siRNA-mediated silencing of Cockayne Cyndrome group B gene potentiates radiation-induced apoptosis and antiproliferative effect in HeLa cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Expression of DNA-PK in hepato- and cholangio-neoplasms and its significance / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To characterize DNA-PKcs and Ku70 expressions in hepato- and cholangio-neoplastic tissues and the association with the degree of malignancy and invasiveness of the tumors.</p><p><b>METHODS</b>The expression of DNA-PKcs and Ku70 was examined in 47 cases of hepato- or cholangio-neoplasm by immunohistochemistry.</p><p><b>RESULTS</b>Ku70 was expressed in all of the neoplastic tissues examined and with a little variation in levels. The highest expression was observed in adenocarcinomas and adenomas. There was no statistically significant association between Ku70 expression level and the degree of their malignancy extent or invasiveness. In contrast to Ku 70, a wide variation in expression levels of DNA-Pkcs was observed among different types of neoplastic tissues. The highest ratio of positive expressing cells was detected in hepatocellular carcinomas (92.1%), which was significantly higher than that in cholangioadeno carcinomas (65.3%) and biliary cystadenocarcinomas (51.9%). Low or no expression level was detected in papillary adenoma cases. DNA-PKcs expression of invasive adenomas and adeno-carcinomas (61.2%) was significantly higher than that of non-invasive adenomas and adeno-carcinomas (30.4%). There was no expression observed in the normal tissues adjacent to the tumors.</p><p><b>CONCLUSION</b>DNA-PKcs is expressed in hepato- and cholangio-neoplasms and its variable level of expression is associated with the types of the tumor and their degree of malignancy and invasiveness. DNA-PKcs could be recognized as a new biomarker for liver neoplasm.</p>