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Studies of RNA Interference for Expression of DNA Polymerase β Gene of Esophageal Carcinoma Cell Line(EC9706)

Author: SunSaZuo
Tutor: DongZiMing
School: Zhengzhou University
Course: Pathology and Pathophysiology
Keywords: DNA polymerase beta RNA interference Esophageal cancer RT-PCR Flow cytometry Trypan blue dye experiment
CLC: R735.1
Type: Master's thesis
Year: 2007
Downloads: 70
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Abstract

The background tumor development is the result of the interaction of environmental factors and host factors, and the repair capacity of the cell sensitivity to carcinogens and mutations directly affect the risk of carcinogenesis. DNA polymerase β (DNA polymeraseβ) is a 39 kD single peptide chain of small molecule protein, the polymerase is highly conserved, and the structure can be divided into two parts: 8KD the amino terminus of the carboxyl terminus, and 31 kd. The DNA polβ is considered to fill a single nucleotide gap in base excision repair (base excision repair, BER), including short segment BER and long fragment BER, DNA polymerase is one of the highest rates of mismatch enzyme, and play an important role in the process of oxidative damage. Due to the high error rate of DNA polymerase β (DNA polβ) when the catalytic base, its expression level is too low, too high, or mutation formation can cause genetic instability caused by the accumulation of mutations promote cell cancer. High expression of the DNA Polβ interfere with a variety of repair system in vivo, resulting in increase in DNA mutation rate and cancer susceptibility; high expression of DNA polβ tumor cell resistance, and genomic instability have a certain relationship. DNA polβ defects will reduce the ability of the cell's DNA base excision repair (BER), resulting in a high sensitivity to methyl methanesulfonate (MMS), methyl methanesulfonate or oxidant can induce the expression of the DNA polβ. In addition to play a role in the base excision repair, DNA polβ involved in cell cycle and cell differentiation, may be involved in DNA replication, recombination, with the maintenance of genome stability, cancer is related to the resistance of certain drugs. RNA interference (RNA interference, RNAi) is discovered in recent years and developed a new gene at the transcriptional level blocking technology is a double-stranded RNA (double-stranded RNA, dsRNA) molecules at the mRNA level Close corresponding sequence of gene expression or its silencing process, in the study of gene function and gene therapy has shown great prospect. The aim of the present study was to investigate the expression vector of RNA interference (small interference RNA expression vector) inhibition of the the DNA polβ gene EC9706 cells by tumor cells in vitro experiments to understand whether the siRNA expression vector can silence to esophageal the cells DNA polβ gene and silencing effect, for the future to improve tumor cell sensitivity to drugs provides a theoretical basis and experimental evidence. The experimental methods offered by Invivogen company computer design software, according to the DNA polβcDNA coding sequence (M13140) from DNA polβmRNA genes to select 2 paragraph 19 bases a specific sequence GGAAGTTTGTAGATGAAGG and TATGAGAGCTCATGCCAAG respectively to design two pairs 66nt oligonucleotide ; 66nt oligonucleotide annealed with Bgl Ⅱ and Hind Ⅲ digested sites of double-stranded form at each end; respectively the the STL1 (annealing double-stranded DNA fragment siRNApolβ448), STL2 (annealing to form double-stranded DNA fragment siRNApolβ954 ) recovered with 2.0% low melting point agarose gel electrophoresis; connection with the pGEM-T easy vector, respectively, to obtain the T-cloning recombinant pGEM-T-STL2 and the pGEM-STL1; using T4 ligase to the target fragment pGEM-T- siRNApolβ448 and pGEM-T-siRNApolβ954 linear expression vectors pSuper.gfp / neo connection; the PCR amplification Screening and Identification of reorganization the siRNA expression the carrier pSuper.gfp/neo-siRNApolβ448 and pSuper.gfp/neo-siRNApolβ954, and its DNA sequence analysis. The expression vector pSuper.gfp/neo-siRNApolβ448, and pSuper.gfp/neo-siRNApolβ954 blank carrier pSuper.gfp / neo were transfected into EC9706 cells, quantitative PCR method to detect non-transfected cells with transfected cells change of of in DNA polβmRNA expression levels; flow cytometry untransfected cells and cells transfected cell cycle changes; using the trypan blue dye assay cisplatin, bleomycin, methylene blue, and hydrogen peroxide under the influence The untransfected cells transfected with the change in the ratio of cell death. Experimental Results 1. Follow siRNA design principles fragment, ultimately determine the DNA polβ448-466 (GGAAGTTTGTAGATGAAGG) 954-972 bits (CCTTCATCTACAAACTTCC) of the target sequence. 2. the siRNA expression the plasmid vector pSuper.gfp/neo-siRNApolβ448 and pSuper.gfp/neo-siRNApolβ954 after bgl Ⅱ, and hind Ⅲ digestion digestion products on a 2% agarose gel electrophoresis, specific bands, with the pre-estimated fragment size consistent. 3. Transfected EC9706 cells can be observed under an inverted fluorescence microscope to green fluorescent protein widely expressed in cells stably expressing cells by G418. 4. DNA polymerase beta mRNA expression level in the experimental group targeted siRNA transfection 1 (the EC9706 cell transfection pSuper.gfp/neo-siRNApo1β448) and the experimental group 2 (transfected EC9706 cells pSuper.gfp/neo-siRNApolβ954 the ) in the control group and the empty vector control group were significantly lower statistically highly significant difference (P <0.01), experimental group 2 mRNA expression than the experimental group was significantly lower statistically there is a significant difference (P <0.05, P = 0.37); blank control group and the empty vector control group the higher level polβmRNA expression, both statistically no significant difference (P> 0.05); The experimental group 1 and experimental group 2 the S phase of the cell with the empty vector group and the control group, there was significant difference (P <0.01) and a significant increase in the proportion of cells in S phase after transfection, cell proliferation rate increased. But no difference between the experimental group and the experimental group 2 (P> 0.05), on the EC9706 the cell DNA polβ expression level reduced to the scope of this study, the rate of cell proliferation is not much. 6. Cisplatin dosing group concentration of less than 60μmol / l, the four groups of cell mortality was no significant difference (P> 0.05), with increasing concentrations of drugs, the mortality rate began a highly significant difference (P <0.01) results are statistically significant. Bleomycin dosing group and methylene blue dosing group from the beginning of each of the initial concentration of the four cell death ratio there is a highly significant difference (P <0.01) had statistically significant results. The hydrogen peroxide dosing set of drug concentrations of less than 120 μmol / l, four groups of cell mortality were compared there was no significant difference (P> 0.05), with increasing concentrations of drugs, the mortality rate began to appear highly significant difference (P <0.01) results are statistically significant. Provide preliminary experimental evidence on the drug sensitivity of to improve tumor cells using RNA interference. Conclusion 1. Successfully constructed the targeted DNA polβ siRNA eukaryotic expression the carrier pSuper.gfp/neo-siRNApolβ448 and the pSuper.gfp/neo-siRNApolβ954, both transfected EC9706 cells can significantly reduce cell polβmRNA the expression instructions to build The siRNA eukaryotic expression vector DNA polβ a highly efficient and specific silencing effect stronger former. 2. Initially confirmed in EC9706 cells DNA polβ low level of expression is not conducive to the maintenance of cell biological characteristics, higher rates of cell proliferation and increased sensitivity to the drug; so expression of DNA polβ in level down to an appropriate level by RNA interference to inhibit tumor The cell has a certain effect.

Studies of RNA Interference for Expression of DNA Polymerase β Gene of Esophageal Carcinoma Cell Line(EC9706)

Abstract

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