title: Silencing of the DEK gene induces apoptosis and senescence in CaSki cervical carcinoma cells via the up-regulation of NF-κB p65.
abstract: The human DEK proto-oncogene has been found to play an important role in autoimmune disease, viral infection and human carcinogenesis. Although it is transcriptionally up-regulated in cervical cancer, its intracellular function and regulation is still unexplored. In the present study, DEK and IκBα [inhibitor of NF-κB (nuclear factor κB) α] shRNAs (short hairpin RNAs) were constructed and transfected into CaSki cells using Lipofectamine™. The stable cell line CaSki-DEK was obtained after G418 selection. CaSki-IκB cells were observed at 48 h after psiRNA-IκB transfection. The inhibitory efficiency of shRNAs were detected by RT (reverse transcription)-PCR and Western blot analysis. The proliferation activity of cells were measured using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, cell apoptosis was measured using an Annexin V/PI (propidium iodide) kit, the cell cycle was analysed by flow cytometry and cell senescence was detected using senescence β-galactosidase staining. The intracellular expression of NF-κB p65 protein was studied by cytochemistry. The expression levels of NF-κB p65, p50, c-Rel, IκBα and phospho-IκBα protein were analysed by immunoblotting in whole-cell lysates, cytosolic fractions and nuclear extracts. The protein expression and activity of p38 and JNK (c-Jun N-terminal kinase) were also assayed. In addition, the NF-κB p65 DNA-binding activity was measured by ELISA. Following the silencing of DEK and IκBα, cell proliferation was inhibited, apoptosis was increased, the cell cycle was blocked in the G0/G1-phase with a corresponding decrease in the G2/M-phase, and cell senescence was induced. All of these effects may be related to the up-regulation of NF-κB p65 expression and its nuclear translocation.
authors.affiliation: Department of Gynecology and Obstetrics, the Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, People's Republic of China. lhb981024@hotmail.com
authors.forename: Kuiran
authors.initials: K
authors.lastname: Liu
authors.forename: Tianda
authors.initials: T
authors.lastname: Feng
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: Ming
authors.initials: M
authors.lastname: Zhong
authors.forename: Shulan
authors.initials: S
authors.lastname: Zhang
mesh_terms: D017209:Apoptosis; D002277:Carcinoma; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016922:Cellular Senescence; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D021381:Protein Transport; D000090063:Proto-Oncogene Mas; D034622:RNA Interference; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D002583:Uterine Cervical Neoplasms
chemical_list: D002868:Chromosomal Proteins, Non-Histone; C084738:DEK protein, human; D020672:I-kappa B Proteins; C000715470:MAS1 protein, human; C000606204:NFKBIA protein, human; D015513:Oncogene Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D000090063:Proto-Oncogene Mas; C498143:RELA protein, human; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha