【作者】 黄琛；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 【NAG7基因的克隆、生物信息学特征及前期研究结果】NAG7基因是我室谢奕博士采用定位候选克隆策略结合生物信息学方法，从最小共同缺失区3p25.3-26.3中成功克隆的与鼻咽癌相关基因，Genbank接受号为AF086709。生物信息学显示，NAG7基因cDNA全长1677bp，编码94个氨基酸的碱性蛋白质，分子量为11023.87道尔顿，预测为一跨膜蛋白质，其N端位于膜内，跨膜区为33-52位氨基酸，含有1个蛋白激酶C(PKC)磷酸化位点和1个肉豆蔻基(Myristyl)化位点。NAG7基因前期功能研究结果表明，NAG7基因在鼻咽癌活检组织和鼻咽癌上皮HNE1细胞系中表达下调甚至缺失，而在正常鼻咽组织中存在明确的表达；NAG7基因编码产物为一胞浆蛋白，NAG7基因在鼻咽癌细胞中的过表达，可导致鼻咽癌细胞HNE1蛋白质表达谱和基因表达谱改变，抑制HNE1细胞和裸鼠皮下移植瘤生长，细胞周期素表达下调，G0／G1期细胞增加，S期细胞减少，凋亡细胞增加。【NAG7基因在恶性肿瘤细胞系中的表达】利用RT-PCR技术分析NAG7基因在七种恶性肿瘤细胞系中的表达，发现NAG7基因在HN1、CNE1、6-10B、SW480、Bel-7402和U251等六种恶性肿瘤细胞系中表达减少，而在5-8F细胞系中呈现高表达。为了明确NAG7基因在恶性肿瘤细胞系中的表达减少是否与其编码区序列的突变相关，进一步从这几种细胞系的基因组DNA中扩增NAG7基因的编码区序列并进行测序分析。结果显示HNE1、CNE1、6-10B、SW480、Bel-7402、U251和5-8F等七种细胞基因组DNA中均可扩增出NAG7基因的编码区序列，测序分析表明HNE1、CNE1、6-10B、SW480、Bel-7402和U251细胞中扩增出来的NAG7基因均在第30位氨基酸的第二个密码子发生了终止突变(NAG7基因编码序列的第86位，T→G)，而5-8F细胞中扩增出来的NAG7基因为正确的编码区序列。因此，NAG7基因在这六种恶性肿瘤细胞系中的表达下调可能为基因突变所致。NAG7基因能明显抑制鼻咽癌HNE1细胞体内、外生长，并且在不同侵袭转移潜能的肿瘤细胞系中存在表达差异(HNE1、CNE1、6-10B、SW480、Bel-7402和U251等六种低／无侵袭转移潜能恶性肿瘤细胞系表达减少，高侵袭转移潜能的5-8F细胞系高表达)和序列突变(无／低侵袭转移潜能细胞系中氨基酸第29位终止突变)。NAG7在不同侵袭转移潜能的恶性肿瘤细胞系，特别是在5-8F和6-10B细胞系中存在差异表达，因此我们提出假设：NAG7基因是否参与了鼻咽癌细胞的侵袭转移?【NAG7基因促进鼻咽癌细胞侵袭转移的表型研究】根据NAG7基因的表达水平和细胞系的侵袭转移潜能，选择3种细胞系作为研究对象：高侵袭转移能力的5-8F、低侵袭转移能力的HNE1和无侵袭转移能力的6-10B。由于NAG7基因序列中未能预测到合适的RNAi序列，不能对5-8F细胞达到有效的干扰效果，因此构建NAG7真核表达载体pEGFP-C2／NAG7，考察NAG7基因在6-10B细胞和HNE1细胞中的过表达对鼻咽癌细胞运动侵袭表型的影响。研究发现，NAG7基因能够增加鼻咽癌细胞HNE1和6-10B与细胞外基质的粘附，增加HNE1和6-10B细胞与内皮细胞粘附，及增强HNE1细胞的侵袭能力和运动迁移能力，但对6-10B细胞作用甚微。NAG7基因不能恢复HNE1和6-10B细胞的细胞间隙连接通讯能力。此外，NAG7基因还可以提高人脐静脉内皮细胞血管生成能力。小鼠肾包膜下移植瘤实验显示，虽未发现小鼠体内出现明显肿瘤转移灶，但NAG7基因转染的HNE1细胞明显向肾内侵袭，范围和深度均显著大于HNE1细胞，NAG7基因能明显促进HNE1细胞向肾内的侵袭。NAG7基因能够明显促进鼻咽癌HNE1细胞的运动侵袭潜能，但是对6-10B细胞作用甚微，说明NAG7基因并不具备启动肿瘤细胞运动侵袭的作用，而是能促进肿瘤细胞的运动侵袭潜能。【NAG7基因通过雌激素受体促进鼻咽癌HNE1细胞运动侵袭的分子机制】利用人肿瘤相关寡核苷酸芯片筛选NAG7基因调控鼻咽癌HNE1细胞的基因差异表达谱，根据芯片位点杂交的信号强度和t检验结合两倍差异的标准，从2747个基因中筛选出差异表达基因共101个，其中上调34个，下调67个基因。选择CAV1和MMP1基因，qRT-PCR验证其在NAG7／HNE1和HNE1细胞中的表达，并与基因芯片结果进行比较，发现表达量的改变与芯片结果较一致，说明芯片结果较准确地反映了基因表达的变化。结合基因功能分类体系Gene Ontology(GO)，对差异表达基因进行相关功能的分类，发现基因主要参与：①细胞周期调控，②细胞骨架，③细胞代谢，④酶的催化反应，⑤信号转导调控，⑥外界刺激反应，⑦细胞定位和基质粘附。KEGG PATHWAY数据库分析，发现42个基因参与54个信号通路，其中9个基因参与MAPK通路，6个基因参与细胞因子和细胞因子受体的相互作用。BIOCARTAPATHWAY数据库检索，发现共有31个基因参与78个信号通路。利用Pubmatrix在线工具，将差异表达基因为检索项，与肿瘤运动侵袭相关词为关键词，检索PubMed文献中abstract部分差异表达基因与关键词并存的文献。发现大多数基因都与这些关键词并存，如40个基因与关键词invasion相关，50个基因与metastasis相关，提示这些基因可能参与了肿瘤的运动侵袭和转移，对随后的基因功能和机制探讨有很好的指导意义。ERα是与NAG7蛋白具有直接交互作用的蛋白，提示其在功能和表达模式上与NAG7表达可能具有一定的相关性。ERα在肿瘤的无限增殖和侵袭转移中具有双重作用，能够明显促进肿瘤细胞的增殖，抑制肿瘤细胞的体外侵袭和体内转移。本实验采用免疫组化技术检测ERα蛋白在鼻咽癌、鼻咽癌旁上皮和鼻咽炎性上皮中的表达，统计分析发现ERα蛋白在鼻咽癌、鼻咽癌旁上皮和鼻咽炎性上皮的表达没有明显差异，ERα蛋白的表达与鼻咽癌患者性别和临床分期无关，而无转移性鼻咽癌中ERα蛋白强阳性表达率明显高于转移性鼻咽癌，提示ERα蛋白在鼻咽癌增殖过程中高表达，而转移进程中表达下降。NAG7蛋白与ERα有直接交互抑制作用，能抑制雌激素刺激下的雌激素受体转录调控作用，使雌激素调控的雌激素反应性基因的表达降低。