NP9基因的克隆及对细胞周期素D1转录活性的影响

Cloning of NP9 Gene and Its Influence on Cyclin D1 Transcription Activity

背景与目的:NP9基因是我们在鼻咽癌中克隆到的一个新的表达下调的基因(GenBank登录号:BF718797),为了进一步研究该基因的功能,我们构建了人NP9基因编码区序列的真核表达质粒,并研究其表达对细胞周期素D1(cyclinD1)的影响。方法:通过核苷酸同源序列比较(BlastN)得到NP9EST的同源cDNA,RT-PCR扩增其编码区序列并构建真核表达重组质粒pRc/CMV2-NP9,脂质体转染后G418筛选出稳定、高效转录NP9基因的细胞克隆;再通过脂质体转染cyclinD1promoterLuc和NF-κB-Luc质粒到稳定表达NP9基因的细胞克隆,测定荧光素酶的活性以确定NP9基因对cyclinD1和NF-κB转录活性的影响;同时亚克隆NP9基因编码序列到载体pEGFP-C1中,脂质体转染细胞以确定NP9蛋白在细胞中的定位。结果:融合的绿色荧光蛋白出现于细胞核。G418抗性克隆中,RT-PCR扩增证实部分克隆表达NP9基因,且强弱不同。表达NP9基因的细胞克隆在瞬间转染cyclinD1promoterLuc和NF-κB-Luc48h后,荧光素酶的活性较其对照组分别下降46%和63%。结论:NP9蛋白在细胞核内表达;NP9基因通过抑制核转录因子NF-κB的转录活性下调cyclinD1的表达。

BACKGROUND &OBJECTIVE:We cloned NP9g ene(GenBank,BF718797)in a previous study which was down-re g ulated in nasopharyng eal carcinoma (NPC).To clarify the function of NP9g ene,we cloned the coding sequence(CDS )of NP9g ene and investig ated the influence of NP9on cyclin D1expression.METHODS :A full-leng th cDNA sequence was obta ined by Blast NP9EST,then the complete CDS was clo ned into an eukaryotic expressing vector pRc /CMV2.The plasmid was tra nsfected into CNE1cells(NPC cells)with lipofectamine and positive cel l clones which stably expressed NP9C DS were established by G418screening .The l uciferase report plasmid with cycli n D1promoter or NF-κB-Luc report plasmid was transfecte d into positive clones and the luciferase activity was detecte d.At last,the NP9CDS was subcloned i nto pEGFP-C1and cellular localization of NP9protein was observed throug h transfecting pEGFP-C1-NP9into CNE1cells.RESULTS :The fusion GFP was located in cell nuclei.The positive cell clones screened by G418expressed NP9CDS at different levels.Compared wi th control,the luciferase activities in NP9positive clones were decreased 46%a nd 63%after 48hours of transfecting the luciferase report plasmid with cycl in D1promoter or NF-κB-Luc plasmid.CONCLUSION:NP9protein is a nuclear protein.NP9g ene can down-reg ulate the transcription activity of cycli n D1and NF-κB.