科研之友
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摘要
Centrosome amplification can drive chromosomal instability (CIN) which is a major source of tumor initiation. The present study aimed to investigate the impact of nuclear factor kappa B (NF-kappa B) on centrosome amplification of Hep-2 cells. Immunofluorescence was performed to display centrosomes. BAY11-7082 was used as an inhibitor of NF-kappa B to assess the inhibition of centrosome amplification, and cyclin-dependent kinase 2 (CDK2), ensuring cell cycle cycle coordination with centrosome cycle was detected by Western blotting. Furthermore, a 1556-bp fragment of the CDK2 promoter was analyzed using the TRANSFAC-TESS software. Luciferase assay, including a series of truncated CDK2 promoters and site mutations, was carried out to determine NF-kappa B binding sites in the CDK2 promoter. Electrophoresis mobility shift and chromatin immunoprecipitation assays were applied to confirm whether NF-kappa B indeed binds to the 5'-promoter region of the CDK2 gene. To reveal the clinical significance of CDK2 expression in laryngeal squamous cell cancer, mRNA and protein levels were assessed by RT-PCR and Western blotting, respectively. We found that the transcription factor NF-kappa B plays a role in centrosome amplification in Hep-2 cells. Centrosome amplification is reduced by inhibition of the NF-kappa B pathway. Moreover, expression of the p65 subunit of NF-kappa B is sufficient to promote centrosome amplification and increase in CDK2 protein levels. We further identified a functional NF-kappa B binding site located in the CDK2 promoter. Single mutation of the NF-kappa B site III (construct mutIII) however resulted in 76 +/- 5% (p<0.01) luciferase activity reduction. Electromobility shift assays and chromatin immunoprecipitaton results suggest that NF-kappa B indeed binds to this responsive element associating with CDK2 expression and centrosome amplification. RT-PCR and Western blotting results revealed that both mRNA and protein levels of CDK2 were significantly higher in tumor tissues than those in paired adjacent normal laryngeal tissues.
