小鼠Mageb18基因克隆、表达分析及其调节黑色素瘤B16-F0细胞恶性表型的初步研究
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摘要
黑色素瘤相关抗原（melanoma-associated antigens，MAGE)是一个多成员基因家族。目前在人、大鼠和小鼠中已经发现的MAGE基因约100个左右。所有的MAGE基因其蛋白序列中都有一个含200个氨基酸左右的结构域，称为MAGE同源结构域（MHD）。根据MAGE基因序列保守性的不同，这些基因可以分为多个不同的亚家族。另外，根据表达谱的不同，MAGE基因还可以分为I型和II型两类。I型MAGE基因由MAGE-A、-B和-C三个亚家族组成，其他的亚家族则都属于II型MAGE基因。已经发现，I型MAGE基因在许多不同类型的肿瘤组织和细胞中表达，但是在正常组织中只有睾丸、卵巢和胎盘表达。正好相反，II型MAGE基因却在许多正常组织中广谱表达。因为睾丸、卵巢和胎盘中HLA分子的表达水平很低，因此I型MAGE基因独特的表达特点使得它们成为肿瘤免疫治疗的理想靶标。目前，针对I型MAGE基因已经开发了许多不同类型的肿瘤疫苗，有的甚至已经开始进入临床试验。但是，由于对I型MAGE基因的表达谱及功能的研究相对较少，因此这在很大程度上限制了MAGE肿瘤疫苗的推广和应用。
虽然有研究表明，I型MAGE基因在正常组织中的表达是睾丸特异的，但是我们前期利用表达序列标签（expressed sequence tag，EST）序列都对应于小鼠Mageb18基因。据此，我们假设认为，小鼠Mageb18基因在正常组织中的表达不是睾丸特异的，而是广谱的。本课题的目的就是克隆小鼠的Mageb18基因，分析它的表达特征以及调控机制，并研究它在调节肿瘤细胞恶性表型方面的功能以及相关分子机制。
为此，我们首先借助生物信息学工具分析和预测了Mageb18基因的结构和功能。结果表明，小鼠Mageb18基因全长2160bp，由5个外显子组成。它最大的开放阅读框为984bp，编码蛋白含327个氨基酸，蛋白的理论分子量和等电点分别为37203.31Da和6.43。MAGEB18蛋白含有一个MAGE N-末端结构域和一个保守的MHD，分别位于Arg~3–Asp82和Ile~(91)–Ala~(289)。亚细胞定位预测表明，MAGEB18蛋白不含亚细胞定位序列，但是可能存在8个磷酸化位点和一个N-豆蔻酰化位点，提示MAGEB18可能是一个胞浆蛋白，并且存在翻译后修饰加工。以前的研究表明，I型MAGE基因主要定位在X染色体，而且同一亚族的基因往往形成相对独立的基因簇。染色体定位分析发现，小鼠Mageb18基因也定位在X染色体的C2-C3区域，并与其他多个MAGE-B亚家族的基因形成基因簇。而且系统进化树分析也表明，Mageb18基因与MAGE-B亚家族基因间的亲缘关系最近。此外，基因同源搜索则发现，Mageb18基因在高等哺乳动物中高度保守，而在哺乳动物以外的物种中不存在。这些结果集中提示，Mageb18基因是一个在高级哺乳动物中高度保守的I型MAGE基因。
接下来，RT-PC分析Mageb18基因的表达谱发现，小鼠Mageb18基因在正常组织中的表达确实是非睾丸特异的。除了睾丸以外，它在胃、大肠、小肠、脾脏、淋巴结、骨髓和外周淋巴细胞都有较高水平表达。另外，我们发现Mageb18基因在小鼠睾丸中的表达是发育相关的。它在出生后第1天的睾丸中开始表达，而且随着时间的进行它的表达逐渐增高，在第21天左右达到最高，此后一直维持在较高的表达水平，提示Mageb18可能与睾丸的发育和精子的形成有关。继续分析Mageb18基因在小鼠细胞株中的表达谱发现，Mageb18在成纤维瘤细胞L929、黑色素瘤细胞B16-F0、肝癌细胞MM45T.Li、乳腺癌细胞4T1、胚胎成纤维细胞NIH3T3和巨噬细胞RAW264.7中表达，而在肝癌细胞H22和脑胶质瘤细胞C6中不表达。但是DNA甲基转移酶抑制剂5-氮-2’-脱氧胞苷和组蛋白去乙酰化酶抑制剂曲古霉素A能够激活Mageb18阴性细胞H22和C6中Mageb18基因的表达，提示表观遗传机制调节Mageb18的转录激活。
然后，利用Western印迹分析HEK293中瞬时表达的HA-MAGEB18融合蛋白表明，小鼠Mageb18基因编码的蛋白大小约为46kDa。进一步分析小鼠正常组织中内源性MAGEB18蛋白则发现，MAGEB18蛋白在小鼠的胃、大肠、小肠、脾脏、淋巴结、骨髓和血液淋巴细胞中表达，而在脑、心脏、肺、肝脏和肾脏中不表达，提示在正常组织中MAGEB18蛋白的表达谱与其mRNA的表达谱完全一致。另外，在组织中全长MAGEB18蛋白大小也为46kDa左右，但是它可能存在翻译后的剪切加工，从而产生一条大小约为26kDa的片段。奇怪的是，小鼠细胞株中的内源性MAGEB18蛋白只存在大小为46kDa一种形式。这些结果提示，MAGEB18蛋白在小鼠组织和细胞株中可能存在不同的翻译后修饰，从而导致形成大小不同的蛋白产物。利用间接免疫荧光分析MAGEB18蛋白的亚细胞定位表明，小鼠MAGEB18蛋白主要定位在细胞浆中，但在不同细胞株的细胞核里也有或多或少的定位。免疫组化分析进一步发现，在睾丸组织中MAGEB18蛋白主要在具有增殖能力的初级和次级精原细胞的细胞浆中表达，但是在成熟的精子中不表达。这些结果提示，MAGEB18蛋白是一个胞浆蛋白，它可能与细胞的增殖表型有关。
接下来，为了阐明Mageb18基因的功能，我们通过siRNA技术干扰内源性MAGEB18基因的表达，定量RT-PCR和Western印迹分析表明siRNA能够有效抑制细胞中Mageb18基因在mRNA和蛋白水平的表达。随后我们通过生长曲线分析、平板克隆形成实验以及皮下成瘤实验证明，干扰Mageb18基因的表达能够显著抑制B16-F0细胞和4T1细胞体外和体内的增殖。Annexin-V-APC/7-AAD细胞凋亡分析发现，干扰Mageb18能够显著增强B16-F0细胞的凋亡。Western印迹分析则表明，这个细胞凋亡增强的过程可能与TP53通路有关，因为干扰MAGEB18蛋白的表达能够提高TP53的蛋白水平，同时激活TP53的靶基因p21和Bax。
最后，进一步分析Mageb18基因在小鼠细胞株中的表达发现，MAGEB18蛋白在高成瘤和高转移的肿瘤细胞株，包括肿瘤干细胞和前癌干细胞中表达，提示Mage18基因不仅与细胞增殖表型有关，而且可能还调节细胞的转移特性。为此，我们通过划痕实验、基质胶侵袭实验以及体内肺转移动物模型分析了Mageb18基因对B16-F0细胞运动性的影响。结果表明，干扰MAGEB18蛋白的表达能够显著抑制B16-F0细胞的迁移、侵袭和转移能力。基质金属蛋白酶家族在肿瘤的迁移和转移中扮演重要角色。我们通过RT-PCR分析发现，B16-F0细胞表达多种不同类型的MMPs。但是，干扰Mageb18基因以后只能特异性地下调MMP2和MMP9的表达，而其他MMP的表达却没有明显的差异。这些结果提示，Mageb18基因确实可以通过MMP2和MMP9调节B16-F0细胞的迁移、侵袭和转移。
总之，我们的工作首次证明小鼠Mageb18基因一个非睾丸特异表达的I型MAGE基因。我们的结果提示，某些I型MAGE基因，至少是Mageb18基因的表达并不像传统观点所认为的那样是睾丸特异的，而是广谱的。由于MAGE家族基因是目前肿瘤疫苗研发中使用最多的靶标，因此我们的结果表明，以MAGE基因为靶标开发肿瘤疫苗之前，为了提高疫苗的有效性和安全性，非常有必要对该MAGE基因在正常组织中的表达谱和功能进行深入的研究。
Melanoma-associated antigens (MAGE) gene family consists of more than120genes and pseudogenes in the human, mouse and rat genomes. All the MAGEproteins shared a central and conserved region named the MAGE homology domain(MHD) which was composed of about200amino acid residues. On the basis of thedifference in sequence similarity and chromosome location, the MAGE genes aredivided into several subfamilies. They can also be classified into type I or type IIgenes on the basis of their expression pattern and functions. The type I MAGE genesare composed of the MAGEA, MAGEB and MAGEC subfamilies, whereas the othersubfamilies belong to the type II MAGE genes. Type I MAGE genes have been foundto be expressed in many different tumors, but their expression in normal tissues isrestricted to germline tissues such as placenta, ovary and testis, which express smallamounts of HLA molecules. In contrast, type II MAGE genes are expressedubiquitously in somatic cells of different tissues. These unique expression propertieshighlight the type I MAGE genes as superior candidates for tumor immunotherapy.To date, various vaccines targeting type I MAGE genes have been developed andhave shown great clinical benefit to treat patients with melanoma, multiple myeloma,breast cancer and so on. However, the study about the expression and functions oftype I MAGE genes is relatively less, which greatly restrains the application ofMAGE cancer vaccines.
Some previous studies indicated that expression of type I MAGE genes wererestricted to germ cells in normal tissues and to different malignancies; however, inour attempt to screen the expression profile of all mouse type I MAGE genes usingthe EST (expressed sequence tag) database in GenBank, we found that, althoughmost of the mouse ESTs identified were cloned from cDNA libraries that containedmouse testis, ovary or embryonic tissues, three of these ESTs were cloned fromcerebellum or adipose tissues. Moreover, these three ESTs reflected the same gene,Mageb18(melanoma antigen family B18). These results led us to assume that the expression of Mageb18gene was not testis-specific, but ubiquitous in normal tissues.
To address this hypothesis, we first used the bioinformatics tools to analyze andpredict the structure and function of mouse Mageb18gene. The results indicated thatthe full length of Mageb18mRNA was2160bp and contained five exons. The largestORF (open reading frame) of mouse Mageb18was984bp and encoded a predictedprotein that consist of327amino acids with an estimated molecular mass of37203.31Da and a theoretical pI of6.43. The MAGEB18protein contained a MAGEN-terminal domain and a conserved MHD (MAGE homology domain), which arelocated in Arg~3–Asp82and Ile~(91)–Ala~(289)respectively. TargetP1.1predicted that theMAGEB18protein carried no subcellular localization sequences; however, eightpotential phosphorylation sites and one N-myristoylation site are present in theprotein，which suggested that the MAGEB18protein may localize in the cytoplasmand undergo some post-translational modifications.
Previous studies have indicated that the type I MAGE genes are located on the Xchromosome and form several gene clusters. Chromosome location analysis indicatedthat mouse Mageb18is also located on chromosome XC2-C3region, which is closelylinked to another seven MAGEB genes. Phylogenetic analysis of the mouse MAGEgenes on chromosome X indicates that Mageb18gene is formed in the late stages ofthe MAGEB subfamily. Meanwhile， other14apparently orthologous proteinsequences in higher mammals have been identified with mouse MAGEB18protein(GenBank accession number NP_776144.1) as a query to screen the non-redundantprotein sequence database. Sequence alignment indicated that the MHD ofMAGEB18proteins among these mammals are highly conserved. To date, no orthologouspredicted protein sequences have been identified in any other species. These resultscollectively suggested that mouse Mageb18belongs to the type I MAGE geneconserved in higher mammals.
