OBJECTIVE: @entity632 is widely used as an effective anti- @entity5 drug clinically treating a number of @entity1 @entity5 , but the effect of @entity632 is limited by drug resistance. The various kinds of investigations indicated that the anti- @entity5 activity of @entity632 resulted from drug-induced free radical formation. The free radicals could lead to oxidative DNA damage, and the lesion would be repaired by base excision repair (BER) pathway. @entity1 @entity25469 DNA glycosylase 1 ( @entity9552 ) is a key enzyme on BER pathway. To study the influence and biological mechanism of the @entity9552 to @entity632 drug-sensitivity, the eukaryotic expression vector with gene of hammerhead ribozyme targeting @entity9552 mRNA would be constructed and identified, and then the change of drug-sensitivity in @entity2 A549 cells would be investigated. METHODS: According to computer design, two specific restriction site BamH I and EcoR I were added to both ends of the ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1 (+). The positive recombinants were screened by @entity869 resistance, 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. The recombinants were transiently transfected into A549 cells. The positive recombinants were identified by reverse transcription-polymerase chain reaction (RT-PCR) targeting to NEO gene, which was a @entity5154 resistance gene for selection of stable cell lines and only existed in vectors. The changes of @entity9552 mRNA in A549 cells were detected by RT-PCR. Then the cellular sensitivity to @entity632 was tested by comparison between untransfected cells and transfected cells by @entity98 assay. The @entity632 -induced DNA damage was investigated by comet assay or single cell gel electrophoresis (SCGE) between untransfected and transfected cells. RESULTS: The recombinants containing the ribozyme gene were successfully selected by restriction endonuclease digestion and agarose gel electrophoresis, and were further proved by DNA automatic sequencing. A549 cells containing the recombinants were identified by RT-PCR, because NEO genes were amplified only in cells transfected successfully. The expression of @entity9552 mRNA in A549 transfected with ribozyme gene was 36% significantly less than in control cells (P < 0.05). @entity98 assay showed that the sensitivity of transfected cells to @entity632 were significantly increased in comparison with untransfected cells (P < 0.05). The comet assay showed that the extent of DNA damage induced by @entity632 was worse in transfected cells than unrtransfected cells (P < 0.05), but there was no time-dependent reaction correlation observed in cells. CONCLUSION: The eukaryotic expression vector with gene of hammerhead ribozyme targeting @entity9552 mRNA was constructed successfully, and effectively inhibited the expression of @entity9552 gene in @entity2 A549 cells, and increased the cellular sensitivity to @entity632 , and will help to study deeply the functions of base excision repair genes- @entity9552 .

Constructing the eukaryotic expression vector to study preliminarily the functions of hammerhead ribozyme targeting base excision repair gene XXXX .

@entity632 :: ('MESH:D004317', 'Chemical') :: ['Adriamycin', 'adriamycin'] @entity1 :: ('9606', 'Species') :: ['human', 'Human'] @entity25469 :: ('MESH:C024829', 'Chemical') :: ['8-oxoguanine'] @entity5 :: ('MESH:D009369', 'Disease') :: ['tumor', 'cancers'] @entity2 :: ('MESH:D008175', 'Disease') :: ['lung cancer'] @entity98 :: ('CHEBI:53233', 'Chemical') :: ['MTT'] @entity5154 :: ('MESH:D009355', 'Chemical') :: ['neomycin'] @entity869 :: ('MESH:D000667', 'Chemical') :: ['ampicillin'] @entity9552 :: ('4968', 'Gene') :: ['HOGG1']
@entity9552 :: (4968,Gene) :: ['HOGG1']
