siRNA-mediated silencing of Cockayne Cyndrome group B gene potentiates radiation-induced apoptosis and antiproliferative effect in HeLa cells.
BACKGROUND
Cockayne syndrome (CS) is a rare human genetic disorder characterized by increased UV sensitivity, developmental abnormalities and premature aging. Cells isolated from individuals with CS have a defect in transcription-coupled DNA repair. Despite the repair defect, there is no any increased risk of spontaneous or UV-induced cancer for CS individuals. The strategy of RNA interfering was used here to explore the potential radiosensitizing and anticancer activity of targeting CS group B (CSB) gene.


METHODS
The vectors encoding CSB-specific siRNAs were constructed by inserting duplex siRNA encoding oligonucleotides into the plasmid P(silencer TM 3.1). The cell lines expressing the CSB-siRNA were generated from HeLa cells transfected with the above vectors. Colony-forming ability was used to assay cell survival. Cell cycle was analyzed by FACScan flow cytometry. The apoptosis was measured by detecting the accumulation of sub-G(1) population as well as by fluorescence staining assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to semi-quantify mRNA expression. Protein level was detected by Western blotting analysis.


RESULTS
Two constructs encoding CSB-specific siRNA were generated, both of them resulted in remarkable suppression on CSB expression in HeLa cells, and led to an increased sensitivity to (gamma-ray and UV light. siRNA-mediated silencing of CSB decreased cell proliferation rate, increased spontaneous apoptosis as well as the occurrence of UV- or cisplatin-induced apoptosis by 2 to 3.5 fold. A significant S phase blockage and a remarkable reduction of G(1) population were induced in control HeLa cells at 18 hours after being exposed to 10 J/m(2) of UV light. The S phase blockage was also observed in UV-irradiated CSB-siRNA transfected HeLa cells, but the extent of increased S phase population was lower than that in the UV-irradiated control cells. No or a relative weak reduction on G(1) phase population was observed in UV-irradiated CSB-siRNA transfected HeLa cells. In addition, siRNA-mediated silencing of CSB promoted the elimination of G(2)/M phase cells after UV light radiation.


CONCLUSIONS
siRNA-mediated silencing of CSB causes cells to proliferate more slowly, sensitize cells to genotoxicants, and modify UV radiation-induced cell cycle changes. siRNA-mediated inactivation of CSB could be an attractive strategy for ameliorating cancer therapy, which can be fulfilled via the combination of gene therapy and sensitization of radiotherapy or chemotherapy.
Liu
Feng
F
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
Yu
Zi-jian
ZJ
Sui
Jian-li
JL
Bai
Bei
B
Zhou
Ping-kun
PK
D017209:Apoptosis / Q000528:radiation effects*; D002453:Cell Cycle / Q000528:radiation effects; D049109:Cell Proliferation / Q000528:radiation effects; D002945:Cisplatin / Q000494:pharmacology; D003057:Cockayne Syndrome / Q000235:genetics*; D020868:Gene Silencing; D015316:Genetic Therapy; D006367:HeLa Cells / Q000528:radiation effects*; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics*; D011836:Radiation Tolerance; D014466:Ultraviolet Rays
D034741:RNA, Small Interfering; D002945:Cisplatin