RT–PCR analysis with total RNA from normal mouse tissues indicated thatMageb18is indeed expressed in stomach, large intestine, small intestine, spleen,lymph node, bone marrow lymphocytes and blood T-lymphocytes, as well as testis.However, no expression was observed in the brain, heart, lung, liver and kidney. Theresult suggested that the expression of Mageb18is ubiquitous in mouse normal tissues. Further analyzing the expression of Mageb18mRNA in testes with differentage showed that Mageb18expression was detected from the first day of birth, andfound to increase steadily in the first3weeks of life. Mageb18mRNA reached fullexpression between14and21days and had a stable expression level between21and56days. The expression of Mageb18in testis was throughout this age range andreached a maximum expression in early puberty, indicating a role for Mageb18inboth testis development and spermatogenesis. RT–PCR was also used to analyze theexpression of Mageb18in eight mouse-derived cell lines, and the result indicated thatMageb18mRNA expression was detected in B16-F0(melanoma),4T1(breastcancer), L929(fibroblasts), NIH3T3(embryonic fibroblasts), MM45T.Li (livercancer) and RAW264.7(macrophages) cells, but not in hepatocellular carcinoma cellline H22and glioma cell line C6. However, treatment of H22and C6cells with DNAmethylation inhibitor5-aza-2-deoxycytidine and/or histone deacetylation inhibitortrichostatin A can reactivate the expression of Mageb18gene. These results suggestedthat DNA demethylation and histone acetylation certainly play important roles inregulating Mageb18gene expression.
Western blotting analyzing the HA-tagged MAGEB18fusion protein transientlyexpressed in HEK293cells indicated that the Mageb18gene encodes a protein with amolecular weight of46kDa. The protein extracts from brain, heart, lung, liver,stomach, large intestine, small intestine, kidney, spleen, lymphoid node, bone marrowlymphocyte, blood T-lymphocyte and testis were further immunodetected using ananti-MAGEB18antibody. The results indicated that, no specific signal was observedin the brain, heart, lung, liver and kidney tissues. However, a strong signal wasdetected at approximately46kDa in the stomach, large intestine, small intestine,spleen, lymphoid node, bone marrow lymphocyte, blood T-lymphocyte and testis.These results are consistent with the Mageb18mRNA expression profile as indicatedusing RT-PCR analysis. Meanwhile, another band at approximately26kDa can alsobe detected in all MAGEB18-positive tissues, but not in MAGEB18-negative tissues.Surprisingly, the MAGEB18protein in mouse-derived cell lines only existed uniqueband at approximately46kDa. These results indicated that the endogenousMAGEB18protein in tissues and cell lines may undergo different post-translational modifications and result in the formation of products with different sizes.
Subcellular localization analysis using indirect immunofluorescent stainingindicated that the endogenous MAGEB18protein was predominantly localized in thecytoplasm. However, the nuclear localization of MAGEB18protein can also beobserved more or less in different type of cells. Further immunohistochemicalstaining with mouse testis sections indicated that endogenous MAGEB18protein wasalso observed in cytoplasm of the primary and secondary spermatocytes, but less soin spermatids. These results collectively demonstrated that the Mageb18gene encodea cytoplasmic protein which may mediate cell proliferation.
To further characterize the function of MAGEB18in regulating cancer cellmalignant phenotypes, we first used siRNA technology to knockdown endogenousMAGEB18in cells. Real time RT-PCR and Western blotting indicated that thesiRNA can effectively suppressed the expression of Mageb18gene both in mRNAand in protein level. Subsequent functional assays using growth curves, colonyformation and in vivo subcutaneous tumorigenesis experiment indicated thatknockdown of MAGEB18significantly inhibited the growth of B16-F0and4T1cellsboth in vitro and in vivo. Meanwhile, APC–annexin V/7-AAD staining followed byFACS analysis indicated that knockdown of Mageb18in B16-F0cell enhanced itsapoptosis. TP53has been reported to be a vital regulator of cell apoptosis. Westernblotting analysis showed that knockdown of MAGEB18indeed increased the proteinlevels of TP53and its target gene p21and Bax. Additionally, the level of anotherpro-apoptotic protein, caspase-3, was also activated in MAGEB18-knockdown cells.These results collectively suggested that knockdown of MAGEB18indeed inhibitedcell proliferation and induced cell apoptosis, and that TP53played an important rolein this process.