Western blot和细胞免疫组化检测发现，NAG7基因能够明显下调HNE1细胞中ERα的表达，10 nM的17β-雌二醇刺激时，HNE1细胞和NAG7／HNE1细胞的ERα表达微弱增加。不同浓度的雌激素刺激，对NAG7／HNE1和HNE1细胞的细胞外基质、内皮细胞粘附和运动迁移能力没有影响，可以降低人脐静脉内皮细胞ECV304血管生成的能力，但对NAG7／ECV304组没有影响。NAG7基因抑制鼻咽癌细胞增殖和促进鼻咽癌细胞运动侵袭可能是通过ERα调控双重作用而现实的，但与雌激素刺激无关。芯片结果生物信息学分析得知，有9个差异表达的基因参与了MAPK信号转导通路，于是重点考察了MAPK通路相关分子以及雌激素刺激下这些分子的改变。Western-blot检测发现，NAG7基因在HNE1细胞中的过表达可以明显增加JNK2、c-Jun表达，下调c-Fos，而对ERK，p-ERK、P53和NF-κB的表达没有影响。不同浓度的雌激素刺激，对这些蛋白的表达均没有影响。NAG7基因能上调MAPK通路的上游Ras／Raf分子H-ras和p-c-Raf蛋白的表达，但对K-ras、N-ras和c-Raf总蛋白没有影响，提示NAG7基因通过激活H-ras／p-c-Raf调控JNK／AP-1通路，增加AP-1的启动子活性。NAG7基因在HNE1细胞中的过表达还可以上调AKT／p70s6K通路、增加MMP1和Ezrin蛋白的表达。综上所述，NAG7基因可以通过抑制HNE1细胞中ERα表达，激活H-ras／p-c-Raf、JNK／AP-1和AKT／p70s6K等信号转导通路调控CAV1、MMP1和Ezrin的表达，促进HNE1细胞的运动侵袭潜能。NAG7基因作为我室自主克隆的鼻咽癌相关基因，深入研究该基因的双重功能，将对探讨基因功能的复杂性，及鼻咽癌细胞的发生发展机理提供极具优势的理论及实验依据。更多还原

【Abstract】 【Background of NAG7】By using positional candidate cloning strategy, Dr. Yi Xie isolated anovel nasopharyngeal carcinoma (NPC)-related gene located in3p25.3-26.3, named NAG7. Its GenBank accession number wasAF086709.Bioinformatics revealed that the full-length of NAG7 cDNA is 1677bp, which encodes a protein of 94 amino acids with a predicatedmolecular weight of 11 kDa. NAG7 protein contains a transmemberaneregion which N-terminal side is inside membrane, a Protein kinase Cphsophorylation site and an N-myristoylation site.Previous studies showed that NAG7 was expressed in very low orundetectable level in NPC biopsies and NPC cell line HNE1, whileexpressed in high level in normal nasopharyngeal epithelial tissues.Subcellular localization of NAG7 protein is mostly in cytoplasm by usinga GFP-NAG7 fusion protein in HNE1 cells. NAG7 re-expression inducedthe changes of protein expression profile and gene expression profile,reduced the proliferation rate of HNE1 cells, and inhibited growth of xenografts after the transfected cell was injected into nude mice in vivo.It also down-regulated the expression of cyclins, increased the G0/G1phase cells arrest and decreased the S phase cells, and induced theapoptotic cells increase.【Expression of NAG7 in malignant cell lines】We found that the expression of NAG7 is very low in six malignantcell lines (HNE1, CNE1, 6-10B, SW480, Bel7402 and U251) by RT-PCRassay, but is high in 5-8F cell line. Sequencing of PCR products generatedfrom gDNA of HNE1, CNE1, 6-10B, SW480, Bel7402 and U251 celllines showed that they all contained point mutation at the same position(ORF of NAG7: T89G), which is a termination codon mutation.Sequence of PCR products generated from gDNA of 5-8F is precise. Thedownexpression of NAG7 in these six cell lines may be caused by genemutation.NAG7 gene suppresses the proliferation of HNE1 cells in vitro andin vivo obviously, and has the differential expression and point mutationin the various potential of invasion and metastasis of cell lines, whichdecreased expression and point mutation in low/none potential cells andincreased expression and right sequence in high potential cell. For NAG7expression varys with different invasive potential tumor cells, especiallyin 5-8F and 6-10B cells, so we propose the hypothesis: does NAG7 participate in the invasion and metastasis of NPC cells besides thesuppressive function on cell growth?【NAG7 promotes migration and invasion of NPC】In order to investigate the effect of NAG7 gene on invasion andmetastasis of NPC, we planned to use three cell lines with differentinvasion and metastasis potential: high potential cell line 5-8F, lowpotential cell line HNE1 and none potential cell line 6-10B. As a result ofabsent appropriate RNAi sequences and lacking of effective RNAi effectin 5-8F cells, NAG7 gene was introduced into HNE1 and 6-10B cells byliposome transfection and the stable cell lines pEGFP-C2/NAG7-HNE1and pEGFP-C2/NAG7-6-10B were established. Our data demonstrate thatNAG7 significantly enhances cell-matrix adhesion, cell-endothelial celladhesion, cell invasion and cell migration in HNE1 cells, but has triflingeffect on 6-10B cells. NAG7 could not restore the Gap JunctionalIntercellar Communication (GJIC) ability of HNE1 and 6-10B cells.Moreover, NAG7 also promote angiogenesis of human umbilical veinendothelial cells (HUVEC). The metastasis tumor of the xenograft ofNAG7/HNE1 and HNE1 cells implanted to the subrenal capsule ofBABL/c mice are undiscovered, while the invading depth and area ofNAG7/HNE1 cell in mice’s kidney are lager than that of HNE1 cells.NAG7 gene can promote the migration and invasion potential of HNE1 cells rather than 6-10B cells, which means NAG7 do not have the abilityto launch the migration and invasion potential of cancer, but only boostthe potential.【Molecular mechanism on NAG7 promote migration and invasionof NPC cells by estrogen receptor】The different gene expression profile of NAG7 regulated HNE1cells were tested using the human cancer oligonucleotide array. Amongthe total 2747 tested genes, the expression of 34 genes were up-regulatedand 67 genes were down-regulated in NAG7/HNE1 cells as comparedwith HNE1 cells. The expression of CAV1 and MMP1 gene inNAG7/HNE1 and HNE1 cells determined by qRT-PCR were consistentwith the expression in array. The result of array accurately reflects thechanges of genes.The main groups of different expression gene functionalclassification based on Gene Ontology as follows:①Regulate cell cycle,②Cellular framework,③Cellular