[Construction of nucleotide excision repair gene XPB antisense RNA expression plasmid and its functions].
OBJECTIVE
Nucleotide excision repair is an important pathway for cellular DNA damage repair. The drug resistance of tumor cell is often companied with the enhanced expression of DNA repair genes. Down-regulation of DNA repair capacity by antisense strategy can increase the drug sensitivity of tumor cells. The aim of this study was to construct the eukaryotic expression plasmid pcDNA-XPB/AS (XPB: xeroderma pigmentosum B) and to investigate the function of XPB gene and its roles in chemotherapeutic drug sensitivity in lung cancer A549 cell.


METHODS
The XPB cDNA (69-520 bp) fragment amplified by reverse transcription polymerase chain reaction (RT-PCR) was inserted into pcDNA3.1/His plasmid with an inverted orientation. The recombinant plasmid was transiently transfected into A549 cells. The Adriamycin-induced DNA damage was compared between the transfected and the untransfected cells by single cell gel electrophoresis assay (SCGE). The cellular sensitivity to Adriamycin of the transfected and the untransfected cells was determined by MTT assay.


RESULTS
The successful construction of antisense plasmid was proved by restriction map and sequence analysis. RT-PCR results showed that the XPB mRNA expression was inhibited in transfected A549 cells. SCGE showed that the cellular damage repair ability induced by 4.0 microg/ml Adriamycin was suppressed in transfected cells. MTT assay showed the sensitivity of the transfected cells to Adriamycin was different from the untransfected cells but without statistical meaning.


CONCLUSIONS
The antisense plasmid constructed by the authors can down-regulate the expression of XPB mRNA in the transfected cells and inhibit the cellular DNA damage repair ability, providing a basis to further study the gene function of XPB.
Zhou
Li-Cheng
LC
Public Health College, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, PR China.
Jiang
Yi
Y
Wu
Xiao-Ming
XM
Hao
Qiao-Ling
QL
Ren
Shu
S
Zhou
Yi-Kai
YK
D001483:Base Sequence; D045744:Cell Line, Tumor; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D004317:Doxorubicin / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms / Q000628:therapy; D008969:Molecular Sequence Data; D010957:Plasmids; D016372:RNA, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004268:DNA-Binding Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; C072022:XPBC-ERCC-3 protein; D004317:Doxorubicin; D004265:DNA Helicases