Constructing the eukaryotic expression vector to study preliminarily the functions of hammerhead ribozyme targeting base excision repair gene HOGG1.
OBJECTIVE
Adriamycin is widely used as an effective anti-tumor drug clinically treating a number of human cancers, but the effect of adriamycin is limited by drug resistance. The various kinds of investigations indicated that the anti-tumor activity of adriamycin 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. Human 8-oxoguanine DNA glycosylase 1 (HOGG1) is a key enzyme on BER pathway. To study the influence and biological mechanism of the HOGG1 to adriamycin drug-sensitivity, the eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA would be constructed and identified, and then the change of drug-sensitivity in lung cancer 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 ampicillin 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 neomycin resistance gene for selection of stable cell lines and only existed in vectors. The changes of HOGG1 mRNA in A549 cells were detected by 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 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 HOGG1 mRNA in A549 transfected with ribozyme gene was 36% significantly less than in control cells (P < 0.05). MTT assay showed that the sensitivity of transfected cells to adriamycin were significantly increased in comparison with untransfected cells (P < 0.05). The comet assay showed that the extent of DNA damage induced by adriamycin was worse in transfected cells than unrtransfected cells (P < 0.05), but there was no time-dependent reaction correlation observed in cells.


CONCLUSIONS
The eukaryotic expression vector with gene of hammerhead ribozyme targeting HOGG1 mRNA was constructed successfully, and effectively inhibited the expression of HOGG1 gene in lung cancer A549 cells, and increased the cellular sensitivity to adriamycin, and will help to study deeply the functions of base excision repair genes-HOGG1.
Zhang
Zun-Zhen
ZZ
Department of Environmental Health, West China School of Public Health, Sichuan University, Chengdu 610041, China.
Zhang
Qin
Q
Wu
Mei
M
D001483:Base Sequence; D045744:Cell Line, Tumor; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005057:Eukaryotic Cells / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D004317:Doxorubicin; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human