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Published on: August 18, 2010
[Reversing multidrug resistance in breast cancer cell line MCF-7/ADR by small interfering RNA]
Chen-Bin Li1, Feng Zhang, Yu-Rong Shi
1Oncology Center Laboratory, Cancer Hospital, Tianjin Medical University, Tianjin 300060, P.R. China. chenbin_li@sina.com
Background & Objective:
Multidrug resistance of tumor cells often leads to failure of chemotherapy. The over-expression of P-glycoprotein (P-gp), encoded by multidrug resistance 1 (mdr1) gene, plays an important role in multidrug resistance of breast cancer. This study was to explore the feasibility of silencing mdr1 gene by small interfering RNA (siRNA) in drug resistant breast cancer cell line MCF-7/ADR.
Methods:
The siRNA oligonucleotides strand designed previously was inserted into pSilencer3.1-H1 Hygro vector, the plasmid was transformed into E.coli. After amplification, the plasmid was purified, and sequenced to determine whether the ligation between siRNA insert and the vector was correct, then transfected into MCF-7/ADR cells, and relevant sensitive MCF-7 cells. MCF-7/ADR cells were screened by hygromycin, surviving cells were cultured. The positive rate of P-gp was detected by flow cytometry, and positive rate of mdr1 gene was detected by real-time relatively quantitative polymerase chain reaction (PCR). Adriamycin (ADM) resistant experiment was performed on MCF-7/ADR cells with siRNA.
Results:
Positive rate of P-gp in MCF-7/ADR cells was decreased from 99.8% (before siRNA transfection) to 12.3% (after siRNA transfection). Real- time PCR revealed that the threshold cycle value of MCF-7/ADR cells increased from 25.22 to 30.64 after transfected with siRNA. The IC(50) of ADM for MCF-7/ADR cells transfected with siRNA was 0.51 micromol/L, while that for MCF-7/ADR cells without transfection was 17.88 micromol/L.
Conclusion:
siRNA can silence mdr1 gene in MCF-7/ADR cells, may become a new, effective medical technique.
Insights
Small interfering RNA (siRNA) effectively silenced the multidrug resistance 1 (mdr1) gene in drug-resistant breast cancer cells. This approach shows promise for overcoming chemotherapy resistance by targeting P-glycoprotein expression.
Area of Science:
- Molecular Biology
- Cancer Research
- RNA Interference
Background:
- Multidrug resistance (MDR) in tumor cells is a major cause of chemotherapy failure.
- Overexpression of P-glycoprotein (P-gp), encoded by the multidrug resistance 1 (mdr1) gene, is crucial for MDR in breast cancer.
Purpose of the Study:
- To investigate the feasibility of silencing the mdr1 gene using small interfering RNA (siRNA).
- To evaluate the efficacy of siRNA-mediated mdr1 gene silencing in the drug-resistant breast cancer cell line MCF-7/ADR.
Main Methods:
- Constructed and validated siRNA oligonucleotides targeting the mdr1 gene.
- Transfected MCF-7/ADR cells with the constructed siRNA plasmid.
- Assessed P-glycoprotein expression via flow cytometry and mdr1 gene levels via real-time PCR.
- Determined adriamycin (ADM) resistance by measuring IC50 values post-transfection.
Main Results:
- siRNA transfection significantly reduced P-gp positive rate from 99.8% to 12.3%.
- Real-time PCR indicated successful mdr1 gene silencing, with threshold cycle values increasing from 25.22 to 30.64.
- The IC50 of ADM decreased dramatically from 17.88 micromol/L to 0.51 micromol/L in siRNA-transfected cells.
Conclusions:
- siRNA is effective in silencing the mdr1 gene in MCF-7/ADR cells.
- This gene silencing strategy holds potential as a novel therapeutic approach for overcoming multidrug resistance in breast cancer.
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