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[Construction of nucleotide excision repair gene XPB antisense RNA expression plasmid and its functions]
Li-Cheng Zhou1, Yi Jiang, Xiao-Ming Wu
1Public Health College, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, PR China.
Background & 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.
Conclusion:
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.
Insights
Researchers created an antisense plasmid to inhibit XPB gene expression in lung cancer cells. This approach reduced DNA repair capacity, offering potential for enhancing chemotherapy drug sensitivity.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Context:
- Nucleotide excision repair is crucial for cellular DNA repair.
- Enhanced DNA repair gene expression correlates with tumor drug resistance.
- Antisense strategies can down-regulate DNA repair, potentially increasing drug sensitivity.
Purpose:
- To construct a eukaryotic expression plasmid, pcDNA-XPB/AS, targeting the xeroderma pigmentosum B (XPB) gene.
- To investigate the function of the XPB gene in lung cancer A549 cells.
- To assess the role of XPB in modulating sensitivity to chemotherapeutic drugs.
Summary:
- An antisense plasmid targeting XPB mRNA was successfully constructed and transfected into A549 lung cancer cells.
- Transfected cells exhibited inhibited XPB mRNA expression and reduced DNA damage repair capacity following Adriamycin treatment.
- While Adriamycin sensitivity differed between transfected and untransfected cells, the difference lacked statistical significance.
Impact:
- The study demonstrates that the constructed antisense plasmid can effectively down-regulate XPB mRNA expression.
- Inhibition of XPB expression leads to suppressed cellular DNA damage repair ability.
- This provides a foundation for further research into XPB gene function and its therapeutic implications in lung cancer.