Further analyzing the expression of Mageb18in various types of mouse-derivedcell lines found that Mageb18is over-expressed not only in cancer cell lines withhighly tumorigenic and metastatic properties, but also in cancer stem cell line andprecancerous stem cell lines, which indicates that the Mageb18gene may alsomediate the migration phenotype as well as proliferation and apoptosis. Therefore, wecombined the wound healing assay, transwell invasion assay and in vivo lung metastasis mouse model to investigate the impact of Mageb18gene on the mobility ofB16-F0cells. Our results indicated that knockdown of MAGEB18not only inhibitedthe in vitro migration and invasion of B16-F0cells, but also suppressed theirmetastasis to lung in vivo. It is well-known that MMPs plays important roles inregulating the migration and metastasis of cancer cells. As revealed by RT-PCRanalysis, we showed that parental B16-F0cells expressed various types of MMPs;however, knockdown of MAGEB18resulted in specifically down-regulating theexpression of gelatinases MMPs2and9. These results collectively indicated thatMageb18indeed mediated migration, invasion and metastasis phenotype of B16-F0cells through MMPs2and9.
In summary, we have cloned and identified a member of MAGE gene family,Mageb18, and firstly characterized it as a non-testis-specific type I MAGE gene. Theresults of the present study thus reveal an important phenomenon that the expressionof some type I MAGE genes, at least for Mageb18, is not testis-specific, butubiquitous. Because the various type I MAGE genes are the most frequently usedtargets in tumor immunotherapy nowadays, our results therefore also suggest thenecessity to study further the expression pattern of these type I MAGE genes innormal tissues prior to using them to develop more effective and safer cancervaccines.
虽然有研究表明，I型MAGE基因在正常组织中的表达是睾丸特异的，但是我们前期利用表达序列标签（expressed sequence tag，EST）序列都对应于小鼠Mageb18基因。据此，我们假设认为，小鼠Mageb18基因在正常组织中的表达不是睾丸特异的，而是广谱的。本课题的目的就是克隆小鼠的Mageb18基因，分析它的表达特征以及调控机制，并研究它在调节肿瘤细胞恶性表型方面的功能以及相关分子机制。
为此，我们首先借助生物信息学工具分析和预测了Mageb18基因的结构和功能。结果表明，小鼠Mageb18基因全长2160bp，由5个外显子组成。它最大的开放阅读框为984bp，编码蛋白含327个氨基酸，蛋白的理论分子量和等电点分别为37203.31Da和6.43。MAGEB18蛋白含有一个MAGE N-末端结构域和一个保守的MHD，分别位于Arg~3–Asp82和Ile~(91)–Ala~(289)。亚细胞定位预测表明，MAGEB18蛋白不含亚细胞定位序列，但是可能存在8个磷酸化位点和一个N-豆蔻酰化位点，提示MAGEB18可能是一个胞浆蛋白，并且存在翻译后修饰加工。以前的研究表明，I型MAGE基因主要定位在X染色体，而且同一亚族的基因往往形成相对独立的基因簇。染色体定位分析发现，小鼠Mageb18基因也定位在X染色体的C2-C3区域，并与其他多个MAGE-B亚家族的基因形成基因簇。而且系统进化树分析也表明，Mageb18基因与MAGE-B亚家族基因间的亲缘关系最近。此外，基因同源搜索则发现，Mageb18基因在高等哺乳动物中高度保守，而在哺乳动物以外的物种中不存在。这些结果集中提示，Mageb18基因是一个在高级哺乳动物中高度保守的I型MAGE基因。
接下来，RT-PC分析Mageb18基因的表达谱发现，小鼠Mageb18基因在正常组织中的表达确实是非睾丸特异的。除了睾丸以外，它在胃、大肠、小肠、脾脏、淋巴结、骨髓和外周淋巴细胞都有较高水平表达。另外，我们发现Mageb18基因在小鼠睾丸中的表达是发育相关的。它在出生后第1天的睾丸中开始表达，而且随着时间的进行它的表达逐渐增高，在第21天左右达到最高，此后一直维持在较高的表达水平，提示Mageb18可能与睾丸的发育和精子的形成有关。继续分析Mageb18基因在小鼠细胞株中的表达谱发现，Mageb18在成纤维瘤细胞L929、黑色素瘤细胞B16-F0、肝癌细胞MM45T.Li、乳腺癌细胞4T1、胚胎成纤维细胞NIH3T3和巨噬细胞RAW264.7中表达，而在肝癌细胞H22和脑胶质瘤细胞C6中不表达。但是DNA甲基转移酶抑制剂5-氮-2’-脱氧胞苷和组蛋白去乙酰化酶抑制剂曲古霉素A能够激活Mageb18阴性细胞H22和C6中Mageb18基因的表达，提示表观遗传机制调节Mageb18的转录激活。
然后，利用Western印迹分析HEK293中瞬时表达的HA-MAGEB18融合蛋白表明，小鼠Mageb18基因编码的蛋白大小约为46kDa。进一步分析小鼠正常组织中内源性MAGEB18蛋白则发现，MAGEB18蛋白在小鼠的胃、大肠、小肠、脾脏、淋巴结、骨髓和血液淋巴细胞中表达，而在脑、心脏、肺、肝脏和肾脏中不表达，提示在正常组织中MAGEB18蛋白的表达谱与其mRNA的表达谱完全一致。另外，在组织中全长MAGEB18蛋白大小也为46kDa左右，但是它可能存在翻译后的剪切加工，从而产生一条大小约为26kDa的片段。奇怪的是，小鼠细胞株中的内源性MAGEB18蛋白只存在大小为46kDa一种形式。这些结果提示，MAGEB18蛋白在小鼠组织和细胞株中可能存在不同的翻译后修饰，从而导致形成大小不同的蛋白产物。利用间接免疫荧光分析MAGEB18蛋白的亚细胞定位表明，小鼠MAGEB18蛋白主要定位在细胞浆中，但在不同细胞株的细胞核里也有或多或少的定位。免疫组化分析进一步发现，在睾丸组织中MAGEB18蛋白主要在具有增殖能力的初级和次级精原细胞的细胞浆中表达，但是在成熟的精子中不表达。这些结果提示，MAGEB18蛋白是一个胞浆蛋白，它可能与细胞的增殖表型有关。
接下来，为了阐明Mageb18基因的功能，我们通过siRNA技术干扰内源性MAGEB18基因的表达，定量RT-PCR和Western印迹分析表明siRNA能够有效抑制细胞中Mageb18基因在mRNA和蛋白水平的表达。随后我们通过生长曲线分析、平板克隆形成实验以及皮下成瘤实验证明，干扰Mageb18基因的表达能够显著抑制B16-F0细胞和4T1细胞体外和体内的增殖。Annexin-V-APC/7-AAD细胞凋亡分析发现，干扰Mageb18能够显著增强B16-F0细胞的凋亡。Western印迹分析则表明，这个细胞凋亡增强的过程可能与TP53通路有关，因为干扰MAGEB18蛋白的表达能够提高TP53的蛋白水平，同时激活TP53的靶基因p21和Bax。
最后，进一步分析Mageb18基因在小鼠细胞株中的表达发现，MAGEB18蛋白在高成瘤和高转移的肿瘤细胞株，包括肿瘤干细胞和前癌干细胞中表达，提示Mage18基因不仅与细胞增殖表型有关，而且可能还调节细胞的转移特性。为此，我们通过划痕实验、基质胶侵袭实验以及体内肺转移动物模型分析了Mageb18基因对B16-F0细胞运动性的影响。结果表明，干扰MAGEB18蛋白的表达能够显著抑制B16-F0细胞的迁移、侵袭和转移能力。基质金属蛋白酶家族在肿瘤的迁移和转移中扮演重要角色。我们通过RT-PCR分析发现，B16-F0细胞表达多种不同类型的MMPs。但是，干扰Mageb18基因以后只能特异性地下调MMP2和MMP9的表达，而其他MMP的表达却没有明显的差异。这些结果提示，Mageb18基因确实可以通过MMP2和MMP9调节B16-F0细胞的迁移、侵袭和转移。
总之，我们的工作首次证明小鼠Mageb18基因一个非睾丸特异表达的I型MAGE基因。我们的结果提示，某些I型MAGE基因，至少是Mageb18基因的表达并不像传统观点所认为的那样是睾丸特异的，而是广谱的。由于MAGE家族基因是目前肿瘤疫苗研发中使用最多的靶标，因此我们的结果表明，以MAGE基因为靶标开发肿瘤疫苗之前，为了提高疫苗的有效性和安全性，非常有必要对该MAGE基因在正常组织中的表达谱和功能进行深入的研究。
Melanoma-associated antigens (MAGE) gene family consists of more than120genes and pseudogenes in the human, mouse and rat genomes. All the MAGEproteins shared a central and conserved region named the MAGE homology domain(MHD) which was composed of about200amino acid residues. On the basis of thedifference in sequence similarity and chromosome location, the MAGE genes aredivided into several subfamilies. They can also be classified into type I or type IIgenes on the basis of their expression pattern and functions. The type I MAGE genesare composed of the MAGEA, MAGEB and MAGEC subfamilies, whereas the othersubfamilies belong to the type II MAGE genes. Type I MAGE genes have been foundto be expressed in many different tumors, but their expression in normal tissues isrestricted to germline tissues such as placenta, ovary and testis, which express smallamounts of HLA molecules. In contrast, type II MAGE genes are expressedubiquitously in somatic cells of different tissues. These unique expression propertieshighlight the type I MAGE genes as superior candidates for tumor immunotherapy.To date, various vaccines targeting type I MAGE genes have been developed andhave shown great clinical benefit to treat patients with melanoma, multiple myeloma,breast cancer and so on. However, the study about the expression and functions oftype I MAGE genes is relatively less, which greatly restrains the application ofMAGE cancer vaccines.
Some previous studies indicated that expression of type I MAGE genes wererestricted to germ cells in normal tissues and to different malignancies; however, inour attempt to screen the expression profile of all mouse type I MAGE genes usingthe EST (expressed sequence tag) database in GenBank, we found that, althoughmost of the mouse ESTs identified were cloned from cDNA libraries that containedmouse testis, ovary or embryonic tissues, three of these ESTs were cloned fromcerebellum or adipose tissues. Moreover, these three ESTs reflected the same gene,Mageb18(melanoma antigen family B18). These results led us to assume that the expression of Mageb18gene was not testis-specific, but ubiquitous in normal tissues.
To address this hypothesis, we first used the bioinformatics tools to analyze andpredict the structure and function of mouse Mageb18gene. The results indicated thatthe full length of Mageb18mRNA was2160bp and contained five exons. The largestORF (open reading frame) of mouse Mageb18was984bp and encoded a predictedprotein that consist of327amino acids with an estimated molecular mass of37203.31Da and a theoretical pI of6.43. The MAGEB18protein contained a MAGEN-terminal domain and a conserved MHD (MAGE homology domain), which arelocated in Arg~3–Asp82and Ile~(91)–Ala~(289)respectively. TargetP1.1predicted that theMAGEB18protein carried no subcellular localization sequences; however, eightpotential phosphorylation sites and one N-myristoylation site are present in theprotein，which suggested that the MAGEB18protein may localize in the cytoplasmand undergo some post-translational modifications.
Previous studies have indicated that the type I MAGE genes are located on the Xchromosome and form several gene clusters. Chromosome location analysis indicatedthat mouse Mageb18is also located on chromosome XC2-C3region, which is closelylinked to another seven MAGEB genes. Phylogenetic analysis of the mouse MAGEgenes on chromosome X indicates that Mageb18gene is formed in the late stages ofthe MAGEB subfamily. Meanwhile， other14apparently orthologous proteinsequences in higher mammals have been identified with mouse MAGEB18protein(GenBank accession number NP_776144.1) as a query to screen the non-redundantprotein sequence database. Sequence alignment indicated that the MHD ofMAGEB18proteins among these mammals are highly conserved. To date, no orthologouspredicted protein sequences have been identified in any other species. These resultscollectively suggested that mouse Mageb18belongs to the type I MAGE geneconserved in higher mammals.
