Study On In Vitro Cleavage Activity And Transient Effects On A549 Cells Of Hammerhead Ribozyme Targeting To Base Excision Repair Gene HOGG1

Objective To study the correlation between pathogenesis of lung cancer and oxidative DNA damage and repair genes, the expression of human 8-oxoguanine DNA glycosylase gene(hOGG1 gene), which could specially excise 8-oxoguanine induced by reactive oxygen species(ROS), was down-regulated in human lung cancer A549 cells by molecular biological and ribozyme technology.Methods According to design by computer, two specific restriction site BamH I and EcoR I were added to both ends of the hammerhead ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1(+). The positive recombinants were screened by tolerance of ampicillin, and plasmids were extracted from the positive recombinants and digested by BamH I and EcoR I, and then were analyzed by agarose gel electrophoresis and DNA sequencing. By the similar means, the conservative hOGGl gene sequence amplified by polymerase chain reaction (PCR) was inserted into in vitro transcription vector pBluescript SK(+) between restriction site Spe Ⅰ and Sac Ⅱ. Then, the ribozyme gene and hOGGl gene were transcribed in vitro respectively. The transcripts of ribozyme and its target RNA sequence with DIG-11-UTP were mixed, followed by a specific hammerhead ribozyme cleaving reaction to hOGG1 mRNA. In succession, the recombinants containing ribozyme DNA were transiently transfected into A549 cells. Thepositive recombinant was identified by reverse transcriptase-polymerase chain reaction (RT-PCR) targeting to Neo gene, which was a neomycin resistance gene for selection of stable cell lines and only appeared in vectors. The changes of hOGGl mRNA in A549 cells were detected by semi-quantitative RT-PCR. Then the cellular sensitivity to adriamycin was tested by comparison between untransfected cells and transfected cells by MTT assay. The adriamycin-induced DNA damage was investigated by comet assay or named single cell gel electrophoresis (SCGE) between untransfected and transfected cells.Results The recombinants containing the ribozyme gene and the recombinants containing hOGGl gene were successfully selected by restriction endonuclease digestion and agarose gel electrophoresis, and were proved by DNA automatic sequencing, respectively. The transcripts of ribozyme and hOGGl were acquired by in vitro transcription, and then cleavage in vitro assay was carried out under different conditions including mole ratio of 1:1, 1:2, 1:4, 1:8 and 1:10, reaction time of 30min, 60min and 90min, reaction temperature of 37°C, 42°C and 50°C, concentration of Mg2+ of lOmmol/L, 15mmol/L and 20 mmol/L. But, under these conditions, the effect of in vitro cleavage of hOGGl mRNA by ribozyme could not be detected. A549 cells containing the recombinants were identified by RT-PCR because Neo genes were amplified only in cells transfected successfully. The expression of hOGGl mRNA in A549 cells transfected ribozyme was 36% significantly less than in control cells(P<0.05). MTT assay showed that on the same concentration, the sensitivity of transfected cells to adriamycin were increased in comparison with untransfected cells. The comet assay showed that the extent of DNA damage induced by 1 |ig/ml adriamycin of transfected cells were worse than unrtransfected cells on the same transfection time (P<0.05), but there was no time-dependent reaction relationship.Conclusion The eukaryotic expression vector with genes of hammerhead ribozyme targeting to hOGGl mRNA and the in vitro transcription vector with itssubstrate hOGGl gene were constructed successfully. Though in vitro cleavage assay could not detect ribozyme's cleavage to its substrate mRNA, it effectively inhibited the expression of hOGGl gene in lung cancer A549 cells, and increased the cellular sensitivity to adriamycin, and it helps to study deeply the functions of base excision repair genes hOGGl.