metabolism,④Catalytic activity,⑤Signal transducer,⑥Response to stimulus,⑦Cellular localization andCell adhesion. The pathway analysis of different expression genes usedKEGG and BIOCARTA database. There are 42 genes participated in 54KEGG pathways, especially 9 genes in MAPK signaling pathway and 6genes in cytokine-cytokine receptor interaction, and 31 genes in 78 BIOCARTA pathways. The relativity of different expression genes andthe key word of migration and invasion of cancer were found byPubmatrix, which searches the abstract of articles in PubMed. The resultshowed a great significant association of the different expression genesand invasion and metastasis of cancer.ERαprotein interacts with NAG7, and has paradoxical actions asboth anti-invasive and proliferative actions in human cancer. Proteinexpression of ERαin the tissue microarray of nasopharyngeal tissues wasscreened by Immunohistochemistry (ICH). It was found that theexpression of ERαin the NPC, adjacent-cancer and benign disease fromthe nasopharynx was similar, and was no relation of gender of patientsand clinical stages of NPC. The strong positive expression ratio of ERαinnon-metastasis NPC group was obviously higher than that in metastasis.It was suggested that the high expression of ERαmight take part in NPCcell proliferation, and the expression of ERαdecreased in the metastasisprocess of NPC.NAG7 protein interacts with ERα, and decreases17beta-estrodial-induced activation of ERαtranscriptional activity inmammalian cells. NAG7 down-regulated the expression of ERαin HNE1,and the faint increase expression of ERαin NAG7/HNE1 and HNE1 cellstreated by 10nM 17beta-estrodial detected by Western blot andimmunohistochemistry. There is no change of cell-matrix adhesion, cell-endothelial cell adhesion and cell migration in NAG7/HNE1 andHNE1 cells treated by 17beta-estrodial. We suggest that the dualfunctions of NAG7, anti-proliferation and promote invasion, actualizedby interact with ERα.In this study, we showed that NAG7 up-regulates JNK2 and c-Junexpression, inhibits c-Fos expression, and invalidates ERK, p-ERK, P53and NF-κB. We also found that 17beta-estrodial treat the cells does notchange the protein expression. NAG7 increases H-ras expression andactivate p-c-Raf, while does not regulate K-ras、N-ras and c-Raf. NAG7regulates H-ras/p-c-Raf and JNK/AP-1 signaling pathway, and increasesthe promoter activity of AP-1. Moreover, NAG7 activates AKT/p70s6Ksignaling pathway and increases MMP1 and Ezrin expression.In a word, NAG7 inhibits ERα, activates H-ras/p-c-Raf, JNK/AP-1and AKT/p70s6K signaling pathway, and upregulates CAV1, MMP1,Ezrin expression, then promotes the migration and invasion of HNE1cells.更多还原