors Quantitative PCR
CLC: R735.1
Type: Master's thesis
Year: 2007
Downloads: 44
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Abstract

Background tumor cell differentiation, proliferation and death mechanisms abnormal results. DNA damage and gene structure abnormalities, as well as on the change in the expression and function of the resulting so-called oncogenes and / or tumor suppressor genes, is the premise of malignant transformation. But not all of the damage cause gene mutations, the reason lies in the DNA repair system cells to clear and repair of DNA damage. The base excision repair (BER) is the main way to clear cellular DNA damage, DNA polymerase β (DNA polymeraseβ, DNA polβ) is a key enzyme of the BER process. DNA polymerase beta is widely distributed in mammalian cells the major eukaryotic cells, DNA damage repair gene, whose DNA sequence is highly conserved, as a housekeeping gene. Its expression level is relatively stable throughout the cell cycle in the range from the proliferation of cell cycle regulation. Normal cell DNA polymerase beta is expressed at low levels. In recent years, many researchers have found DNA polymerase β gene mutation, such as colorectal cancer, stomach cancer, breast cancer, liver cancer, lung cancer, bladder cancer, prostate cancer and esophageal cancer in human tumor tissue samples. In addition, recently there is a high level of expression at the level of genes and proteins found in many tumors polβ, prostate cancer, breast cancer, ovarian cancer, colon cancer and esophageal cancer found that the expression than paraneoplastic polβ normal tissue multiples to the dozens of times. The high expression polβ DNA repair pathway, at the nucleotide level, the cross-injury synthesis and DNA replication mechanism to increase the rate of nucleotide mutations lead to increased spontaneous mutation of cells, promote tumor development. Research results have confirmed polβ mutation and high expression is closely related to the occurrence and development of a variety of tumors. However, what about the polβ low expression and tumor development relationship as well as the impact of the research reported on the biological characteristics of the tumor cells is not yet. The emergence of RNAi technology provides a new way to explore this issue. RNA interference (RNA interference, RNAi) is a conserved biological response caused by a double-stranded RNA (doublestrands RNA, dsRNA), dsRNA after RNA enzyme III nuclease (Dicer) processing, degradation into 21-23 nucleotides siRNA (small interfering RNA), siRNA with the RNA-induced silencing complex (RNA-induced silencing complex, RISC) binding, siRNA solution chain, the chain guide of the antisense RNA-induced silencing complex combination of homologous mRNA, thereby cutting the mRNA in loss of transcriptional information, to achieve specific inhibition of target gene expression results. Research purpose of this experiment is through to build of DNA polβ targeted siRNA expression vector of siRNA expression vector and transfected with wild-type polβ to EC9706 cells, regulation of the expression of the state of polβ. The biological characteristics of EC9706 cells in the DNA polβ different expression level. To appropriate intracellular reveal the DNA polβ gene expression and functional status, and to provide new ideas and therapeutic targets for the prevention of esophageal cancer intervention. Research Methods in accordance with the nucleotide sequence of the DNA polymerase β in GenBank (M13140), with reference to the siRNA design strategy, select the loci of RNAi oligonucleotide designed and synthesized for the coding region of the DNA single strand, is formed after annealing duplexes. And the pSINsi-hU6 to carry hU6 promoter plasmid construct siRNA expression vectors for DNA polymerase beta. In cationic liposome-mediated siRNA expression vector and transfected with wild-type polβ to esophageal cancer EC9706 cell line. The experiment was divided into five groups, respectively to express carrier of the experimental group and the experimental group 2 the transfection wild-type polβ, polβ high-expression group, transfected with empty vector pSINsi-hU6 the transfection with siRNA empty vector and untransfected blank control group, the G418-positive clones were screened to establish cell lines stably expressing. Growth and proliferation of esophageal cancer EC9706 cells in vitro by flow cytometry Observer DNA polβ in different expression levels change; nude mice, nude mice tumor tissues DNA polymerase βmRNA of expression was detected by quantitative PCR methods by nude mice subcutaneously into the tumorigenic ability in esophageal cancer cells in the expression levels of the tumor experiments detect DNA polβ changes to observe the impact of EC9706 cells in vivo proliferation. Experimental Results 1. Successfully constructed the targeted siRNA DNA polβ to to express the the carrier pSINsi-hU6-siRNApolβ288 and pSINsi-hU6-siRNApolβ814. siRNA expression vector and the wild-type polβ liposome method EC9706 cells transfected by G418 positive clone cells. 3. Flow cytometry showed the transfected DNA polβ targeted siRNA expression vector (pSINsi-hU6-siRNApolβ288 and pSINsi-hU6-siRNApolβ814) and the wild type polβ the EC9706 cells with EC9706 cells transfected with empty vector and untransfected phase The ratio of the S phase of the cell cycle frequency was significantly increased (P <0.05). 4. Nude mice experiment results show that compared with the empty vector group and the control group the transfected DNA polβ targeted siRNA expression vector (pSINsi-hU6-siRNApolβ288 and pSINsi-hU6-siRNApolβ814) experimental group 1 and experimental group 2, tumor weight a significant increase compared with the empty vector group and the control group (P <0.01); the transfection wild-type polβ, DNA polβ high expression group, tumor weight was significantly increased (P <0.01). The experimental group the experimental group and DNA polβ of high expression group was no significant difference in tumor weight between. 5. Fluorescence quantitative PCR detection of tumor tissue the INA polβmRNA in expression. Compared with the empty vector group and the control group, the transfected DNA polβ targeted siRNA expression vector the (pSINsi-hU6-siRNApolβ288 and pSINsi-hU6-siRNApolβ814) experimental groups 1 and 2, the experimental group polβmRNA the expression was significantly decreased (P <0.01), the DNA polβ transfection wild-type polβ, high expression the group polβmRNA expression was significantly increased (P <0.01). Show that the construction of siRNA expression vectors DNA polβ silence inhibition and silence the same effect. Conclusion The results show that not only the DNA polβ high expression related to the incidence of esophageal cancer, and low expression of the DNA polβ also may be an important molecular mechanism of esophageal cancer. Therefore, using RNAi technology to inhibit the expression of polβ to the appropriate level, while maintaining polβ repair enzyme activity due to its high expression does not produce low-fidelity replication might be helpful to the intervention of esophageal cancer.

Initial Studies on Regulating the Expression of DNA Polymeraseβ in Esophageal Carcinoma Cell Line(EC9706)

Abstract

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