【作者】 刘真；

【导师】 姚开泰； 方唯意；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 研究背景与目的鼻咽癌（Nasopharyngeal carcinoma）是我国南方省份及东南亚高发的一种恶性肿瘤,具有明显的种族聚集性和地域性。其发生是一个多阶段、多途径、多机制的过程。流行病学调查显示,鼻咽癌的发生涉及到遗传倾向性和环境致癌因素,很可能是遗传易感性个体接受了致癌物的作用而发生的。EB病毒（EBV）、化学致癌物和胚性击中（即遗传因素或自发突变）构成NPC的主要病因,而发病阶段至少在两个以上。各种环境因素、EBV感染、遗传和表遗传学的改变导致瘤基因过表达及抑瘤基因表达下调或缺失,经过癌前病变,最终发生鼻咽癌。肿瘤的生成涉及多种基因和基因以外的变化,任何单独一种基因的改变不足以致瘤,多种基因变化的积累才能引起控制细胞生长和分化的机制紊乱,使细胞的生长失控而瘤变。在这些基因的变化中,最常发生两类基因的异常变化,即瘤基因及肿瘤抑制基因。鼻咽癌的生成同样涉及多基因的表达改变。在过去几十年中,虽然我们对于鼻咽癌发生的分子基础认识已经有了很大的提高,但这些认识还是远远不能完全揭示鼻咽癌的发病机理。因此,鼻咽癌相关新基因的发现和研究仍将有助于进一步揭示鼻咽癌的发病机理。NESG1基因（Genbank AF094758）,官方名为CCDC19（NM₀12337.1）,是本课题组姚开泰教授指导的博士生黎众魁于1999年利用mRNA差异显示法,从人正常鼻咽上皮和软腭口腔上皮中筛选,并结合3’-RACE和5’-RACE技术,分别扩增出长约1.4kb和0.7kb的片段（其中包含了一段174bp的重叠产物以确认扩增的特异性）,拼接后发现的一个全长约1850bp连续的cDNA序列。该基因位于1q22。预测其编码框长1161bp,编码386个氨基酸,分子量为46252Da,蛋白质等电点为9.99,SOSUI和PSORTⅡ网上预测其为可溶性核蛋白。BLAST比对发现,NESG1的cDNA序列与胎儿肺组织cDNA文库和Stratagene肺癌cDNA文库中的两个EST序列同源。多组织（包括鼻咽粘膜、气管、食管、大脑、心脏、膀胱、肝脏、肺脏、胃、肾脏、胸腺和大肠）Northern杂交显示,该基因特异性的表达于鼻咽和气管纤毛柱状上皮。本课题在此研究基础上,对NESG1基因进行重新克隆测序分析及功能鉴定,并寻找NESG1基因相关的信号转导通路,探讨该基因在鼻咽癌中的作用机制,为进一步阐明鼻咽癌发病机理提供参考。研究内容与方法1. NESG1基因的序列修正以及新序列的生物信息学分析对NESG1基因进行了重新克隆、测序分析并对其序列进行修正,重新预测编码框。对新校正序列进行生物信息学分析。2. NESG1基因在鼻咽癌中表达水平检测（1）.NESG1基因mRNA表达水平利用RT-PCR和Real-time PCR检测NESG1 mRNA在鼻咽癌细胞、鼻咽癌组织与非癌鼻咽组织中的表达情况；原位杂交检测NESG1基因的mRNA表达定位。（2）.NESG1兔抗人多克隆抗体制备及NESG1蛋白表达构建pGEX-4T-1-NESG1-GST原核融合表达载体。IPTG诱导NESG1-GST基因融合表达。利用GST抗体纯化融合蛋白用于免疫大白兔,约100天后取兔血上清,并亲和纯化,最后获得所需的NESG1多克隆抗体。利用免疫组化和Western blot检测NESG1蛋白在鼻咽癌组织与非癌鼻咽组织中的表达情况。利用免疫组化检测鼻咽外多组织中NESG1的表达分布。3. NESG1基因在鼻咽癌中的功能初步研究（1）.NESG1过表达对鼻咽癌细胞5-8F生物学特性的影响构建与增强型绿色荧光蛋白融合表达的NESG1慢病毒载体,利用293FT细胞包装成成熟的慢病毒颗粒,感染具有高成瘤和高转移能力的鼻咽癌细胞5-8F。96孔板有限稀释法挑选阳性单克隆细胞,扩大培养,建立稳定过表达NESG1的鼻咽癌细胞株,以空载体同时进行病毒包装及感染鼻咽癌细胞5-8F,获得对照细胞株。利用MTT法、平板克隆形成实验、流式细胞术、Transwell小室迁移实验及Boyden小室侵袭实验等检测NESG1基因在细胞水平对细胞生长、细胞周期、迁移及侵袭能力的影响；检测稳定过表达NESG1的鼻咽癌细胞裸鼠皮下成瘤能力的改变。（2）.抑制NESG1表达对鼻咽癌细胞5-8F生物学特性的影响构建靶向NESG1的shRNA慢病毒干扰载体,稳定干扰过表达NESG1基因的鼻咽癌细胞,利用MTT法、平板克隆形成实验、Transwell小室迁移实验及Boyden小室侵袭实验等观察NESG1表达抑制后细胞生长、迁移及侵袭能力的变化。进一步探讨NESG1基因在鼻咽癌细胞中的基本功能。4. NESG1基因介导的分子基础初步研究（1）.应用Affymetrix全基因组芯片检测NESG1基因稳定导入前后对鼻咽癌细胞基因表达的影响,寻找差异表达基因（2）.利用生物信息学的方法,分析基因芯片差异表达基因介导的信号通路（3）.NESG1抑制细胞周期进展的初步分子机制由于过表达的NESG1能阻滞细胞周期由G1向S期转化,因此,NESG1参与抑制了细胞周期的进展。基于基因芯片的数据,我们利用荧光定量RT-PCR和Western blot验证了S期相关的几个重要基因CCNDl、CCNAl、CDK4、p21、CDK2、CDC2的表达,初步探讨了NESG1基因抑制细胞周期进展的机制。结果1. NESG1基因的序列修正以及新序列的生物信息学分析（1）.NESG1基因的序列修正最近,我们对NESG1基因进行重新克隆并测序分析时发现,测序结果与原提呈的AF094758（NM₀12337.1）序列存在两处差异：A碱基替代了原序列CDS区第1181位的G碱基、原序列第905位点处有一个A碱基的插入,从而导致氨基酸编码框改变。新序列与人类基因组序列比对显示了与其完全的一致性。另外,新序列在与其他的人cDNA序列（CR604695、BC089391）和模式生物cDNA序列如NM₀01024882.1（褐家鼠）、XM₅13919（黑猩猩）、AB168450（食蟹猴）等比对时并未发现1181和905位点有其它类型的碱基改变,这就证实了新克隆NESG1序列的正确性。由于新序列1181碱基由A替代了G以及905位点A碱基的插入,导致NESG1基因的编码序列较前发生了变化。重新预测了编码框,结果显示NESG1基因编码框长1656bp,编码551个氨基酸,这与Macaca fascicularis（食蟹猴）睾丸cDNA中克隆出的NESG1基因（AB168450）的编码框预测长度一致。向NCBI参考序列部提交修改申请后,Genbank数据库接收了修改意见,使其版本由最初的NM₀12337.1升级为NM₀12337.2。（2）.序列修正后NESG1基因的生物信息学分析修正后的NESG1基因编码551个氨基酸,预测分子量为65729,7Da,等电点为8.94。SMART程序预测NESG1主要是一个含卷曲螺旋（coiled-coil）结构的蛋白（157-468aa和498-527aa）。Blast比对和距离树进化分析显示,该基因进化相对保守,与许多模式生物都有很高的同源性,而且进化越高级,同源性越高。PROSITE分析显示该蛋白含有一个N-myristoylation位点、四个Protein kinase C phosphorylation位点、一个N-glycosylation位点、七个Casein kinaseⅡphosphorylation位点、两个cAMP and cGMP-dependent protein kinase phosphorylation位点及两个Amidation位点。