Abstract
DNA ligase IV is one of the key proteins that are associated with DNA double strand break (DNA DSB) repair through the non-homologous end joining (NHEJ) pathway. We designed 21 bp small interfered double stranded RNA (siRNA) on the basis of its specific DNA sequence, and transfected Hela cells with the DNA ligase IV siRNA. The RNA and protein expression levels of DNA ligase IV, cell growth, DNA DSB ratio were examined in the siRNA-transfected and mock-transfected cells. The morphological analysis with a fluorescence microscope was carried out in the cells to examine the mode of cell death. The colony formation of the cells irradiated with X-ray was also investigated. Compared to the cells with mock-transfection, the RNA and protein expressions were remarkably reduced in the siRNA-transfected cells. The expression of XRCC4 protein, which forms the complex with DNA ligase IV in the cells, was also reduced. And a decrease on cell growth and a increase on DNA DSB were observed in the siRNA-transfected cells. The cells exhibited the morphological changes due to the typical necrotic cell death. These results indicate that necrotic cell death could be promoted in human cancer cells by reducing the expression of DNA ligase IV using RNA interference technology. But only slight decrease on cell survival was shown in X-irradiated cells transfected with the siRNA. Further investigation must be done to apply the technology to radiosensitization of cancer cells.
