| backgroundThe interaction between environmental factor and host factor results in the occurrence and development of tumor. Cell's sensitivity to carcinogen and it's rapair ability of self-mutation can influence cell canceration probability. DNA polymerase beta (DNA polβ) is an 39KD single peptide bond micromolecule protein which is heightly conservative. It can be divided in two parts: 8KD amino terminal and 31KD carboxyl terminal. The major role of DNA polymerase beta was thought to be supply gaps of mononucleotide, including short patch base excision repair and long patch base excision repair. It has the highest mismatch rate in DNA polymerases, and produces a marked effect during oxidative damage. Because of the high mismatch rate in catalysis bases, the change of DNA polymerase beta expression or mutation could cause genetic instability, accumulated mutation, then result in corpuscular canceration.The high expression of DNA polymerase beta interfere many restoration systems in vivo, which increase the DNA mutation rate and sensitivity of cancer. The high expression of DNA polymerase beta have related with drug resistance and genomic instability. The defection of DNA polβcan cut down DNA base excision repair ability, which lead to cell's hypersensitivity to methylmethanesulfonate(MMS), meanwhile, MMS or oxidizer can induce DNA polβexpression. Besides playing a role in BER, DNA polβis involved in cell cycle and cell differentiation. It also participate duplication, recombination, and is concerned with genome stability and drug resistance. RNA interference is a new technology which blocks gene expression at the post-transcriptional, and a process in which gene silencing mechanism induced by double-stranded RNA (dsRNA) on molecular level. It has a huge application foreground in studying the gene function and gene therapy.ObjectiveThe aim of our study is to inhibite the expression of DNA polymerase beta in esophageal carcinoma cell line EC9706 by small interfering RNA(siRNA), and observe the depressant effect. Whether RNAi-medicated silencing of DNA polβexpression could influence tumor cells in vivo or not was determined in this study.MethodsAccording to DNA polβcDNA exon(M13140) and the strategy of designing siRNA, we choose two specific sequences contained 19 bases:GGAAGTTTGTAGATGAAGG and TATGAGAGCTCATGCCAAG, and designed two pairs of 66nt oligonucleotide. After annealing, the single-stranded oligos will generate a double stranded (ds) anneal product which have two sites: BglⅡand HindⅢ. We cloned the anneal product into pSUPER.gfp/neo vector to construct two siRNA expression vectors targetting DNA polβ: pSuper.gfp/neo-siRNApolβ448 and pSuper.gfp/neo-siRNApolβ954, then analysing DNA sequence. RNAi expression vector was transfected into esophageal tumor cells(EC9706) by lipofectin reagent and positive clones were selected by G418. We used the method of RT-PCR, flow cytometry and trypan blue exclusion assay to observe the gene expression, the proliferation activity of cells and the death rate.Results1. According to the principle of siRNA design and sequence of DNA polβin GeneBank, we choose two target sequence GGAAGTTTGTAGATGAAGG and CCTTCATCTACAAACTTCC by homologous series index.2. Successfully constructed the siRNA eukaryotic expression vectors pSuper. gfp/neo-siRNApolβ448 and pSuper.gfp/neo-siRNApolβ954, extract the vector and expanding products by PCR, the 2% gel electrophoresis result shown: The PCR products are near 5.4kb and 70bp and accord with the design, results of DNA sequence proved it preciseness.3. RNAi expression vector was transfected into esophageal tumor cells(EC9706) by lipofectin reagent and positive clones were selected by G418.4. GFP expression in transfected Ec9706 cell: 48h after LipofectimineTM 2000 transfecting. The EC9706 cell shown significant green fluorescence implicated the transfectiong was successful.5. RT-PCR analysis revealed that lipofection of DNA polβsiRNA-expressing plasmid into EC9706 cells lead to remarkable suppression of pol beta mRNA expression compared to transfection of the control siRNA group or blank plasmid group. Similarly, RT-PCR showed that the expression of pol beta in EC9706 cells transfected with the STL1 siRNA-expressing plasmid was strongly inhibited. Transfection of a nueleotide control and common EC9706 cell, had no impact on pol beta expression at either the mRNA or the cell cycle level. Taken together, these plasmid vectors expressing siRNA against DNA polβexerted a powerful and specific knockdown effect with respect to endogenous DNA polβexpression in EC9706 cells.6. FCM analysis showed that the transfected groups cells of S stage were increased significantly compared with control groups cells(P＜0.01), therefore, the proliferation rate of the transfected cells increased. But the S stage between EC9706 cells transfected with the STL1 siRNA-expressing plasmid and cells transfected with the STL2 siRNA-expressing plasmid have no variance (P＞0.05), which indicate that the range of the low level of DNA polβexpression can't effect the rate of cell prolification.7. This study adopted trypan blue exclusion assay to evaluate the induction effect on EC9706 cell death rate. When DDP level is less than 60μmol/l, the death rates of four groups cells were similar with each other(P＞0.05). As the DDP level increased, the rates became significantly different(P＜0.01). The death rates of four groups added hydrogen peroxide had the same condition when level exceeded 120μmol/l(P＜0.01). On the contrary, four groups of EC9706 cells added bleomycin and methyl blue showed highly sensitivity from the drugs' initial concentration(P＜0.01).Conclusions1. Successfully construct DNA polβtargeted siRNA eukaryotic expression vectors named pSuper.gfp/neo-siRNApolβ448 and pSuper.gfp/neo-siRNApolβ954 and transfect into EC9706 cell.2. The low level of DNA polβexpression couldn't keep cell's nomal bionomics such as proliferation rate and drug sensitivity increase. It is possible to restrain tumor cell if RNA interference knock down the level of DNA polβexpression properly.
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