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Inhibition of C13orf19 mRNA expression by siRNA Inhibition of C13orf19 mRNA expression by siRNA
in prostate cancer cellsin prostate cancer cells
*e-mail: [email protected]
IntroductionA 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.
Doreen Kunze*, Uta Schmidt, Susanne Fuessel, Axel Meye, Manfred P. WirthDoreen Kunze*, Uta Schmidt, Susanne Fuessel, Axel Meye, Manfred P. Wirth Department of Urology, Technical University of Dresden, GermanyDepartment of Urology, Technical University of Dresden, Germany
FIG.4 Cell cycle distribution.
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.1Treatment scheme of PCa cells in vitro.
ConclusionsThe 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.
ResultsThe 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.
Material & MethodsWe 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.
References1 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.
2 Kraemer, K., Fuessel, S., Schmidt, U., Kotzsch, M., Schwenzer, B., Wirth, M. P., and Meye, A. (2003). Clin Cancer Res 9, 3794-3800.
siRNA-D5
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siRNA-D5 125 nM siRNA-D5 250 nM ns-siRNA 125 nM ns-siRNA 250 nM untreated
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FIG.2 Cell viability.
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FIG.6 Clonogenic survival
FIG.1 Cell viability.
Seeding
Transfection(4 h)
WST-1 assay
Cell countingRNA isolation
Apoptosis detectionCell cycle
distributionClonogenic survival
24 – 72 h 20 – 68 h
