Initial Studies On Regulating The Expression Of DNA Polymeraseβ In Esophageal Carcinoma Cell Line(EC9706)

BackgroundTumors are involved with the abnormality of mechanism on cell differentiation,cell proliferation and cell death.DNA damage,the abnormality of genetic structure and the change in expression and function of oncogene and/or anti-oncogene caused by them are the premises of cell malignant transformation. However,not all damages can result in gene mutation,DNA restoration system of cells can clean and repair DNA damage.Among them,base excision repair is an important pathway to clean DNA damage of cells,then DNA polymeraseβjust is a key enzyme in base excision repair.DNA polymeraseβis an important repair gene for DNA damage in eukaryote cells.As a housekeeping gene,the DNA sequence of DNA polβgene is highly conservative.The main function of DNA polβis to excise and repair DNA base mutation.The expression level is relatively stable in the whole cell cycle without being regulated by proliferation of cell cycle.DNA polymeraseβexpression level is very low in the normal tissues and cells.In recent years,it has been reported that DNA polymeraseβgene mutation has been found in many tumor tissues including colorectal cancer,gastric cancer, breast cancer,liver cancer,lung carcinoma,bladder carcinoma,prostatic carcinoma and esophageal carcinoma.In addition,it has been found that most of tumors exhibited overexpression of DNA polymeraseβin gene and protein,and then the expression of DNA polymeraseβis higher several times in prostatic carcinomas breast cancer,ovarian cancer,colon carcinoma and esophageal carcinoma than it in cancer-adjacent normal tissues.The overexpressed DNA polymeraseβincreases mutation rate of nucleotide through DNA repair system,translesion synthesis and DNA replication,then increases spontaneous mutation of cells and promote the onset and progression of tumors.The findings have confirmed that mutation and overexpression of DNA polymeraseβare closely related with the onset and progression of tumors.However,what effects low expression of DNA polymeraseβhas on the biological characters of tumor cells and the relations between low expression of DNA polymeraseβand the development of tumors have been seldom reported.However,RNAi is exactly a new method to explore this problem.RNA interference(RNAi) is a conserved biological response to double-stranded RNA,which involves generation of active small interfering RNA(siRNA) in vivo through the action of an RNase III endonuclease complex,Dicer.The resulting 21-23nt siRNA is incorporated into a multiprotein RNA-induced silencing complex (RISC),where the siRNA duplex is unwound,leaving the antisense strand to guide RISC to its homologous mRNA targets for endonucleolytic cleavage.RNAi has been successfully utilized to down-regulate the endogenous gene expression.ObjectiveIn this study,siRNA expression vectors targetting DNA polymeraseβare constructed.siRNA expression vectors and wild-type polβare transfected into esophageal tumor cells respectively to regulate the expression of polβ.Biological characters of esophageal carcinoma cell under various expression levels of polβare studied.The objective is to reveal expression suitably and functional status of DNA polymeraseβin cells,and provide a new method for prevention and therapy of esophageal tumor.Method Upon the nucleotide sequence quoted from GenBank of DNA polymeraseβgene and according to the strategy for designing small interference RNA(siRNA),the domain of DNA polymeraseβgene was selected for designing and synthesizing complementary single-strand DNA oligos.After annealing,the single-stranded oligos will generate a double stranded(ds) oligo.We cloned the ds oligo into pSINsi-hU6 vector and constructed two siRNA expression vectors targetting DNA polymeraseβ. siRNA expression vectors and wild-type polβwere transfected into esophageal tumor cells(EC9706) respectively by lipofectin reagent.Five groups were divided,there were experimental group 1 and experimental group 2 transfected with siRNA expression vectors,polβoverexpression group transfected with wild-type polβ,empty vector group and control group.Positive clones were selected by G418.Flow cytometry was used to observe the proliferation activity of esophageal cancer cells in vitro under various expression levels of DNA polymeraseβ;after inoculating nude mice,the DNA polymeraseβexpression of espphageal tumor tissues in nude mice was detected by Fluorescent quantitation PCR,Nude mice xenograft model was used to observe the proliferation activity of esophageal cancer cells in vivo under various expression levels of DNA polymeraseβ.Results1. Two DNA polβtargeted siRNA expression vectors including pSINsi-hU6 -siRNApolβ288 and pSINsi-hU6-siRNApolβ814 were successfully constructed.2. DNA polβtargeted siRNA expression vectors and wild-type polβwere transfected into EC9706 cell by lipofectin reagent respectively and positive clones were selected by G418.3. Flow cytometry analysis showed that compared with untransfected EC9706 and EC9706 cells transfected with empty vector,S-period frequency of cell cycle increased significantly in the cells transfected with wild-type polβand siRNA expression vectors respectively.4. Nude mice xenograft model showed that compared with empty vector group and control group,the weight of tumors increased significantly in experimental group 1 and experimental group 2 transfected with siRNA expression vectors; compared with empty vector group and control group,the weight of tumors increased significantly in polβoverexpression group transfected with wild-type polβ.There is no significantly difference within experimental group 1,experimental group 2 and polβoverexpression group.5. Fluorescent quantitation PCR detected the mRNA expression level of polβin tumors.Compared with empty vector group and control group,the mRNA expression level of polβreduced significantly in experimental group 1 and experimental group 2 transfected with siRNA expression vectors;the mRNA expression level of polβincreased significantly in polβoverexpression group transfected with wild-type polβ.siRNA expression vectors had obvious and similar silencing effects on DNA polβexpression.ConclusionsThe results of studies showed that not only low extremely expression of DNA polβis involved in the occurrence of esophageal carcinoma,but also the overexpression of DNA polβmay be another important mechanism to mediate the tumorgenegsis of esophagus.So RNAi can be used to suppress the expression of DNA polβto suitable level where DNA polβcan maintain the activity of repair enzyme but not participate in low fidelity replication mediated by overexpression.RNAi may be an useful tool for prevention and therapy of esophageal tumor.