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Multidrug resistance protein 1 silencing in osteosarcoma and chondrosarcoma cell lines
Sarah S Freund1, Michael M Bendtsen1, Akmal Safwat2
1Department of Orthopedics, Aarhus University Hospital, Denmark.
Background:
The poor response of metastatic osteo- and chondrosarcomas to chemotherapy could be the result of multidrug resistance (MDR), which may be overcome through the use of small interfering RNA (siRNA). However, several methodologic questions remain unresolved.
Aims:
To test the toxicity of three commonly used siRNA transfection reagents and apply the least toxic reagent to investigate the siRNA-induced MDR1 mRNA knockdown.
Methods:
The toxicity of TransIT-TKO, Lipofectamine 2000, and X-tremeGENE siRNA transfection reagents was investigated on osteosarcoma (MG-63) and chondrosarcoma (SW1353) cell lines. The toxicity was measured at 4 and 24 hours using a MTT toxicity assay. The least toxic transfection reagent was applied to investigate the siRNA-induced MDR1 mRNA knockdown effect using qRT-PCR. Furthermore, five housekeeping genes were assessed in the BestKeeper software to obtain mRNA expression normalization.
Results:
Lipofectamine 2000 was the least toxic transfection reagent, reducing the cell viability only in chondrosarcoma 24 hours following exposure to the highest dose. In contrast, TransIT-TKO and X-tremeGENE transfection reagents displayed a significant reduction in cell viability in both chondrosarcoma after 4 hours and in osteosarcoma after 24 hours. Significant MDR1 mRNA silencing of over 80% was achieved in osteo- and chondrosarcoma using Lipofectamine at a final siRNA concentration of 25 nM. No significant dose response was observed in knockdown efficiency in either Lipofectamine or siRNA concentration.
Conclusion:
Lipofectamine 2000 was the least toxic transfection reagent in osteo- and chondrosarcoma. Successful siRNA-induced MDR1 mRNA silencing of over 80% was achieved.
Insights
Lipofectamine 2000 is the least toxic reagent for delivering small interfering RNA (siRNA) to osteosarcoma and chondrosarcoma cells. This method successfully silenced multidrug resistance (MDR1) mRNA by over 80%.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Metastatic osteosarcomas and chondrosarcomas exhibit poor response to chemotherapy, potentially due to multidrug resistance (MDR).
- Small interfering RNA (siRNA) offers a potential strategy to overcome MDR, but optimal delivery methods require investigation.
Purpose of the Study:
- To evaluate the toxicity of three common siRNA transfection reagents: TransIT-TKO, Lipofectamine 2000, and X-tremeGENE.
- To determine the efficacy of siRNA-mediated knockdown of MDR1 mRNA in osteosarcoma and chondrosarcoma cell lines using the least toxic reagent.
Main Methods:
- Toxicity assessment of transfection reagents on MG-63 (osteosarcoma) and SW1353 (chondrosarcoma) cell lines using MTT assay at 4 and 24 hours.
- Quantitative real-time PCR (qRT-PCR) was employed to measure MDR1 mRNA knockdown.
- BestKeeper software was utilized for normalization of mRNA expression using five housekeeping genes.
Main Results:
- Lipofectamine 2000 demonstrated the lowest toxicity, with minimal impact on chondrosarcoma cell viability at 24 hours.
- TransIT-TKO and X-tremeGENE showed significant cytotoxicity in both cell lines within 4-24 hours.
- Over 80% silencing of MDR1 mRNA was achieved in both cell types using Lipofectamine 2000 at 25 nM siRNA concentration.
Conclusions:
- Lipofectamine 2000 is identified as the optimal and least toxic reagent for siRNA delivery in osteosarcoma and chondrosarcoma.
- Effective silencing of MDR1 mRNA, a key factor in multidrug resistance, was successfully demonstrated using this approach.
