Biochemical and Biophysical Research Communications
RNA interference-triggered reversal of ABCC2-dependent cisplatin resistance in human cancer cells
Section snippets
Materials and methods
Cell lines and cell culture. The human ovarian carcinoma cell line A2780 and its cisplatin-resistant subline A2780RCIS were cultivated in supplemented Leibovitz L-15 medium as described previously [4]. To ensure maintenance of the cisplatin-resistant phenotype, medium of A2780RCIS contained 10 μg/ml cisplatin (GRY-Pharma, Kirchzarten, Germany). Medium used for cultivation of stably transfected cell clones of A2780RCIS contained 400 μg/ml zeocin (Invitrogen, Carlsbad, CA, USA).
Cell proliferation
Modulation of ABCC2 mRNA expression by siRNAs
Two different siRNA constructs, ABCC2-A and ABCC2-B, were used to decrease the mRNA expression of ABCC2 in the cisplatin-resistant human ovarian cancer cell line A2780RCIS. ABCC2-A was designed according to general recommendations of siRNA selection, the target sequence of ABCC2-B was chosen to be homologous to a well-characterized hammerhead ribozyme cleavage site within the ABCC2-specific mRNA. Quantitative real-time RT-PCR experiments were performed to quantify ABCC2 mRNA expression values
Conclusions
In conclusion, RNAi technology is effective to modulate the ABCC2 expression in human ovarian carcinoma cells. The RNAi technology showed a comparable gene-silencing efficiency compared to hammerhead ribozymes targeting the identical site of the ABCC2 mRNA sequence in these cells. Thus, the data demonstrate the utility of the analyzed siRNAs and shRNAs as powerful laboratory tools and indicate that siRNA- and shRNA-mediated RNAi-based gene therapeutic approaches may be applicable in preventing
Acknowledgments
This work was supported by Grant LA 1039/2-3 of the “Deutsche Forschungsgemeinschaft” (DFG), by the “RNA-network” funded by the “Bundesministerium für Bildung und Forschung” (BMBF) and Berlin, and oligene GmbH.
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