An Jing, Xu Qin-Zhi, Sui Jian-Li, Bai Bei, Zhou Ping-Kun
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, P.R. China.
Int J Mol Med. 2005 Sep;16(3):455-62.
DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of a sub-family of phosphoinositol 3-kinases, has been reported overexpressed in various human cancers, but its significance is unclear. In the present study, we generated the stable cell line HeLa(siRNAH1) of silenced DNA-PKcs by transfecting HeLa cells with the siRNA construct targeting the catalytic motif of DNA-PKcs. The expression of DNA-PKcs was markedly suppressed in HeLa(siRNAH1) cells, and eventuating in increased cellular sensitivity to ionizing radiation as well as cisplatin. Microarray assay was used to explore the transcriptional profiling of signal transduction-associated genes. The results demonstrated that 15 genes were up-regulated and eight were down-regulated in HeLa(siRNAH1) as compared with the HeLa(control) cells that transfected with non-specific siRNA construct. Seven of the up-regulated genes are associated with the interferon-signaling events, the others function in the BMP signal pathway, or as regulators of cell cycle and differentiation. The down-regulated genes include IL8, IL10RA, DAPK3, and those involved in nuclear factor of activated T cells (NFAT) signal pathway and endocrine responsiveness. Using the NFAT-driving secreted alkaline phosphatase reporter expression system, we further confirmed that NFAT transcriptional activity was markedly minimized after silencing DNA-PKcs. These results demonstrated that inactivation of DNA-PKcs altered the transcriptional level of certain signal transduction-associated genes related to proliferation and differentiation.
DNA依赖蛋白激酶催化亚基（DNA-PKcs）是磷酸肌醇3激酶亚家族的成员，已有报道称其在多种人类癌症中过表达，但其意义尚不清楚。在本研究中，我们通过用靶向DNA-PKcs催化基序的siRNA构建体转染HeLa细胞，生成了沉默DNA-PKcs的稳定细胞系HeLa(siRNAH1)。DNA-PKcs的表达在HeLa(siRNAH1)细胞中明显受到抑制，最终导致细胞对电离辐射和顺铂的敏感性增加。利用基因芯片分析来探索信号转导相关基因的转录谱。结果表明，与转染非特异性siRNA构建体的HeLa(对照)细胞相比，HeLa(siRNAH1)中有15个基因上调，8个基因下调。上调的基因中有7个与干扰素信号事件相关，其他基因在骨形态发生蛋白信号通路中起作用，或作为细胞周期和分化的调节因子。下调的基因包括IL8、IL10RA、DAPK3，以及那些参与活化T细胞核因子（NFAT）信号通路和内分泌反应的基因。使用NFAT驱动的分泌碱性磷酸酶报告基因表达系统，我们进一步证实沉默DNA-PKcs后NFAT转录活性明显降低。这些结果表明，DNA-PKcs的失活改变了某些与增殖和分化相关的信号转导相关基因的转录水平。