疏水性及跨膜蛋白预测结果显示NESG1是跨膜蛋白的可能性小。SignalP 3.0软件信号肽预测结果显示NESG1没有信号肽序列,因此是分泌蛋白的可能性小。用三种不同软件预测NLS （Nuclear Localization Signal）,均在248aa-258aa,以及503aa-521aa附近发现核定位信号,提示我们NESG1蛋白很可能在细胞核有表达。2. NESG1基因在鼻咽癌中表达水平检测（1）.NESGl基因mRNA表达水平原位杂交显示,NESG1基因表达在鼻咽粘膜纤毛柱状上皮细胞的细胞核、浆内。半定量RT-PCR和荧光定量RT-PCR均显示,与非癌鼻咽组织相比,NESG1基因在鼻咽癌组织和细胞中表达明显下调（t=19.819,P<0.001）。（2）.NESG1兔抗人多克隆抗体制备及NESG1蛋白表达使用新制备的NESG1多克隆抗体,利用Western blot方法在正常鼻咽组织中检测到一条表达很强的约66kD的条带,表明校正后的NESG1确实是正确的基因。Western blot检测各鼻咽癌细胞株、鼻咽癌组织以及非癌鼻咽组织中NESG1在蛋白水平的表达情况,以β-actin为内参,根据各条带的NESG1灰度值与β-actin灰度值比率计算各细胞中NESG1的表达水平,结果表明,与非癌鼻咽组织相比,NESG1在鼻咽癌组织和细胞中表达均显著下调（t=41.116,P<0.001）。免疫组化检测82例鼻咽癌组织和40例非癌鼻咽组织中NESG1的表达情况,结果显示,NESG1主要在鼻咽粘膜柱状、鳞状上皮细胞浆中表达,个别细胞中也可见核表达。与非癌鼻咽组织相比,NESG1在鼻咽癌组织中表达显著降低（Z=6.852,P<0.001）。经统计分析,NESG1的表达与患者的年龄无相关性（Z=0.506,P=0.613和0.474）；与性别也无相关性（Z=0.717,P=0.474）；而与淋巴结转移显著负相关（Z=2.110,P=0.035）；与临床分期显著负相关（χ2=14.660,P=0.001）其他正常组织中的检测结果表明,NESG1主要表达于甲状腺导管上皮、乳腺导管上皮、舌鳞状上皮、细支气管柱状上皮、肠上皮以及胃,在肾小管也有弱表达。肺泡上皮、胸腺、肝、脾、阑尾、子宫内膜、子宫颈鳞状上皮不表达。其中在甲状腺导管上皮、胃小凹以及细支气管柱状上皮中发现有核表达。3. NESG1基因在鼻咽癌中的功能初步研究（1）.NESG1过表达对鼻咽癌细胞5-8F生物学特性的影响将NESG1导入到鼻咽癌细胞系5-8F中,建立了稳定过表达NESG1的细胞系。选取两株单克隆细胞2F4和3D8用于后续功能研究,以空载体单克隆细胞C6作为对照。2F4细胞中NESG1 mRNA的表达量比3D8细胞几乎高2倍,而蛋白表达则高1.76倍。采用MTT法对NESG1过表达后5-8F细胞体外增殖能力的改变进行检测,结果表明,导入NESG1的2F4和3D8细胞比空载C6细胞生长速度减慢,并且NESG1表达相对更高的2F4细胞比3D8细胞生长抑制地更明显。对3D8、2F4、C6、5-8F组细胞的增殖情况用析因设计的方差分析,四组细胞的增殖能力具有显著差异（F=1404.066,P<0.001）,3D8和2F4细胞较C6和5-8F细胞增殖缓慢。平板克隆形成实验的结果同样显示3D8、C6、5-8F三组细胞的克隆形成能力具有显著差异（F=27.559,P<0.001）；2F4、C6、5-8F三组细胞的克隆形成能力也具有显著差异（凡50.148,P<0.001）。两个实验结果均表明NESG1过表达后,抑制了鼻咽癌细胞5-8F的体外增殖。采用流式细胞术分析过表达NESG1的单克隆细胞3D8和2F4的细胞周期分布,结果发现,5-8F、C6、3D8和2F4四组细胞的S期细胞比例有显著差异（F=9.086,P=0.006）。与空载对照单克隆细胞C6以及未处理细胞5-8F相比,3D8细胞S期细胞比例无统计学差异（P=0.796、P=0.536）,而2F4细胞S期细胞比例比C6和5-8F细胞显著下降（P=0.003、P=0.002）,也就是说,在NESG1表达更高的2F4细胞中,细胞出现了G1向S期转化障碍,细胞被阻滞在G1期。采用Transwell小室检测NESG1过表达后细胞体外迁移运动能力的变化,结果发现四组细胞运动能力的差异具有显著性（F=1054.101,P<0.001）。与C6和5-8F细胞相比,2F4和3D8穿过膜的细胞数显著减少,其运动能力明显降低。采用Boyden小室的方法分析NESG1过表达后细胞体外侵袭能力的变化,统计学分析的结果显示四组细胞侵袭能力的差异具有显著性（F=304.640,P<0.001）。与5-8F和C6相比,3D8和2F4细胞穿过基质胶的细胞数显著减少,其体外侵袭能力明显降低。裸鼠皮下成瘤接种后连续观察,于第14天处理3D8／C6组6只裸鼠,第18天处理2F4／C6组5只裸鼠。结果表明,与对照细胞C6相比,3D8细胞组裸鼠皮下成瘤重量有显著差异（F=8.478,P=0.005）,体积也有显著差异（F=22.199,P=0.004）；同样,与对照细胞C6相比,2F4细胞组裸鼠皮下成瘤重量有显著差异（F=2.904,P<0.001）,体积也有显著差异（F=7.957,P=0.010）。NESG1基因导入5-8F细胞后,能显著抑制细胞在裸鼠体内的生长。（2）.抑制NESG1表达对鼻咽癌细胞5-8F生物学特性的影响构建靶向NESG1的shRNA慢病毒干扰载体,稳定干扰过表达NESG1基因的鼻咽癌细胞株2F4,选取两株单克隆细胞1C9和1D10用于后续功能研究,以空载体细胞C1作为对照。荧光定量检测1C9干扰效率为97.7%,1D10干扰效率为97.8%。采用MTT法对NESG1基因沉默后2F4细胞体外增殖能力的改变进行检测,结果发现三组细胞的体外增殖能力具有显著差异（F=3764.751,P<0.001）。与空载对照单克隆细胞C1相比,干扰NESG1后的单克隆细胞1C9、1D10增殖速度显著加快。平板克隆形成实验的结果显示三组细胞的克隆形成能力具有显著差异（F=140.525,P<0.001）,干扰NESG1后的单克隆细胞1C9、1D10的克隆形成能力较空载对照单克隆细胞C1强。两个实验结果均表明干扰NESG1表达后,鼻咽癌细胞的体外增殖能力增强。采用Transwell小室检测NESG1干扰后细胞体外迁移运动能力的变化,结果发现三组细胞运动能力的差异具有显著性（F=1162.190,P<0.001）。与空载体对照单克隆C1细胞相比,干扰NESG1后的单克隆细胞1C9、1D10穿过膜的细胞数显著增加,其运动能力明显增强。采用Boyden小室的方法分析NESG1沉默后细胞体外侵袭能力的变化,统计学分析的结果显示三组细胞侵袭能力的差异具有显著性（F=887.155,P<0.001）。与空载体对照单克隆C1细胞相比,干扰NESG1后的单克隆细胞1C9、1D10穿过基质胶的细胞数显著增加,其体外侵袭能力明显增强。4. NESG1基因介导的分子基础初步研究（1）. Affymetrix全基因组表达谱芯片对NESG1-2F4和C6空载对照细胞进行Affymetrix全基因组表达谱芯片检测,一共检测到了2400多个差异表达基因,其中上调基因797个,下调基因1707个。将这些差异表达基因进行了初步的Pathway分析,结果显示NESG1基因参与多条信号转导途径,其中包括Tight junction、MAPK通路和Cell cycle通路等。（2）.NESG1抑制细胞周期进展初步机制研究根据基因芯片数据,我们采用荧光定量RT-PCR和Western blot检测NESG1过表达后S期相关基因的表达变化。荧光定量RT-PCR结果显示,CCNAl在2F4、3D8和C6中的表达有显著差异（Welch=213.053,P=0.001）；CDK4在2F4、3D8中的表达与对照细胞C6无统计学差异（F=0.579,P=0.589）；CCND1在2F4、3D8中的表达与对照细胞C6无统计学差异（F=0.017,P=0.983）；与空载对照细胞C6相比,p21在2F4、3D8中表达显著增高（F=35.675,P<0.001）；CDK2在2F4、3D8中的表达与对照C6无统计学差异（Welch=0.092,P=0.914）；CCD2在2F4、3D8中的表达与对照细胞C6无统计学差异（F=0.769,P=0.504）。Western blot结果表明,与空载C6相比,CCNA1在2F4、3D8中表达显著下调（Welch=155.736,P=0.001）；CDK4在2F4、3D8中的表达与对照细胞C6无统计学差异（F=0.470,P=0.646）；CCND1在2F4、3D8中的表达与对照细胞C6无统计学差异（F=0.558,P=0.600）；与空载细胞C6相比,p21在2F4、3D8中表达显著增高（F=86.359,P<0.001）。我们的结果提示,NESG1可能通过间接抑制CCNA1的表达和上调p21的表达,从而阻碍了细胞周期的进展。结论1.修正了NESG1基因的序列,使其在Genbank数据库中的版本由最初的NM₀12337.1升级为NM₀12337.2。2.NESG1稳定导入鼻咽癌细胞5-8F后,细胞的体内、外增殖、迁移和侵袭能力显著下降,在NESG1表达更强的2F4细胞中,细胞出现了G1向S期转化障碍,细胞阻滞在G1期。在抑制NESG1表达后,细胞增殖能力、迁移和侵袭能力显著恢复。这提示,NESG1在鼻咽癌中是一个肿瘤抑制相关基因。NESG1通过抑制CCNA1和上调p21的表达,参与了鼻咽癌细胞周期进展的调控。本研究创新之处1.修正了NESG1基因的序列,更新了NESG1在Genbank数据库中的版本号。2.对NESG1基因进行了表达定位,在mRNA和蛋白水平证实了NESG1基因在鼻咽癌中表达下调,为理解和研究鼻咽癌发病的分子机制提供了线索。3.初步证实NESG1基因与鼻咽癌的发生发展密切相关,能够抑制鼻咽癌细胞的增殖、迁移和侵袭能力,并且将细胞阻滞在G1期。4.基因芯片分析显示,NESG1参与介导了Tight junction、MAPK和Cell cycle等多条信号通路,这为研究NESG1在鼻咽癌中介导的信号通路提供了关键的线索。5.NESG1抑制CCNA1的表达和上调p21的表达,阻碍了鼻咽癌细胞的细胞周期进展。6.NESG1基因是本课题组克隆及修正的鼻咽癌候选抑瘤基因,具有独立的知识产权。对该基因的研究能进一步阐明鼻咽癌发病机理,有重要的研究价值。更多还原

【Abstract】 Background and ObjectivesNasopharyngeal carcinoma (NPC) is one of the commonest carcinomas with a high degree of malignant phenotype in Southern China and Southeast Asia. Both the geographic pattern and familial aggregation of incidence are two main characteristics of NPC. The carcinogenesis of nasopharyngeal epithelium is a multi-stage, multi-path and multi-mechanism process, which is involved in the effects of several factors including genetic predisposition, enviromental carcinogen and Epstein barr virus (EBV). The changes of various environmental factors, EBV infection, genetics and epigenetics cause the overexpression of oncogenes and downregulated or null expression of tumor suppressors in nasopharyngeal epithelium, which will lead to the transition of epithelium to precancerous lesion, and finally be transformed to NPC.Carcinogenesis is associated with the participation of expression alteration of multiple genes. The expression change of any single gene does not have the ability to drive the malignant tranformation of normal epithelium. In the past few years, although the knowledge of molecular mechanisms about NPC has greatly increased, its pathogenesis remains to be clarified.NESG1 gene (Genbank AF094758), official name CCDC19 (NM-012337.1), was found by Dr Zhongkui Li who is guided by Professor Kaitai Yao in 1999. Dr Li used mRNA differential display combined with the techniques of 3’-RACE and 5’-RACE to amplify respectively the 1.4kb and 0.7kb fragments (There were 174bp overlap sequence in the two fragments, which was utilized to confirm the specificity of amplification).After the two fragments were spliced, a full-length cDNA with consecutive 1850bp fragment was discovered. This gene is located at the chromosome 1q22. Predicted coding sequence (CDS) of this gene was 1161 bp and encoded 386 amino acids. Molecular weight of this protein was 46252Da and its isoionic point was 9.99. Online prediction of SOSUI and PSORTⅡshowed that deduced NESG1 was a soluble nucleoprotein. Using NCBI’s BLAST tool, he found that cDNA of NESG1 was homologous to two ESTs from human fetal lung cDNA library and Stratagene lung carcinoma cDNA library, respectively. Northern blot analysis of NESG1 transcript in human multi-tissues including nasopharyngeal epithelium (NE), trachea (TR), esophagus (ES), cerebrum (CR), heart (HE), bladder (BL), large intestine (IN), liver (LI), stomach (ST), kidney (KI), lung (LU) and thymus (TH) has revealed that NESGl is specifically expressed in nasopharyngeal epithelium and columnar ciliated epithelium of trachea.Based on the above investigation, we re-analyzed the sequence of NESG1 and revised the CDS region of NESGl. Further, we identified the function of this revised gene and analyzed its molecular basis in NPC preliminarily. This may provide new clues for further elucidating the NPC pathogenesis.Contents and methods1. Sequence-revision of NESG1 gene and its bioinformatics analysisOriginal NESG1 was recloned and analyzed by sequence identification. Its sequence was revised and CDS was repredicted. Then, revised NESG1 gene was analyzed by bioinformatics.2. Detection of NESG1 expression level in NPC(1). Expression level of NESGl mRNANESG1 mRNA expression was measured by RT-PCR and Real-time RT-PCR in NPC cells, NPC tissues and noncancerous nasopharyngeal tissues. In situ hybridization was used to locate the mRNA expression of NESG1 in nasopharyngeal tissues.(2). Preparation of rabbit anti human NESG1 poly-clonal antibody and analysis of NESG1 protein expressionThe open reading frame of revised NESG1 was cloned into prokaryotic expression vector pGEX-4T-1. Subsequently, recombinant GST-NESG1 fusion protein expression was induced using IPTG and the resulting protein was purified using GSH-sepharose. The purified protein was used to immunize rabbits. After 100 days, serum of rabbit was taken and the antibody was purified by affinity chromatography. The antigenicity of this fusion protein was validated using western blotting assay with rabbit anti-GST antibody. Subsequently, protein expression of NESG1 was determined in NPC and noncancerous nasopharyngeal tissues by Western blot and immunohistochemistry using NESG1 antibody. Furthermore, the expression pattern of NESG1 protein was also determined in other human tissues by immunohistochemistry.3. Functional analysis of NESG1 in NPC(1). Effects of NESG1 overexpression on biological characteristics in 5-8F NPC cellsThe NESG1 open reading frame was cloned into a pGC-FU-GFP lentivirus vector. The resulting lentivirus vector together with pHelperl and pHelper2 vectors were co-transfected into 293FT cells to generate lentiviral stock. An "empty" vector pGC-FU-GFP was utilized as a negative control. Lentiviral particles were used to infect NESG1-negative 5-8F cells, an NPC cell line with high tumorigenic and metastatic potential. Colonies with GFP expression were selected by limiting dilution assay of 96-well plate to expand culture. Finally,5-8F NPC cells with NESG1 overexpression and negative empty vector 5-8F cells were respectively established for further investigation. MTT, colony formation, FACSCaliber cytometry, Transwell and Boyden chamber were utilized to determine the ability of cell growth, cell cycle, cell migration and invasion in vitro. In vivo subcutaneous tumorigenesity in nude mice was used to evaluate the effect of overexpressed NESG1 on NPC cells.(2). Effects of suppressing the expression of NESG1 on biological characteristics in NESG1-overexpressed NPC 5-8F cellsNESG1-shRNA lentivirus interference vector was constructed and NPC cell with NESG1 silenced by stable RNA interference was established. MTT, colony formation, Transwell and Boyden chamber assay were utilized to detect the alterations of the ability of cell growth, migration, and invasion in NESG1 silencing.4. Priliminary investigation of molecular basis mediated by NESG1 in NPC(1). Affymetrix genome-wide microarray was employed to measure the differentially expressed genes after NESG1 gene was introduced into NPC cells(2). Bioinformatics was used to analyze the signal pathways mediated by these differentially expressed genes(3). Preliminary molecular mechanisms of NESG1 in inhibiting cell cycle progressionOwing to the role of overexpressed NESG1 in blocking the transition of cell cycle from G1 to S phase, we believed that NESG1 is involved in inhibiting cell cycle progression. Based on the microarray data, we verified the expression of several significant genes associated with S phase including CCND1, CCNA1, CDK4, p21, CDK2 and CDC2 and preliminarily explored the molecular mechanism of NESG1 in suppressing cell cycle progression.Results1. Sequence revision of NESG1 and its bioinformatics analysis(1). Sequence revision of NESG1The sequence analysis of newly cloned NESG1 showed that, comparing to formerly suggested NESG1 sequence, a G base in 1181 position was replaced by an A base and an A base inserted in 905 position led to a frame shift mutation. Furthermore, newly cloned sequence also displayed the consistency with the human genome sequence, some human cDNA clone sequences (CR604695, BC089391) and model organism sequences, such as NM-001024882.1 (Rattus norvegicus), XM-513919 (Pan troglodytes) and AB168450 (Macaca fascicularis) et al at 1181 and 905 positions. Based on these data, we updated NESG1 sequence and repredicted its CDS. The CDS of NESG1 was 1656bp and encoded 551aa, which is the same with the prediction of NESG1 of Macaca fascicularis testis cDNA clone (AB168450) in CDS region. Subsequently, we submitted the application of revised NESG1 sequence to department of NCBI reference sequence. They accepted our request and updated the NESG1 version from NM 012337.1 to NM 012337.2(2). Bioinformatics analysis of revised NESG1 geneRevised NESG1 encoded 551aa and its predicted molecular weight was 65729.7Da, the isoionic point was 8.94. Using the prediction of SMART program, we found that NESG1 is a coiled-coil structural protein (157-468aa and 498-527aa) Using blast and distance tree evolution analysis, we found that human NESG1, a relatively conserved gene in evolution, is highly homologous to the sequence of some other model organisms. Furthermore, human NESG1 sequence shares a higher homology with other model organisms with higher evolution level. PROSITE analysis showed that NESG1 contains one N-myristoylation site, four Protein kinase C phosphorylation sites, one N-glycosylation site, seven Casein kinase II phosphorylation sites, two cAMP and cGMP-dependent protein kinase phosphorylation sites and one amidation site. Prediction of hydrophobicity and transmembrane protein indicated that the probability of transmembrane protein of NESG1 was very small. Signal peptide prediction by SignalP 3.0 software displayed that there was no signal peptide sequence in NESG1, so NESG1 is not a secretory protein. Using three different softwares to predict Nuclear Localization Signal of NESG1, we found that there all existed Nuclear Localization Signal in 248aa-258aa and 503aa-521aa. This hinted that NESG1 could be expressed in cell nucleus.2. Measurement of NESG1 expression level in NPC(1). mRNA expression level of NESG1In situ hybridization showed that NESG1 mRNA was expressed in nucleus and cytoplasm of human nasopharyngeal epithelial cells. Using reverse transcription PCR and real-time PCR, we found that comparing to normal nasopharyngeal tissues, NESGl expression was down-regulated in NPC tissues and all of 8 NPC cell lines (t=19,819, P<0.001)(2). Preparation of rabbit anti human NESG1 polyclonal antibody and analysis of NESG1 protein expressionUsing the newly prepared anti-NESG1 rabbit polyclonal antibody, we investigated NESGl protein expression in NPC, NP and other human tissues. Western blotting assay showed a protein band with apparent molecular weight of approximate 66 kD, which is in agreement with the calculated molecular weight based on the revised NESG1 ORF sequence. Western blot revealed that protein expression of NESG1 was significantly downregulated in NPC tissues and NPC cells comparing with noncancerous nasopharyngeal tissues (t=41.116, P<0.001). Immunohistochemistry staining was performed for 40 normal nasopharyngeal tissues and 82 NPC tissues. NESG1 protein was abundantly expressed in nasopharyngeal epithelial cells and mainly localized in cytoplasm (sometimes expressed in nucleus). In contrast, NESGl expression was significantly down-regulated in NPC tissues (Z=6.852, P<0.001). No significant associations were found between NESG1 expression and age (Z=0.506, P= 0.613)and gender (Z=0.717, P=0.474) of NPC patients. Interestingly, we observed that NESGl expression was negatively correlated with N classification (Z=2.110,P=0.035) and clinical stage (x2=14.660, P=0.001) of NPC patients. Furthermore, we also performed the expression pattern of NESG1 protein in other human tissues by immunohistochemistry. The result showed that NESG1 was mainly expressed in cytoplasm of thyroid duct epithelium, breast duct epithelium, squamous epithelium of tongue, bronchiole columnar epithelium, intestinal epithelium and gastric epithelium, furthermore, weak expression of NESG1 was also showed in distal convoluted tubules. Among these, intranuclear expression of NESG1 was also seen in thyroid duct epithelium, gastric pits and bronchiole columnar epithelium. However, NESG1 expression was undetectable in the other tissues including alveolar epithelium, thymus gland, liver, spleen, vermiform appendix, endometrium and squamous epithelium of cervix.3. Functional investigation of NESG1 in NPC(1). Effects of NESG1 overexpression on biological characteristics in 5-8F NPC cellsAfter NESG1 was introduced into NPC cells, we established stable NESG1 overexpressed NPC cells. Two single cell clones 2F4 and 3D8 were chosen for further functional study. Empty vector C6 was selected as a negative control. In 2F4 and 3D8 cells, expression level of NESG1 mRNA in 2F4 cells was about 2 folds higher than that in 3D8 cells, and protein expression level of NESG1 in 2F4 cells was 1.76 folds higher than that in 3D8 cells. Compared to C6 control cells, both two clonal cells showed to be inhibited markedly in MTT cell proliferation assay (F=1404.066, P<0.001), colony formation assay (2F4:F=50.148, P<0.001; 3D8:F=27.559, P<0.001), Transwell migration assay (F=1054.101, P< 0.001) and Boyden invasion assay (F=304.640, P<0.001). In vivo subcutaneous tumorigenesity in nude mice showed that tumor weight and volume of 3D8 and 2F4 were significantly decreased comparing with C6 (weight of 3D8:F=8.478, P=0.005; weight of 2F4:F=2.904, P< 0.001; volume of 3D8:F=22.199, P=0.004, volume of 2F4:F=7.957, P=0.010). Furthermore, cell cycle analysis showed that the S phase population decreased significantly in 2F4 cells in comparison to the NESG1-negative C6 cells (P=0.003). However, in 3D8 cells the change in S phase population was not significant (P=0.796). This might be associated with the fact that 2F4 cells had higher levels of NESG1 than 3D8 cells did.(2). Effects of NESG1 expression suppressed on biological characteristics in 5-8F NPC cellsAfter shRNA lentivirus interference vector targeting NESG1 was constructed, we established 2F4 NPC cells with NESG1 silenced by stable RNA interference. Two stably cell clones 1C9 and 1D10 were chosen for functional exploration. Interference efficiency of 1C9 and 1D10 was 97.7% and 97.8% respectively. Compared to a negative control C1 cell, the proliferation ability of shRNA-NESGl 1C9 and1D10 cells was significantly increased (F=3764.751, P<0.001). Furthermore, the ability of migration (F=1162.190, P<0.001) and invasion (F=887.155,P<0.001) in shRNA-NESG1 1C9 and1D10 cells was also markedly increased compared to control cells.4. Preliminary study of molecular basis mediated by NESG1(1). Affymetrix genome-wide expression profile microarrayAffymetrix genome-wide expression profile microarray was used to determine the differential gene expression between NESG1-2F4 NPC cells and control C6 NPC cells. The results showed that 2400 genes were differentially expressed. Among them, 797 genes were upregulated and 1707 genes were downregulated. We analyzed NESG1-mediated pathways by comparing gene expression profiles in 5-8F NPC cells with or without NESG1 expression. Interestingly, there were many pathways determined by KEGG Pathways Database, including Tight junction (P=3.0E-6), MAPK signaling pathway (P=4.0E-6), regulation of Actin cytoskeleton (P=2.42E-4), and Cell cycle (P=3.8E-3) etc. (2). Preliminary molecular mechanisms of NESG1 in inhibiting cell cycle progressionWe verified the expression of several important genes associated with S phase progression by real-time PCR and Western blot based on microarray data. The results showed that the mRNA levels of CCND1 (F=0.017, P=0.983),CDK4 (F=0.579, P=0.589),CDK2 (Welch=0.092, P=0.914), and CDC2 (F=0.769,P=0.504) did not change in spite of the restored expression of NESG1. However, we found that CCNA1 mRNA expression was inhibited in both two clonal cells, especially in 2F4 cell (Welch=213.053, P=0.001). Furthermore, p21 expression level was also increased in 2F4 and 3D8 (F-35.675, P<0.001). Western blot also showed that the protein level of CCND1 (F=0.558,P=0.600) and CDK4 (F=0.470, P=0.646) in 2F4 and 3D8 cells have no significant change after the overexpression of NESG1, accompanied with the decreased CCNA1 (Welch=155.736, P=0.001) and increased p21 (F=86.359, P<0.001) protein expression level. The suppressive effect of NESG1 on cell cycle is greater in 2F4 cells than in 3D8 cells. Considering the fact that 2F4 cells express higher levels of NESG1 than 3D8 cells do, these data suggest that the effect of NESGl on cell cycle is dose-dependent. Taking together, our studies suggest that NESGl is able to retard the progression of cell cycle, and this effect is possibly mediated by the inhibited CCNAl expression and the upregulated p21 expression.Conclusion1. After NESG1 sequence was revised, its version in Genbank database was updated from NM 012337.1 to NM 012337.2.2. Funtional investigations showed that, after NESG1 was introduced into NPC 5-8F cells, the cell proliferation in vivo and in vitro, cell migration and invasion were markedly reduced. Furthermore, in higher NESG1-expressed 2F4 cells, cell cycle progression was blocked from G1 to S phase. After NESG1 expression was inhibited, the cell proliferation, migration and invasion was significantly recovered, which suggest that NESG1 should be a tumor-suppressive associated gene.NESG1 was involved in the regulation of cell cycle progression of NPC cell by inhibiting the expression of CCNA1 and upregulating the expression of p21.Discoveries and innovations of this investigation1. NESG1 gene was revised and its version in Genbank database was updated.2. NESG1 location was confirmed in human tissue. Furthermore, mRNA and protein expression level of NESG1 was down-regulated in NPC. This may provide clues for understanding and investigating the molecular mechanism of NPC pathogenesis.3. NESGl was closely associated with the genesis and development of NPC. Overexpressed NESGl could suppress the ability of cell proliferation, cell migration and cell invasion. Furthermore, Overexpressed NESG1 blocked the cell cycle progression from G1 to S phase.4. Microarray analysis showed that NESGl mediated multi-pathway, such as Tight junction, MAPK and Cell cycle. This may provide key clues for studying the signal pathway mediated by NESGl.5. NESG1 retarded the progression of cell cycle by inhibiting CCNA1 expression and upregulating p21 expression. 6. As a new candidate tumor suppressor, NESG1 was cloned and revised by our own research group. So we possesed the knowledge property right of NESG1 ourselves. Furthermore, we would continue to investigate its role in NPC, which might laid the basis for further elucidating the NPC pathogenesis.更多还原