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Stable suppression of MDR-1 gene using siRNA expression vector to reverse drug resistance in a human uterine sarcoma
Jun Hua1, David G Mutch, Thomas J Herzog
1Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Washington University School of Medicine, Box 8064, 4911 Barnes Hospital Plaza, St. Louis, MO 63110, USA.
Objective:
Chemotherapy is highly effective in treating a number of gynecologic malignancies; however, its effectiveness diminishes with repeated exposures due to the emergence of multi-drug resistance (MDR). The aim of this study was to establish a permanent MDR gene knockdown model via infection with the siRNA-hairpin expression vector. The impact of transfecting the RNAi upon MDR-1 mRNA and P-glycoprotein expression as well as resultant chemotherapy resistance was assessed.
Methods:
Multi-drug resistant cell line MES-SA/DX5 was transfected with the siRNA-hairpin expression vector (pSMDR-HYG) designed to target MDR-1 mRNA. A negative control was established utilizing a vector lacking the anti-sense component (pSCON-HYG). The LD(50) of doxorubicin for the stable transfectants was determined utilizing a cytotoxic MTT assay. The mRNA expression of MDR-1 gene among those cell lines was evaluated by semi-quantitative RT-PCR. The product of P-glycoprotein (P-gp) was examined by Western blotting hybridization and immunostaining.
Results:
Two stable transfected cell lines: MES-SA/DX5-M (with pSMDR-HYG) and MES-SA/DX5-C (with pSCON-HYG) were established. The cell line MES-SA/DX5-M was nearly 7 times more sensitive to doxorubicin than MES-SA/DX5-C and its parent cell line MES-SA/DX5 (P < 0.01). The mRNA expression of the MDR-1 gene in MES-SA/DX5-M was also statistically significantly lower than in the other 2 cell lines (P < 0.01) as assessed by semi-quantitative RT-PCR. A barely detectable signal for P-gp (170 kDa) was observed in MES-SA/DX5-M. The vast majority of MES-SA/DX5-M cells were immunohistochemically negative for P-gp.
Conclusions:
Stable, sequence-specific MDR-1 gene silencing can be demonstrated by inducing the endogenous expression of hairpin siRNA. Hairpin-siRNA-based MDR-1 gene silencing correlated with decreased levels of MDR-1 mRNA and P-gp, thereby restoring permanent native chemosensitivity. This methodologic strategy may have significant clinical impact in reversing chemo-resistance, especially the multi-drug-resistant phenotype, in the treatment of gynecologic malignancies.
Insights
This study established a gene knockdown model to overcome multi-drug resistance (MDR) in gynecologic cancers. Silencing the MDR-1 gene restored chemotherapy sensitivity by reducing MDR-1 mRNA and P-glycoprotein levels.
Area of Science:
- Gynecologic Oncology
- Molecular Biology
- Cancer Therapeutics
Background:
- Chemotherapy resistance is a major challenge in treating gynecologic malignancies.
- Multi-drug resistance (MDR) diminishes treatment efficacy with repeated exposures.
Purpose of the Study:
- To create a permanent gene knockdown model for MDR using siRNA-hairpin expression vectors.
- To assess the impact of MDR-1 gene silencing on chemotherapy resistance.
Main Methods:
- Transfected MES-SA/DX5 cells with an MDR-1 targeting siRNA vector.
- Utilized a control vector lacking anti-sense component.
- Assessed doxorubicin sensitivity (LD50) via MTT assay.
- Quantified MDR-1 mRNA by RT-PCR and P-glycoprotein by Western blotting and immunostaining.
Main Results:
- Established stable cell lines MES-SA/DX5-M (siRNA) and MES-SA/DX5-C (control).
- MES-SA/DX5-M showed ~7-fold increased sensitivity to doxorubicin (P < 0.01).
- Significantly lower MDR-1 mRNA and P-glycoprotein levels were observed in MES-SA/DX5-M cells.
Conclusions:
- Inducible hairpin siRNA enables stable, sequence-specific MDR-1 gene silencing.
- MDR-1 gene silencing reduces MDR-1 mRNA and P-glycoprotein, restoring chemosensitivity.
- This strategy holds potential for reversing chemo-resistance in gynecologic cancers.
