Abstract
NF-κB activating protein (NKAP) is a highly conserved protein involved in transcriptional repression, immune cell development, maturation, acquisition of functional competency and maintenance of hematopoiesis. In the present study, the function of NKAP in the progress of Ewing sarcoma (ES) was investigated. It was identified that NKAP is highly expressed in ES cells when compared with human mesenchymal stem cells (MSCs). NKAP was knocked-down in human ES cell lines A673 and RD-ES using small interfering (si)RNA transfection. The effectiveness of transfection was then verified using reverse transcription-quantitative PCR and western blot analysis to determine mRNA and protein levels, respectively. The results of the proliferation assays indicated that the knockdown of NKAP inhibited the proliferation and clonogenic abilities of human ES cells. Transwell assays further indicated that cell invasion and migration were significantly inhibited by NKAP knockdown, which may be mediated by downregulation of matrix metalloproteinase (MMP)-9 activity. Gain-of-function analysis also demonstrated the positive role NKAP played in the proliferation, invasion and migration of ES cells. Cell apoptosis was evaluated by flow cytometry, which identified that apoptotic cells were significantly increased when NKAP was silenced. In addition, downregulation of NKAP increased the levels of Bax and cleaved caspase 3, but decreased Bcl2 levels, which suggested that the mitochondrial apoptosis pathway was activated. To explore the action mechanism of NKAP, the status of the AKT signaling pathway in NKAP-silenced A673 and RD-ES cells was investigated. Results indicated that NKAP knockdown led to decreased phosphorylation of AKT and expression of cyclin D1, a down-stream effector of the AKT signaling pathway, suggesting inactivation of the AKT signaling pathway. In conclusion, the present study revealed that NKAP promoted the proliferation, migration and invasion of ES cells, at least partly, through the AKT signaling pathway, providing new approaches for the therapeutic application of NKAP in ES.