Silencing of DNA-PKcs alters the transcriptional profile of certain signal transduction genes related to proliferation and differentiation in HeLa cells.
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.
An
Jing
J
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, P.R. China.
Xu
Qin-Zhi
QZ
Sui
Jian-Li
JL
Bai
Bei
B
Zhou
Ping-Kun
PK
D002454:Cell Differentiation; D049109:Cell Proliferation; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation / Q000235:genetics
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases