NAG7基因转染对鼻咽癌细胞生长的影响

为了探讨鼻咽癌表达下调基因NAG7对鼻咽癌细胞系HNE1生长的影响, 构建了NAG7基因的真核表达载体pcDNA3.1(+)/NAG7, 并采用脂质体转染技术将真核重组体pcDNA3.1(+)/NAG7质粒和真核空载体pcDNA3.1(+)质粒分别导入HNE1细胞, 经G418筛选后获得稳定转染细胞克隆, RT-PCR和RNA印迹检测NAG7基因的表达, 并通过细胞生长曲线、裸鼠接种和流式细胞等方法对转染细胞的生物学行为进行检测.结果显示：转染NAG7基因后,基因表达增加,细胞生长倍增时间较空载体转染和HNE1明显延长,流式细胞技术检测表明,NAG7可延缓细胞由G0～G1期进入S期;裸鼠接种实验显示转染NAG7基因后的HNE1细胞致瘤性受到抑制.上述结果表明：NAG7基因转染后鼻咽癌细胞生长受到抑制,提示NAG7基因是一鼻咽癌相关的抑瘤基因候选者.     

应用二维电泳和质谱技术初步研究NAG7基因的功能

为了进一步研究鼻咽癌相关基因NAG7的功能,NAG7基因编码框的cDNA片段被亚克隆至pcDNA3.1( )的表达载体,通过脂质体转染入鼻咽癌细胞系HNE1。抽提HNE1细胞和已转染了NAG7基因的HNE1细胞总蛋白质,用二维凝胶电泳分离蛋白质,获得二维凝胶图谱,PDQuest软件包分析后,确立表达上调的蛋白质斑点进行质谱分析,得到的肽质指纹经蛋白质数据库分析鉴定蛋白质。共分析了12个蛋白质点,其中3个未有质谱结果,已鉴定的9个蛋白质包括生长捕获特异蛋白、DNA结合蛋白、c-myc启动子结合蛋白及胱冬肽酶（caspase)6等蛋白质。通过对这些蛋白质性质和功能的讨论,发现它们参与了细胞周期调控、转录调节及细胞凋亡等重要事件。因此,VAG7很可能是通过介导上述蛋白质的表达上调而发挥作用。     

鼻咽癌相关基因NAG7编码产物的表达和定位分析

为了研究NAG7基因编码产物的特性和在活细胞内定位与表达,首先利用生物信息学分析NAG7编码蛋白的一般性质并预测其定位,再通过构建增强型绿色荧光蛋白（Enhance green fluorescent protein,EGFP)与NAG7融合基因的真核表达载体pEGFP-C2-NAG7,通过脂质体介导分别转染非洲绿猴肾细胞系COS7和人鼻咽癌细胞系HNE1,瞬时表达后荧光显微镜观察NAG7基因编码蛋白的活细胞内定位及表达,研究结果表明NAG7的编码产物可以COS7细胞中高表达并定位于细胞浆,而在HNE1细胞中虽也定位于胸浆,但仅有极少数细胞存在表达,因此NAG7编码产物在HNE1中的表达差异是否是NPC发生的原因之一有待深入研究。     

NAG7基因转染HNE1细胞后下调蛋白质的鉴定及其意义(英文)

NAG7基因是我室克隆的与鼻咽癌相关的肿瘤抑制候选基因 .将NAG7编码框的cDNA片段克隆至pGEM3.1(+ )的表达载体 ,经脂质体转染入HNE1细胞 ,经G4 18筛选 ,并运用PCR技术证实 .建立含NAG7基因稳定转染的细胞系 ,抽提细胞总蛋白质 ,双向凝胶电泳分离蛋白质 ,对表达下调的蛋白质点进行质谱分析 ,获得的肽质指纹经SWISS PROT数据库分析以鉴定蛋白质点 .鉴定出的 7个下调蛋白质包括纤溶酶原、收缩蛋白、Ras 相关蛋白Rab 36及ARF 相关蛋白等 .通过对蛋白质性质和功能的分析 ,发现这些蛋白质参与了细胞信号的转导、蛋白质的转运及细胞代谢等众多事件 .因此 ,NAG7基因很可能是通过介导这些蛋白质的表达下调而发挥其功能     

新基因BRD7对鼻咽癌蛋白质表达谱影响的初步研究

BRD7基因是与鼻咽癌相关的候选抑瘤基因.为了进一步研究该基因的作用机制,将pcDNA3.1(+)/BRD7的表达质粒经脂质体导入HNE1细胞,RNA斑点杂交筛选出阳性克隆,通过双向电泳分离过表达BRD7的HNE1细胞内蛋白质,筛选出差异表达的蛋白质点,并进行质谱分析鉴定.鉴定出10种表达上调的蛋白质点,包括精氨(基)琥珀酸裂解酶,TSA(thio-specific antioxdant), Proteaseome activator28 beta subunit(PA28),金属蛋白酶抑制因子-2前体等,这些蛋白质涉及细胞生长,细胞代谢等很多相关事件.这些结果说明BRD7基因可能通过多种途径影响鼻咽癌的发生发展.     

鼻咽癌相关基因 BRD7 对鼻咽癌细胞CNE1的影响

为了研究BRD7基因对鼻咽癌细胞CNE1的影响,通过脂质体转染方法,将BRD7基因导入NPC细胞株CNE1细胞中.通过细胞生长曲线发现该基因能够抑制CNE1细胞的生长.为了探讨可能的作用机制,进而采用蛋白质组技术研究该基因对鼻咽癌蛋白质表达谱的影响,从而研究该基因在CNE1中的地位和作用.通过对过表达BRD7基因后鼻咽癌细胞系CNE1的蛋白质表达谱改变的研究,鉴定出19个差异表达蛋白,这些蛋白质包括:BCCIP (BRCA2 and CDKN1A(p21(Waf1/Cipl)),FHL2(four and a half LIM domains 2),Chloride channel regulatory protein;Hin-1(high-in-normal-1),WISP-1(connective tissue growth factor related protein),SREC-4(scavenger receptor expressed by endothelial cells-2),folate receptor.这些差异蛋白涉及到基因表达调控、细胞黏附等众多的事件.从另一个侧面研究了BRD7基因与鼻咽癌的关系,扩展了BRD7基因的研究范围,并进一步充实了该基因做为鼻咽癌候选抑瘤基因的证据.     

NAG11和NAG12基因转染对鼻咽癌细胞生长的影响

为了考察鼻咽癌表达下调／缺失基因NAG11和NAG12对鼻咽癌细胞系HNE1生长的影响,构建了NAG11和NAG12基因真核表达载体pcDNA3.1（＋）／NAG11和pcDNA3.1（＋）／NAG12,采用脂质体转染技术将真核重组质粒和空载体质粒分别导入HNE1细胞,观察转染后HNE1细胞生物学特性的变化,结果显示,NAG11重表达对HNE1细胞生长和细胞周期没有明显的影响,而NAG12重表达对HNE1细胞有生长抑制作用,与空载体转染组相比,倍增时间由24.1h延长至31.1h,停滞于G0－G1期细胞数由51.42%增加至68.14%。以上实验进一步说明鼻咽癌是多基因改变的疾病,NAG12的重表达有助于处国咽癌恶性表型的逆转。     

两个鼻咽癌负相关新基因的分离与特性

8个通过 c DNA代表差异分析法 ( c DNA representational difference analysis,c DNA RDA)分离的新 c DNA序列中 ,经 RT- PCR验证 ,发现其中一 c DNA序列 (登录号 :AF0 91 51 7)在 40 %的鼻咽癌活检组织中存在表达缺失和下调 .Northern杂交显示 ,AF0 91 51 7代表转录本为 1 .1 kb和 1 .4kb大小的两个基因 ,进而采用文库筛选 ,成功分离出 3′端完全不同的两个基因 ,命名为 NAG1 1和 NAG 1 2 (登录号分别为 AF 1 70 30 7和 AF 1 94971 ) .经过计算机预测 ,NAG 1 1编码 87个氨基酸组成的跨膜蛋白 ,NAG1 2编码 1 36个氨基酸组成的可溶性的核蛋白 ,两者无任何同源性 .NAG 1 1蛋白含有 3个 ATP结合区、两个蛋白激酶 C磷酸化位点和两个 N-肉豆寇酸化位点 ,NAG 1 2含有POU结构域和多个功能位点 .结果说明 NAG1 1和 NAG1 2的表达的缺失与下调可能参与了鼻咽癌的进程 ;NAG1 1基因产物可能与 ATP的跨膜转运有关 ;NAG1 2基因产物可能与转录翻译有关 .     

Identification of differential proteins in nasopharyngeal carcinoma cells with p53 silence by proteome analysis

Although mutation of p53 tumor-suppressor gene is rare in nasopharyngeal carcinoma (NPC), NPC has a high frequency of overexpression of p53 protein. There seem to be complex mechanisms of inactivation and stabilization of p53 in NPC. To detect proteins associated with the function of p53 in high throughout screening, we succeeded in establishing p53 knockdown human NPC CNE2 cell line (CNE2sip53) using stable RNA interference, and compared the proteomic changes between CNE2sip53 and control cell line CNE2/pSUPER using two-dimensional gel electrophoresis. Twenty-two differentially expressed proteins between the two cell lines were identified by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3sigma, etc.), and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, several differential proteins including HSP27, HSP70, GRP75 and GRP78 were verified as p53 interacting proteins in NPC by immunoprecipitation and Western blot analysis, and the suppression of HSP27 expression by HSP27 antisense oligonucleotides could decrease the p53 protein level. Our data suggest that these differential proteins may be associated with the function of p53 in NPC, and provide new clues to elucidate the mechanisms of inactivation and stabilization of p53 in NPC.     

抑瘤基因NGX6对鼻咽癌细胞株HNE1细胞生长的影响(英文)

鼻咽癌是我国南方多发恶性肿瘤 ,它的发生发展与遗传因素密切相关 .采用定位候选克隆策略在 9p上克隆出一个候选抑瘤基因 ,命名为NGX6 .为了进一步研究它的功能 ,将NGX6基因的全长cDNA片段亚克隆至pcDNA3.1(+ )的表达载体中 ,通过脂质体转染入鼻咽癌细胞系HNE1中 ,Northern杂交方法筛选高效表达NGX6的细胞株 ,并借助细胞生长曲线、软琼脂糖集落形成实验、裸鼠体内接种实验和流式细胞仪对转染细胞的生物学行为进行了检测 .结果显示 ,转染了NGX6基因的HNE1细胞的生长速度明显减慢 ,在软琼脂中集落形成率较对照组显著下降 (P〈0 0 5 ) ,裸鼠体内成瘤的时间较对照组明显延长 ,瘤体的大小和重量较对照组明显减少 ,流式细胞仪检测发现细胞的凋亡率无明显变化 .为了明确NGX6蛋白在细胞中发挥作用的部位 ,进一步将NGX6的开放阅读框架完整正确地克隆到pEGFPC1的荧光载体中 ,转染到COS7细胞中 ,用荧光显微镜观察细胞中荧光的分布 ,发现荧光主要分布在细胞浆中 ,说明NGX6蛋白可能是一种胞浆蛋白 .该研究表明 ,NGX6在NPC的发生发展中起重要作用 ,为全面阐述NGX6的功能提供重要的信息 ,为进一步的功能研究打下基础     

Proteomic detection of changes in protein synthesis induced by NGX6 transfected in human nasopharyngeal carcinoma cells

In the previous study, we cloned a new gene, named NGX6, related to nasopharyngeal carcinoma (NPC) at 9p. To study its function in the pathogenesis of NPC, we have investigated changes in protein synthesis between NPC cell line HNE1 and that transfected with the gene. Using high-resolution two-dimensional electrophoresis, we found that 22 protein spots showed variations that were significant and reproducible. Analysis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searches identified seven proteins that were upregulated and seven proteins that were downregulated. These proteins included Fas, zinc-finger protein (ZNF), RAB, and Ah receptor-interacting protein (AIP). The functional implications of the identified proteins are discussed.     

Identification of the proteins related to p53-mediated radioresponse in nasopharyngeal carcinoma by proteomic analysis

Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), and p53 is closely associated with the radiosensitivity of cancer, but the molecular mechanisms of p53-mediated radioresponse in NPC remains unclear. We previously established NPC CNE2sip53 cell line with p53 knockdown and paired control cell line CNE2/pSUPER, which provides a cell model system to investigate mechanisms of p53-mediated radioresponse in NPC. In this study, we first compared the radiosensitivity of CNE2sip53 and CNE2/pSUPER by a clonogenic survival assay, cell growth assay, and Hoechst 33258 staining and flow cytometry analysis of apoptotic cells. The results showed that the radiosensitivity of CNE2sip53 was significantly lower than that of CNE2/pSUPER, indicating that p53 plays a role in mediating NPC radiosensitivity. To search for the proteins associated with the p53-mediated radioresponse in NPC, a proteomic approach was performed to identify the radioresponsive proteins in CNE2sip53 and CNE2p/SUPER, respectively, and then the difference of radioresponsive proteins in CNE2sip53 and CNE2p/SUPER was compared. As a result, 14 differential radioresponsive proteins were identified in the two cell lines, 4 proteins of which were conformed by Western blot. Among them, 9 and 5 proteins were identified solely from CNE2p/SUPER and CNE2sip53, respectively. Furthermore, protein-protein interaction analysis showed that 7 differential radioresponsive proteins identified only in CNE2p/SUPER were related to p53 protein. Our results suggest that the differential radioresponsive proteins unique to CNE2p/SUPER may be involved in p53-mediated radioresponse in NPC, which will be helpful for elucidating the mechanisms of p53-mediated NPC cellular response to radiotherapy.     

鼻咽癌下调新基因NOR1相互作用蛋白的筛选和鉴定

NOR1基因是新的鼻咽癌相关基因,该基因在鼻咽癌细胞系HNE1和鼻咽癌组织中表达下调．在鼻咽癌细胞HNE1中恢复NOR1基因表达抑制了鼻咽癌细胞的生长和增殖能力．为了探讨NOR1基因的生物学功能,以NOR1基因为诱饵运用酵母双杂交技术在人胎脑文库中筛选其交互作用蛋白,挑选阳性克隆,进行DNA序列分析和同源检索,阳性克隆编码7个不同的蛋白质,其中一个阳性克隆编码线粒体ATP合成酶亚基OSCP蛋白．瞬时转染pCMV-myc-NOR1质粒进入鼻咽癌5-8F细胞,通过密度梯度离心法分离线粒体蛋白,Western blot检测表明myc-NOR1蛋白分布于线粒体与胞浆．免疫荧光检测表明在鼻咽正常上皮细胞NP69中内源性NOR1蛋白与线粒体存在明显共定位．随后采用特异性酵母双杂交、免疫荧光共定位、免疫共沉淀技术证实了NOR1与OSCP在线粒体内存在交互作用．提示,NOR1是一个新的线粒体蛋白,可能通过结合OSCP蛋白调控细胞能量代谢,为深入探讨其功能提供了重要线索．     

NGX6基因转染对鼻咽癌细胞基因表达谱的影响

鼻咽癌是我国南方的常见肿瘤 .NGX6是新近克隆的定位于 9p2 1 2 2 ,在鼻咽癌组织中表达下调的基因 .初步的研究结果显示 ,NGX6基因转染鼻咽癌细胞HNE1能够延缓其生长速度 ,使肿瘤细胞更多地停留在G0 G1期 .为探索NGX6基因在鼻咽癌发病机制中的作用 ,建立了高表达NGX6的鼻咽癌细胞系 .利用包含 14 0 0 0个基因的cDNA微阵列分析了NGX6基因转染对HNE1细胞基因表达谱的影响 ,发现NGX6基因的转染能够上调p2 9、APC7、NEU1、RNasek6、αE catenin和TFⅡEα等基因的表达 ;同时也下调properdinP因子、G0S2、BAZ2B、ZHX1,OS4和PBX3等基因的表达 .研究结果提示 ,NGX6基因对鼻咽癌细胞的生物学行为的影响可能与它对一些细胞周期调控因子和转录调控因子的影响相关 .作为一种高通量的分析技术 ,cDNA微阵列为新基因的功能研究提供了重要的线索     

Proteomic features of potential tumor suppressor NESG1 in nasopharyngeal carcinoma

We previously defined the recently revised NESG1 gene as a potential tumor suppressor in nasopharyngeal carcinoma (NPC). Here, we further used proteomics technology to globally examine NESG1‐controlled proteins in NPC cells. Twenty‐six proteins were found to be deregulated by NESG1 using proteomics analysis while enolase 1 (alpha) (ENO1), heat shock protein 90 kDa beta (Grp94), member 1 (HSP90B1), and cathepsin D (CTSD) proteins were differentially expressed by Western blot. Interestingly, a‐enolase (ENO1), an overexpressed gene in NPC, was confirmed as a NESG1‐regulated protein in NPC cells. Overexpressed ENO1 not only restored cell proliferation and cell‐cycle progression, but also antagonized the regulation of NESG1 to cell‐cycle regulators p21 and CCNA1 expression as well as induced the expression of C‐Myc, pRB, and E2F1 in NESG1‐ovexpressed NPC cells. Real‐time PCR and immunohistochemistry analysis showed that NESG1 expression is negatively correlated with ENO1 expression in NPC tissues. Our observations suggest that ENO1 downregulation plays an important role in NESG1‐induced growth inhibition of NPC cancer cells.     

定位于染色体9p21-22的一个新基因UBAP1的克隆测序及在鼻咽癌中的表达分析

 在染色体 9p2 1 2 2鼻咽癌杂合性丢失 (lossofheterozygosity,LOH)高频区 ,应用EST介导的定位 侯选克隆策略 ,用RT PCR及Northern杂交检测了 2 2个表达序列标记 (expressedsequencetag ,EST)在鼻咽癌细胞株HNE1和原代培养的正常鼻咽上皮细胞中的表达差异 ,并对其中一个在鼻咽癌细胞株HNE1中表达下调的EST检测了在鼻咽癌活检组织中的表达 .用生物信息学方法获得其全长cDNA序列 ,GenBank登录号AF2 2 2 0 4 3.该基因cDNA全长 2 70 1bp ,其开放阅读框 (openreadingframe ,ORF)编码一个含 50 2个氨基酸、分子量为 55kD的碱性蛋白质 ,在蛋白羧基端含有 2个连续的重要UBA功能域 (ubiquitinassociateddomain) ,属于遍在蛋白相关蛋白家族的一个新成员 ,经国际人类基因命名委员会同意 ,将其命名为UBAP1 (ubiquitinassociatedprotein 1 ) .Northern表达分析显示UBAP1在所检测的人组织中广泛表达 ,但在人的心脏、骨骼肌及肝脏中的表达较强 .UBAP1基因在63 2 % ( 1 2 1 9)的鼻咽癌活检组织中表达下调 .UBAP1基因作为一个遍在蛋白相关蛋白家族的新成员 ,结合其在 9p的重要定位信息 ,有必要进一步研究其表达下调参与鼻咽癌发生发展的可能机制 .     

NGX6 gene inhibits cell proliferation and plays a negative role in EGFR pathway in nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is a common cancer in South China but is rare in other parts of the world. A novel NPC-related gene was isolated by location candidate cloning strategy, whose expression was down-regulated in NPC. This gene was designated human NGX6 (Genbank accession AF188239) and encoded a predicted protein of 338 amino acids that harbors an EGF-like domain. The effects of NGX6 on cells from human NPC cell line HNE1 were investigated. The cells transfected with NGX6 had a markedly high expression of NGX6, leading to significant decrease in cell proliferation and the capability to form colonies in soft agar, delaying the G0-G1 cell cycle progression. Flow cytometry assay indicated that the expression of cyclin D1 significantly decreased in NGX6-transfected HNE1 cells as well as cyclin A and E. There was a delay in tumor formation and a dramatic reduction in tumor size when cells transfected with NGX6 were injected into nude mice. In another way, we found NGX6 played a negative role in EGFR Ras/Mek/MAPK pathway. We propose that NGX6, as an EGF-like domain gene, could delay cell cycle G0-G1 progression and thus inhibit cell proliferation by negatively regulating EGFR pathway in NPC cells and down-regulating the expression of cyclin D1 and E.     

p53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1.

The Drosophila seven in absentia (sina) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/Raf pathway and targets other proteins for degradation via associations with a ubiquitin-conjugating enzyme. Recently, a mammalian sina homologue was reported to be a p53-inducible gene in a myeloid leukemia cell line. To explore the function of human SINA-homologous (Siah) proteins, expression plasmids encoding Siah-1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis. We discovered that BAG-1, a ubiquitin-like Hsp70/Hsc70-regulating protein, is a negative regulator of Siah-1A. Siah-1A was identified as a BAG-1-binding protein via yeast two-hybrid methods. Specific interaction of BAG-1 with Siah-1A was also demonstrated by in vitro binding experiments using glutathione S-transferase fusion proteins and co-immunoprecipitation studies. Siah-1A-induced growth arrest in 293 and GM701 cells was abolished by co-transfection of wild-type BAG-1 with Siah-1A but not by a C-terminal deletion mutant of BAG-1 that fails to bind Siah-1A. Over-expression of BAG-1 significantly inhibited p53-induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids. BAG-1 also prevented growth arrest following UV-irradiation-induced genotoxic injury without interfering with accumulation of p53 protein or p21(waf-1) expression. BAG-1 functions downstream of p53-induced gene expression to inhibit p53-mediated suppression of cell growth, presumably by suppressing the actions of Siah-1A. We suggest that Siah-1A may be an important mediator of p53-dependent cell-cycle arrest and demonstrate that Siah-1A is directly inhibited by BAG-1.     

转染人乳头瘤病毒16型E6E7重组基因及感染模型细胞建立

人乳头瘤病毒(human papillomavirus,HPV)是性传播疾病的主要病原之一,高危型人乳头瘤病毒感染和宫颈癌的关系已被肯定[1],在约50%~90%的宫颈癌组织中可检出HPV16 DNA。为了解与HPV相关疾病的的分子生物学致病基础,尚待深入研究其生物特性和病理特征,方能逐步解决临床简便易行的试验诊断方法和抗HPV感染的治疗问题。目前已经证实HPV16 E6/E7基因是转化基因,它们编码的蛋白可分别使抑癌蛋白P53和PRb失活,在宫颈癌的发生中起着重要作用[2],被认为是宫颈癌的始动因子。本文选择HPV16 E6E7作为研究对象,利用基因重组技术构建EGFP-…