摘要
近年来细胞和分子遗传学研究发现：鼻咽癌基因组存在高频率3p、9p、11q、13q和14q染色体的等位基因的杂合性丢失。我室阳剑波博士采用定位候选克隆的方法从9p区域克隆了一个新基因NGX6,Genbank登录号为AF188239,它的表达在鼻咽癌中明显下调,可能为一个与鼻咽癌相关的肿瘤抑制候选基因。
生物信息学显示：NGX6基因cDNA全长2134bp,编码338aa,预测分子量37kDa,含有2个跨膜结构域,胞外区含有1个表皮生长因子样结构域(EGF-like domain)和3个糖基化位点,胞内区较短,含有1个酪氨酸激酶磷酸化位点。
NGX6基因前期功能研究结果表明：NGX6在鼻咽癌活检组织和鼻咽癌上皮细胞株中表达下调甚至缺失,而在正常鼻咽组织中存在高表达；NGX6基因在结直肠癌中亦存在不同程度的表达下调,伴有淋巴结转移的结直肠癌样本中NGX6表达下调的比例(15／16)远高于无淋巴结转移的结直肠癌样本(25／34)(P<0.05)。利用高通量的组织芯片进行原位杂交实验发现,NGX6基因的表达在有转移的肿瘤组织下调更明显,且与临床分期相关。NGX6基因分别转染鼻咽癌和结肠癌细胞之后可使鼻咽癌和结肠癌细胞的恶性表型得到部分逆转；可通过Ras/Raf/MAPK途径负调控EGFR增殖信号通路；免疫共沉淀实验发现NGX6可与作为细胞运动和转移调节者的Ezrin发生交互作用,CYTO是NGX6和Ezrin发生交互作用发生交互作用的关键结构域。NGX6高表达使5-8F细胞体外侵袭能力减弱,细胞黏附率增加,恢复间隙连接细胞间通讯；成功建立了scid鼠体内自发转移模型,NGX6高表达能明显抑制鼻咽癌5-8F细胞在scid鼠体内的成瘤和肺转移灶形成。EGF和CYTO结构域是NGX6调节细胞黏附的重要功能域,缺失该区域的突变体细胞黏附能力较NGX6转染组降低；CYTO是调节细胞运动,迁移,细胞生长增殖的关键功能域。EGF能诱导鼻咽癌5-8F细胞发生运动和迁移,并发生一系列的分子水平事件如EGFR, PI3K, RhoA表达上调,RhoA由胞浆向胞膜移位,侵袭能力增强,细胞通讯能力减弱。NGX6能部分抑制EGF诱导的鼻咽癌5-8F细胞发生运动和迁移,侵袭能力减弱,而且使EGFR, PI3K, RhoA表达维持诱导前水平,RhoA由胞浆向胞膜移位减少,细胞通讯能力增强。
综上所述,NGX6的生物信息学特征和初步研究结果提示NGX6基因可能在鼻咽癌的侵袭与转移中发挥抑制性的作用。
抗体是基因功能研究重要的工具。为了制备NGX6多克隆抗体,采用pET原核表达系统表达并纯化NGX6△TM2蛋白(第二跨膜区缺失)。将重组蛋白免疫新西兰兔,制备获得抗NGX6蛋白多克隆抗体。该抗体特异性好,效价高,可应用于Western blot (1:3000)、免疫组化(1：1000)以及免疫荧光(1：500)检测。应用该抗体研究以及数据库比对发现,NGX6蛋白存在两个蛋白亚型a和b,分别来自3个不同的转录本。其NCBI参考序列号分别为：NM_001042589(转录本1),NM_001042590(转录本2),NM_016446(转录本3)。其中,转录本1和2的ORF框一致,即编码完全一样的a蛋白亚型(NGX6a)。转录本3即NM 016446,也就是我们前期研究的NGX6基因序列,编码b蛋白亚型(NGX6b)。a蛋白亚型(NGX6a)是新发现的蛋白亚型,我们从人胎脑文库中克隆出了NGX6a的ORF序列。Western Blot和免疫组织化学检测显示：NGX6a普遍表达与多种组织和器官中,在神经系统、鼻咽、脂肪、膀胱表达很高,主要定位于上皮组织和神经元细胞；而NGX6b主要表达于神经系统中,心,肾,鼻咽,肺,脂肪组织有很弱的表达。NGX6a在正常组织中的表达远高于NGX6b,是占主要地位的蛋白亚型。生物信息学预测NGX6a蛋白全长476个氨基酸,和NGX6b蛋白(338AA)高度同源,两者之间前272个氨基酸完全一致。a蛋白亚型和b蛋白亚型一样具有一个共同的表皮生长因子样结构域(EGF-like domain)。不一样的是a蛋白亚型具有7个跨膜区。通过Western blot检测NGX6a在各种鼻咽癌细胞株中表达下调,免疫组织化学检测含有308例鼻咽癌样品的鼻咽癌组织芯片,NGX6a在鼻咽癌中表达下调,并且和转移相关。
将NGX6a转染入鼻咽癌细胞5-8F、6-10B、HNE1,构建稳定转染细胞系。在这些细胞株中NGX6a的mRNA水平上调20倍以上；而在蛋白水平,利用Western Blot检测不出有目的蛋白表达。在使用蛋白酶体抑制剂MG-132处理细胞后,在蛋白水平可以检测出NGX6a蛋白的表达,表达水平随着MG-132浓度的增高而增高。同时,利用免疫荧光技术检测到,在用10微摩尔以上浓度的MG-132处理细胞24小时以后可以看到细胞中有荧光出现,强度随MG-132浓度的增高而增高。将pCMV-myc-NGXa转染进5-8F细胞,瞬时表达NGXa蛋白,利用偶联了myc单克隆抗体的琼脂糖珠将NGXa蛋白免疫沉淀下来,用泛素抗体检测,却检测不到任何信号。结果表明：NGXa蛋白在鼻咽癌细胞内发生了降解,这种降解途径是不依赖于泛素化的,经蛋白酶体的降解。
利用免疫荧光、免疫共沉淀证实了NGX6a和Ezrin具有交互作用。Ezrin在鼻咽癌细胞株中表达明显高于正常鼻咽上皮细胞株和鼻咽上皮组织,和NGX6a的表达相反。在鼻咽癌组织芯片中,免疫组织化学证明了Ezrin在鼻咽癌活检组织中表达上调,并且在有淋巴结转移的鼻咽癌组织中Ezrin表达水平高于没有淋巴结转移的癌组织,这种分布模式和NGX6a相反。同样的,Ezrin在正常人组织表达分布情况也和NGX6a相反。比如：在神经系统,NGX6a表达于神经元细胞,而Ezrin表达在一些胶质细胞中,在神经元中不表达。在小脑的切片中,Ezrin表达于小脑分子层浅层的星形细胞及其神经纤维,在分子层深层的神经纤维和蒲肯野细胞层没有表达；而NGX6a表达于分子层深层的神经纤维和蒲肯野细胞内。利用过表达和RNAi技术证明了Ezrin介导了NGX6a在细胞中的降解。通过构建NGX6a一系列突变体,证实了七次跨膜结构域是NGX6a被Ezrin降解的关键部位。
划痕实验和基质胶侵袭实验证实了,NGX6a可以明显抑制鼻咽癌细胞5-8F的伤口愈合和侵袭运动能力。用RNAi技术抑制Ezrin表达后,鼻咽癌5-8F细胞的侵袭能力下降；而抑制Ezrin表达的同时进一步提高NGX6a的表达水平可以进一步抑制鼻咽癌5-8F细胞的侵袭能力,和单独抑制Ezrin表达相比具有统计学差异。使用SP1抑制剂光辉霉素处理鼻咽癌细胞5-8F,可以使转录因子SP1表达下调,并且下调Ezrin的表达,上调NGX6a的表达水平,从而抑制了5-8F细胞的侵袭转移能力。在使用光辉霉素抑制Ezrin表达的同时,加用蛋白酶体抑制剂MG132进一步抑制NGX6a的降解,可以明显抑制鼻咽细胞5-8F的侵袭转移能力,和分别单用两种药物相比具有统计学差异。这表明,NGX6a位于Ezrin的下游,被Ezrin降解,是Ezrin促进肿瘤转移的关键因素。
综上所述,本研究通过制备NGX6的抗体,检测NGX6蛋白在组织中的分布情况,发现了NGX6a这个新的蛋白亚型,NGX6a主要分布在上皮组织和神经元细胞中。通过大样本鼻咽癌标本检测,NGX6a在鼻咽中表达下调并和转移相关。NGX6a在鼻咽细胞中发生降解；Ezrin在鼻咽癌组织中表达上调,有淋巴结转移的上调更为明显；Ezrin在组织内的分布情况和NGX6a相反；Ezrin在细胞内和NGX6a发生交互作用并且介导了NGX6a的降解；NGX6a七次跨膜结构域是其发生降解的关键部位；降低鼻咽癌细胞中Ezrin的表达水平,提高NGX6a的表达水平可以明显抑制鼻咽癌细胞的侵袭转移能力。
Recently, nonrandom loss of heterozygosity at 3p,9p, 11q,13q and 14q in NPC has been detected by comparative genomic hybridization (CGH) and genome-wide analysis with microsatellite allelotyping. By using positional candidate cloning strategy, Dr. Jian-Bo Yang isolated a novel NPC-related gene which was down-regulated in NPC. This gene was designated human nasopharyngeal carcinoma associated gene6 (NGX6) (GenBank accession number:AF188239).
