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Inhibition of C13orf19 mRNA expression by siRNA in prostate cancer cells

siRNA-D5. 3.9. 1.6. ns-siRNA. 3.6. 1.3. WST-1 assay Cell counting RNA isolation Apoptosis detection Cell cycle distribution Clonogenic survival. Transfection (4 h). Seeding. 24 – 72 h. 20 – 68 h. untreated. 3.5. 0.8. Inhibition of C13orf19 mRNA expression by siRNA

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Inhibition of C13orf19 mRNA expression by siRNA in prostate cancer cells

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  1. siRNA-D5 3.9 1.6 ns-siRNA 3.6 1.3 WST-1 assay Cell counting RNA isolation Apoptosis detection Cell cycle distribution Clonogenic survival Transfection (4 h) Seeding 24 – 72 h 20 – 68 h untreated 3.5 0.8 • Inhibition of C13orf19 mRNA expression by siRNA • in prostate cancer cells Doreen Kunze*, Uta Schmidt, Susanne Fuessel, Axel Meye, Manfred P. Wirth Department of Urology, Technical University of Dresden, Germany *e-mail: doreen.kunze@gmx.de Material & Methods We compared the downregulation of the C13orf19 mRNA expression by 5 different siRNA duplexes (D1: nt 2245‑2263; D2: nt 2215‑2233; D3: nt 1024‑1042; D4: nt 440‑458; D5: nt 1288‑1306) by DOTAP-mediated transfection of the PCa cell line PC-3. The PC-3 cells showed higher C13orf19 mRNA expression compared to other PCa cell lines (Du145, LNCap, 22RV1). We optimized the transfection conditions and selected the most efficient siRNA. The mRNA expression of C13orf19 and the PBGD reference gene were measured by quantitative PCR. The relative expression values were normalized to the non-silencing siRNA-control. Cellular viability was analyzed by WST‑1 viability assay and apoptosis by annexin V-propidium iodide staining. Also, cell cycle distribution and clonogenic survival were examined. In addition, the effects of C13orf19 down-regulation in combination with chemotherapy on overall cell survival were examined. Introduction A high proportion of bladder cancer (BCa) progress from superficial to Human C13orf19 (NM 017569) was previously identified to be down-regulated in prostate cancer (PCa). Until now its function is unknown. We therefore inhibited the C13orf19 mRNA expression by lipid-mediated siRNA transfection. FIG.1 Cell viability. FIG.2 Cell viability. FIG.5 Apoptosis in PC-3 cells after siRNA transfection. The percentages of early apoptotic cells (annexin V-positive, PI-negative; lower right) and cells died by apoptosis (annexin V-positive, PI-positive; upper right) are shown. FIG.4 Cell cycle distribution. FIG.6 Clonogenic survival Results The application of these siRNAs showed no obvious effects on doubling time and cellular morphology. After 24 h siRNA D5 inhibits the C13orf19 mRNA expression down to 16%. After 72 h there is still an inhibition to 31%. Cell cycle distribution, clonogenic survival, apoptosis and cell viability showed no alterations as compared to the controls. Conclusions The C13orf19 mRNA inhibition by D5 has no effects on cellular growth properties. We suppose that the inhibition leads to reduced apoptosis in PCa cells. Therefore, studies on the potential chemoprotective effects of siRNA D5 are underway. FIG.1 Treatment scheme of PCa cells in vitro. References 1 Sharma, G. G., Gupta, A., Wang, H., Scherthan, H., Dhar, S., Gandhi, V., Iliakis, G., Shay, J. W., Young, C. S., and Pandita, T. K. (2003). Oncogene 22, 131-146. 2Kraemer, K., Fuessel, S., Schmidt, U., Kotzsch, M., Schwenzer, B., Wirth, M. P., and Meye, A. (2003). Clin Cancer Res 9, 3794-3800.

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