The Initial Study On Expression Of DNA Polβ MRNA In BGC-823 Cell Silenced By SiRNA

Background and objectivesDNA polymerase beta (DNA polβ) is a house-keeping gene and one of the most important polymerase in base excision repair (BER). The sequence of DNA polβis highly conservative among species. DNA polβgene exists extensively in mammalian cells. It plays a very important role in repairing damaged DNA. As an important repair enzyme, it can supply a gap with 1-4 nucleotide in BER. However it is the most inaccurate DNA polymerase in mammalian, there are many mismatch in the processing of nucleotide incorporation. The expression level of DNA polβis relatively lower and stable in the whole cell cycle without being regulated by proliferation of cell cycle in mammalian cells. So many endocelluar/extracellular stimulating factors, such as DNA damage agent, can induce its expression. The increasing absolutely or relatively expression of DNA polβwill interfere or displace the normal function of other DNA polymerase. Recently, it has been reported that tumorigenesis is closely related with DNA damage. And as one of the most important DNA repair enzyme, DNA polβ's mutation is relevant with the tumorigenesis. And the high expression of mutated polβis detected in many human tumor tissues, such as colorectal cancer, prostatic cancer, liver cancer, lung cancer, bladder cancer, esophageal cancer, gastric cancer and nasopharyngeal cancer with varieties of mutation. High expression of polβis also found in the tumor cells which have been drug resistant.Our study group have found that both the mRNA level and protein expression of DNA polβare significantly higher in gastric tumor tissues and the gastric tumor cell lines than in normal gastric tissues. Overexpression of DNA polβcan result in more low fidelity of DNA synthesis, which could cause the genome instable and higher spontaneous mutation rate. At last the incident of tumorigenesis and anticancer drug resistance occur. So it is important and meaningful for the prevention and therapy of cancer to search for an effective way to interfere the pol 3 overexpression and make it express properly.RNA interference (RNAi) is a posttranscriptional gene silencing phenomenon (PTGS) induced by double-stranded RNAs (dsRNAs). The gene-silencing technique can effectively knock down the expression of posttranscriptional gene, even can replace gene knock-out., which is an effective pathway to research the gene regulation at present. In our study we plan to construct siRNA expression vector targeting directly pol 3 gene and transfect it into cells of gastric cancer cell lines (BGC-823), and then detect the silence effect of the expression of pol 3 , and meanwhile , observe. the influence on the cells biological behaviour.Methods:According to the principle of designing siRNA sequence, we used the Takara, Ambion and promega siRNA sequence designing and analysis software to scan human pol 3 cDNA exon (M13140). siRNA sequences targeting pol 3 with 19 bases (WLSI1, WLSI2) were selected and definited by BLAST homology analysis and a contrast sequence (Con) was randomly assorted. Afterwards, two pairs of DNA oligonucleotides targeting directly pol 3 and a pair of irrelevant DNA oligonucleotides, which coded short hairpin RNA sequence were synthesized respectively, then annealed into double strands DNA. After this, the annealed products were purifed and cloned into siRNA expression vector(pRNAT-U6.1) that has been cut by BamHI and XhoI restriction enzyme; The positive ones (pRNAT-U6.1-sipol 3 1, pRNAT-U6.1-sipolβ2, pRNAT-U6.1-Con) were identified by PCR, and then sequenced. Eventually, the positive recombinants (pRNAT-U6.1-sipolβ1, pRNAT-U6.1-sipolβ2, pRNAT-U6.1-Con) were transfected into human gastric tumor cell line (BGC-823),while empty vector group and untransfected group cellswere taken as control groups. The expression level of polβmRNA in every group cells were detected by real-time PCR; cell cycle and cell proliferation rate were detected by Flow cytometry. At last, the BGC-823 cells in every group were injected into athymic mouse respectively, then the speed of cell proliferation was detected in vivo, while the expression level of polβmRNA in every group tumors were detected by real-time PCR.Results:1. 27 target siRNA oligonucleotides were obtained by preliminary screening. After retrieveing and optimizing homologization sequence, two target siRNA sequences with 19 bases were ensured. WLSI1: GAACGTGAGCCAAGCTATC (274-292); WLSI2: GATTCGGCAGGATGATACG (661-679) .2. After annealing the hairpin DNA of siRNA, there were three obvious stripes whose molecular weight were similar to the design, about 70bp in agar gel electrophoresis.3. The pRNAT-U6.1-sipolβ1, pRNAT-U6.1-sipolβ2 and pRNAT-U6.1-Con were constructed. And after sequenced, the target sequences were coincident completely with the design.4. A great quantity of GFP can be observed in transfected BGC-823 cell by fluorescence microscope 48 hours later. And cell clones resistanting to G418 were selected.5. The mRNA level of polβin experimental group transfected by pRNAT-U6.1 -sipolβ1 and pRNAT-U6.1-sipolβ2 was obviously reduced, and the silence effect of pRNAT-U6.1-sipolβ2 is better than pRNAT-U6.1 -sipolβ1, nearly completely inhibited. The mRNA level of polβwas still high in irrelevant siRNA control group, empty vector control group and untransfected group.6. The different cell ratio in period G₀-G₁,G₂-M,S and cell proliferation rate were no significance(P>0.05) in irrelevant siRNA control group, empty vector control group and untransfected group. Contrast with irrelevant siRNA control group, empty vector control group and untransfected group, S period proportion and cell proliferation rate cut down in BGC-823 cells transfected with pRNAT-U 6.1-sipolβ1; On the contrary, S period proportion and cell proliferation rate step up in BGC-823 cells transtected with pRNAT-U6.1-sipolβ2 .7. Every group cells were respectively injected into the subcutaneous tissue of athymic mouse. The growth speed, size and weight of the tumor in the experimental group 1 is lower than that in other control groups and the experimental group 2 (P < 0. 05), whereas the growth speed, size and weight of the tumor in the experimental group 2 is higher than that in other group. The results of mRNA level of polβin every group tumors were coincident with the results in every group cells.Conclusions:1. The siRNA vector pRNAT-U6.1-sipolβ1 and pRNAT-U6.1-sipolβ2 targeting DNA polβwas constructed successfully.2. The mRNA level of polβwas obviously decreasing after transfecting with target siRNA vectors in BGC-823 cell line.3. Neither high nor extremely low expression of polβis benefit to maintain the cell's physiological functions; the expression of polβwas silenced to the proper level by siRNA, which plays a important role in inhibiting tumorigenesis.