Bioinformatics reveals that the full-length of NGX6 cDNA is 2134 bp, which encodes a protein of 338 amino acids with a predicated molecular weight of 37 kDa. NGX6 protein contains two transmemberane regions, an EGF-like domain and three potential N-glycosylation sites in the extracellular domain. The short cytoplasmic region contains a tyrosine residue that is a potential phsophorylation site of tyrosine kinase.
Previous studies showed that NGX6 was expressed in high level in normal nasopharyngeal epithelial tissues, while expressed in very low or undetectable level in nasopharyngeal carcinoma biopsies and cell lines. It was also found that NGX6 was down-regulated in colorectal carcinomas, and that the down-regulation rate of NGX6 in colorectal carcinoma tissues with lymph-node or distance metastasis (15/16) was significantly higher than that without metastasis (25/34) (P<0.05). Transfection of NGX6 into nasopharyngeal and colorectal carcinoma cells could induce the reversion of some malignant phenotypies, and down-regulate the expression of key molecules of the EGFR pathway. It was found that NGX6 could interact with Ezrin by immunoprecipitation assay in COS7 cells. NGX6 could decrease the invasive ability, increase the adhesive rate and improve communication ability of 5-8F cells. There was a delay in tumor formation and metastasis in lung when cells with NGX6 overexpression were injected into scid mice. The EGF-like domain and CYTO region are important to modulate cell adhesion. The role of△EGF or△CYTO to increase the adhesion ability decreased compared with NGX6, indicating that EGF and CYTO were involved in the modulation of the adhesion ability of 5-8F cells; the CYTO domain of NGX6 also is critical for in the modulation of cell invasion, migration, proliferation and growth. Epidermal growth factor could induce the invasion and mobility of 5-8F cells. The expression of EGFR, PI3K and RhoA were increased as the concentration and duration time of EGF induction increased, and RhoA was translocated from cytosol to membrane. While NGX6 could partly inhibit the EGF-induced invasion and migration of 5-8F cells by EGFR-PI3K-IKK-NF-κB pathway.
These previous studies illuminated that NGX6 gene may be a suppressor in the invasion and migration of NPC.
To better understand the cellular role of the NGX6 gene, in this study, His-NGX6△TM2 recombinant protein were expressed in E.coli and Polyclonal anti-NGX6 antibody was produced by immunizing New Zealand white rabbits with the purified His-NGX6△TM2 recombinant protein. The specificity of the antibody was identified by Western blot and immunofluorescence. With the prepared NGX6 antibody, we detected the distribution of NGX6 in the human fetus. The result of western blot showed the protein of NGX6 had two type of isoforms, isoform a (NGX6a) and isoform b (NGX6b). Searched for the NCBI nucleinic acid and protein database, there are three mRNA transcript variants of NGX6. Transcript variant 1 (NM_001042590) and variant 2 (NM_001042589) encode the same NGX6 protein isoform a (NP_001036055 or NP_001036054) which is composed with 472 amino acids with a calculated molecule weight of 52 kDa, and transcript variant 3(NM_016446)encodes isoform b. The isoform a is composed with 472 amino acids with a calculated molecule weight of 52 kDa, while the isoform b is composed with 338 amino acids with a calculated molecule weight of 37 kDa. It is predicated that there is an EGF (epidermal growth factor) domain in N-terminal of both a and b isoforms, and seven transmembrane domains in NGX6a, but only two transmembrane domains in NGX6b. The expression level of NGX6a was higher than NGX6b in human fetus tissues. Obvious high expression of NGX6a protein presents in human fetus nervous system and epithelial tissues, but the NGX6b protein (37 kDa) is mainly expressed in the nervous system. We further analyzed tissue microarray (TMA) contained 308 nasopharyngeal carcinoma biopsies and 140 non-NPC biopsies and found that NGX6a was significantly down-regulated in the nasopharyngeal carcinoma and associated with tumor metastasis.
We analyzed the expression of NGX6a in several human nasopharyngeal carcinoma cell lines using Western blot. A high expression of NGX6a at the protein level was detected in normal nasopharyngeal epithelial cell line NP69 and normal nasopharyngeal epithelial tissue. However, all of the nasopharyngeal carcinoma cell lines showed downexpression of NGX6a compared with NP69. To investigate whether over-expression of NGX6a in cancer cells leads to suppression of cancer cells growth, NGX6a was stablely transfected into 5-8F,6-10B, HNE1, cell line. In these cells, the mRNA levels of NGX6a were increased more than 20 folds, but it can't be detected any signals in the protein levels. After treated with proteasomes inhibitor MG132, NGX6a protein could be detected to increase as the concentration a of MG132 induction increased by using Western Blot and immunofluorescence. Transfect pCMV-myc-NGXa vector into 5-8F cell and transient expressed NGX6a protein, then isolate and purify NGX6a protein by immunoprecipitation and used anti-ubiqutin antibody to procee to Western Blot. The result showed NGX6a protein didn't be ubiqutinated. These results demonstrated that NGX6a protein was degraded in NPC cell through proteasomes but not ubiqutin pathway.
Previous studies illuminated that NGX6b protein interacted with ezrin. The present study showed that NGX6a protein also could interact with Ezrin by immunoprecipitation assay and immunofluorescence test. Ezrin, a linker between membrane protein and cytoskeleton, played important role in the cell morphology, cytoskeleton reorganization, adhesion, invasion and metastasis. Detected by tissue-microarray (TMA) and western blot, the overexpression of Ezrin in NPC and in NPC cell lines contrast to NGX6a suggested that Ezrin was involved in the progression and invasion of NPC. Another interesting fact is that the distribution of Ezrin is contrary to NGX6a in the human fetus tissues. For example, in the nerve system ezrin was detected predominantly in astrocytes, while NGX6a was expressed in neurons, no ezrin was detected in neurons in most tissues. In human fetus cerebellum, very strong ezrin staining was detected in the top of the molecular layer. However, Purkinje cells were ezrin-negative, while NGX6a was expressed in the purkinje cells and nerve fibers in the molecular layer. Further, using over-express and RNAi technology, we demonstrated that Ezrin Facilitates degradation of NGX6a protein in cell. We also demonstrated that seven-transmemberane domain was critical region for the degradation of NGX6a in cell by using mutant technology.
NGX6a could inhibit the invasion and mobility of NPC cells detected by matrigel migration assay, scraping test. Further invesgation showed that expression silence of Ezrin by RNAi could reduce the invasive ability of the 5-8F cells by matrigel migration assay. Simultaneously knocking-down Ezrin expression level and over-expressing NGX6a could make this inhibitory effect more significant. Using mithramycin which is SP1 inhibitor to treat 5-8F cells could down-regulated the expresson of SP1 and Ezrin and up-regulated NGX6a, which reduced the invasion and mobility of 5-8F cells. Combined use of mithramycin and MG132 to treat 5-8F cells could further inhibit the degradation of NGX6a protein, and is more effective to reduce the invasion and metastasis of 5-8F cells than single use of mithramycin or MG132 respectively.
Taken together, we generated a specific NGX6 antibody and investigated the distribution of NGX6 protein in human fetus. We found a novel subtype protein of NGX6:NGX6a; Obvious high expression of NGX6a protein presents in human fetus nervous system and epithelial tissues. NGX6a was significantly down-regulated in the nasopharyngeal carcinoma and associated with tumor metastasis; NGX6a was degraded in NPC cells. Ezrin was over-expressed in the nasopharyngeal carcinoma and associated with tumor metastasis. The distribution pattern of Ezrin was contrary to NGX6a in tissues and cells; Ezrin could interact with NGX6a and facilitates degradation of NGX6a protein; The seven-transmemberane domain of NGX6a was critical region for the degradation of NGX6a; Decreasing the expression level of Ezrin and increasing the expression level of NGX6a can reduce the ability of invasion and migration of 5-8F cells significantly.
引文
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