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Objective: There is growing interest in using RNA interference (RNAi) as a therapeutic modality. However, the functional impact of low and high expression levels of the RNAi processing proteins, Dicer and Drosha, is not clear and the focus of the current study.
 Methods: Dicer and Drosha mRNA (real-time PCR) and protein (Western blot) levels were measured in 8 ovarian cancer cell lines (HeyA8, SKOV3ip1, EG, 222, OVCAR3, OVCAR420, IGROV, A2780-Par) and a non-transformed ovarian surface epithelial cell line (HIO-180). DNA mutational analysis was performed in cell lines with high and low Dicer levels. Protein expression levels of Galectin-3 were compared following siRNA vs. shRNA transfections in vitro. Results: Dicer mRNA and protein levels were decreased (mRNA: 2.03 to 12.5 fold and 1.20 to 5.33 fold in 50% and 63% of cells lines, respectively) and increased (mRNA: 2.01 to 3.41 fold and 1.16 to 3.38 fold in 50% and 37% of cancer cell lines, respectively). Drosha mRNA and protein levels were decreased (mRNA: 1.10 to 15.3 fold and 1.09 to 2.23 fold in 50% and 37% of cells lines, respectively) and increased (mRNA: 2.01 to 3.41 fold and 1.13 to 2.11 fold in 50% and 63% cell lines, respectively). Genomic DNA from cell lines with low (HeyA8 and SKOV3ip1) and high (OVCAR3 and A2780-PAR) Dicer and Drosha levels was analyzed for mutations. Two synonymous single nucleotide polymorphisms were discovered in both Dicer and Drosha sequencing in all four cell lines. Two different non-synonymous mutations were noted in the Drosha sequencing of OVCAR3 and A2780-PAR cell lines. A splice-site mutation was discovered and confirmed for Drosha in the SKOV3ip1 cell line. Gene silencing with siRNA targeting Galectin-3 was superior to shRNA in cell lines with low Dicer (78-95% vs. 4-8% Galectin-3 silencing compared to non-targeting sequences). No difference was observed in cell lines with high Dicer (62-73%).
 Conclusions: To our knowledge, this is the first evidence demonstrating the functional significance of RNAi machinery in ovarian carcinoma. These findings support the use of small-interfering RNA (siRNA) constructs that do not require endogenous Dicer and Drosha for therapeutic applications in ovarian cancer.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA