02 BOOK OF ABSTRACTS .indd

Electrically-assisted delivery of siRNA targeting p53:
an in vitro study on human tumor cell lines with
different p53 status
Suzana Mesojednik, Gregor Ser{a, Alenka Gro{el, Simona Kranjc, Gregor
Tev`, Maja ^ema`ar
Institute of Oncology, Department of Experimental Oncology, Zalo{ka 2, SI-1000 Ljubljana,
Slovenia
Background: Silencing of oncogenes or other genes contributing to tumor malignancy
or progressson by 20–25- nucleotide dsRNA, better known as siRNA, offers a promising
approach to treat tumors. High transfection efficiencies are of crucial importance for
gene silencing experiments. Electroporation is currently receiving much attention as a
way to increase siRNA delivery.
Aim: The aim of our study was to determine the transfection efficiency and induction of
siRNA-mediated silencing of p53 in two colorectal (HT-29 and LoVo) and two prostatic
(PC-3 and DU145) carcinoma cell lines with different p53 status by varying voltage of
electric pulses in in vitro electroporation protocol for siRNA transfection.
Materials and methods: Two commercial plasmids (Invivogen) expressing siRNA
were used, therapeutic (psiRNA-hp53 directed against the p53 mRNA) and control
(psiRNA-GL3 directed against the luciferase mRNA). Both of them were, together with
siRNA, expressing GFP, which allowed us to assess transfection efficiency. Plasmids
were delivered to cells by electroporation. Mixture of cells (1 × 10 6 ) and plasmid
(10 µg) as well as cells alone were exposed to eight square wave electric pulses
(600 - 1000 V/cm, 5 ms, 1 Hz). Electric pulses were delivered through two parallel
stainless steel plate electrodes with 2 mm distance between them and were generated
by an electroporator GT-1. To qualitatively evaluate the siRNA effects against p53
in different tumor cell lines with different p53 status the western blot analysis was
performed. Cell survival after treatment was determined by clonogenic assay. GFP was
detected by invert fluorescence microscopy.
96p24
Results: Transfection efficiency of electrically-assisted delivery of plasmids expressing
siRNA varied depending on the cell line used and voltage of electric pulses applied.
The optimal voltage of applied electric pulses were 700 V/cm for PC-3 and DU145
cell lines and 900 V/cm for HT29 and LoVo cell lines which resulted in 10 - 40 %
(PC-3), 1 - 9 % (DU145), 1 - 4 % (HT29 ) and 1 – 7 % (LoVo) transfection efficiency.
The GFP expression was detected already at 6 hours after electroporation. It remained
present during 1 week with a peak in fluorescence emission at day 1 or 2. Western blot
analysis showed that siRNA directed against p53 mRNA resulted in up to 40% lower
p53 protein level in Du145 (p53mt) cells. PC-3 (p53null) cells were negative control.
In accordance with our expectation, no silencing of p53 mRNA by control siRNA was
detected. Treatment of cells with electrically-assisted delivery of siRNA targeting p53
did not result in reduced cell survival compared to the treatment with control siRNA.
Conclusions: Our results showed that electroporation is feasible and effective method
for delivering of siRNA to tumor cells. Electrically-assisted delivery of siRNA directed
against p53 resulted in lower p53 protein content in cell lines used in this study but it
did not reduce cell survival compared to the treatment with control siRNA.