Background. Increased expression of PCNA gene was detected in chronic myeloid leukemia (CML) patients in our
laboratory. The gene may participate in the disease development. The aim of the study was to develop appropriate
conditions for PCNA-siRNA transfection into K562 and MOLM-7 cell lines (which both have the up-regulated PCNA
gene) and to silence the increased expression.
Methods and Results. Key parameters of successful siRNA delivery into the cells are type and quantity of
transfection reagent, cells and siRNA concentration or cultivation time before an expression analysis. Transfection
reagents ExGene 500 (Fermentas), Metafectene (Biontex), Oligofectamine (Qiagen) and siPORT Amine (Ambion)
were tested. Transfection efficiency was monitored by fluorescence microscopy of fluorescein labeled siRNA. Gene
silencing was determined at mRNA level by real-time PCR and at protein level by western blots. As the most suitable
reagent was chosen Oligofectamine, which achieved 70% decrease of PCNA mRNA level. Further, 50 nM siRNA
concentration, 1x106 cells/ml and amount of Oligofectamine 4 µl per 1 ml of transfected cells were selected. The best
cultivation time after siRNA delivery was 48 h.
Conclusions. Based on the results of this study, transfection method for siRNA delivery into the K562 and MOLM-7
cell lines was proposed. The procedure can be transferred also on further selected genes potentially involved in CML
and afterwards it will be possible to monitor the impact of siRNA-inhibition on expression profile. In the future siRNAs
against some over-expressed genes would be used for gene therapy of CML.
siRNA, chronic myeloid leukemia, PCNA, gene expression.