[Cloning of NP9 gene and its influence on cyclin D1 transcription activity].
OBJECTIVE
We cloned NP9 gene (GenBank, BF718797) in a previous study which was down-regulated in nasopharyngeal carcinoma (NPC). To clarify the function of NP9 gene, we cloned the coding sequence (CDS) of NP9 gene and investigated the influence of NP9 on cyclin D1 expression.


METHODS
A full-length cDNA sequence was obtained by Blast NP9 EST, then the complete CDS was cloned into an eukaryotic expressing vector pRc/CMV2. The plasmid was transfected into CNE1 cells (NPC cells) with lipofectamine and positive cell clones which stably expressed NP9 CDS were established by G418 screening. The luciferase report plasmid with cyclin D1 promoter or NF-kappaB-Luc report plasmid was transfected into positive clones and the luciferase activity was detected. At last, the NP9 CDS was subcloned into pEGFP-C1 and cellular localization of NP9 protein was observed through transfecting pEGFP-C1-NP9 into CNE1 cells.


RESULTS
The fusion GFP was located in cell nuclei. The positive cell clones screened by G418 expressed NP9 CDS at different levels. Compared with control, the luciferase activities in NP9 positive clones were decreased 46% and 63% after 48 hours of transfecting the luciferase report plasmid with cyclin D1 promoter or NF-kappaB-Luc plasmid.


CONCLUSIONS
NP9 protein is a nuclear protein. NP9 gene can down-regulate the transcription activity of cyclin D1 and NF-kappaB.
Liu
Qi-Cai
QC
Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong, 510060, PR China.
Fang
Yan
Y
Li
Xiao-Yan
XY
Liang
Wei-Jiang
WJ
Zeng
Yi-Xin
YX
D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D019938:Cyclin D1 / Q000235:genetics*; D015536:Down-Regulation; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics; D009303:Nasopharyngeal Neoplasms / Q000235:genetics*; D014158:Transcription, Genetic
D016328:NF-kappa B; D019938:Cyclin D1