RT–PCR analysis with total RNA from normal mouse tissues indicated thatMageb18is indeed expressed in stomach, large intestine, small intestine, spleen,lymph node, bone marrow lymphocytes and blood T-lymphocytes, as well as testis.However, no expression was observed in the brain, heart, lung, liver and kidney. Theresult suggested that the expression of Mageb18is ubiquitous in mouse normal tissues. Further analyzing the expression of Mageb18mRNA in testes with differentage showed that Mageb18expression was detected from the first day of birth, andfound to increase steadily in the first3weeks of life. Mageb18mRNA reached fullexpression between14and21days and had a stable expression level between21and56days. The expression of Mageb18in testis was throughout this age range andreached a maximum expression in early puberty, indicating a role for Mageb18inboth testis development and spermatogenesis. RT–PCR was also used to analyze theexpression of Mageb18in eight mouse-derived cell lines, and the result indicated thatMageb18mRNA expression was detected in B16-F0(melanoma),4T1(breastcancer), L929(fibroblasts), NIH3T3(embryonic fibroblasts), MM45T.Li (livercancer) and RAW264.7(macrophages) cells, but not in hepatocellular carcinoma cellline H22and glioma cell line C6. However, treatment of H22and C6cells with DNAmethylation inhibitor5-aza-2-deoxycytidine and/or histone deacetylation inhibitortrichostatin A can reactivate the expression of Mageb18gene. These results suggestedthat DNA demethylation and histone acetylation certainly play important roles inregulating Mageb18gene expression.
Western blotting analyzing the HA-tagged MAGEB18fusion protein transientlyexpressed in HEK293cells indicated that the Mageb18gene encodes a protein with amolecular weight of46kDa. The protein extracts from brain, heart, lung, liver,stomach, large intestine, small intestine, kidney, spleen, lymphoid node, bone marrowlymphocyte, blood T-lymphocyte and testis were further immunodetected using ananti-MAGEB18antibody. The results indicated that, no specific signal was observedin the brain, heart, lung, liver and kidney tissues. However, a strong signal wasdetected at approximately46kDa in the stomach, large intestine, small intestine,spleen, lymphoid node, bone marrow lymphocyte, blood T-lymphocyte and testis.These results are consistent with the Mageb18mRNA expression profile as indicatedusing RT-PCR analysis. Meanwhile, another band at approximately26kDa can alsobe detected in all MAGEB18-positive tissues, but not in MAGEB18-negative tissues.Surprisingly, the MAGEB18protein in mouse-derived cell lines only existed uniqueband at approximately46kDa. These results indicated that the endogenousMAGEB18protein in tissues and cell lines may undergo different post-translational modifications and result in the formation of products with different sizes.
Subcellular localization analysis using indirect immunofluorescent stainingindicated that the endogenous MAGEB18protein was predominantly localized in thecytoplasm. However, the nuclear localization of MAGEB18protein can also beobserved more or less in different type of cells. Further immunohistochemicalstaining with mouse testis sections indicated that endogenous MAGEB18protein wasalso observed in cytoplasm of the primary and secondary spermatocytes, but less soin spermatids. These results collectively demonstrated that the Mageb18gene encodea cytoplasmic protein which may mediate cell proliferation.
To further characterize the function of MAGEB18in regulating cancer cellmalignant phenotypes, we first used siRNA technology to knockdown endogenousMAGEB18in cells. Real time RT-PCR and Western blotting indicated that thesiRNA can effectively suppressed the expression of Mageb18gene both in mRNAand in protein level. Subsequent functional assays using growth curves, colonyformation and in vivo subcutaneous tumorigenesis experiment indicated thatknockdown of MAGEB18significantly inhibited the growth of B16-F0and4T1cellsboth in vitro and in vivo. Meanwhile, APC–annexin V/7-AAD staining followed byFACS analysis indicated that knockdown of Mageb18in B16-F0cell enhanced itsapoptosis. TP53has been reported to be a vital regulator of cell apoptosis. Westernblotting analysis showed that knockdown of MAGEB18indeed increased the proteinlevels of TP53and its target gene p21and Bax. Additionally, the level of anotherpro-apoptotic protein, caspase-3, was also activated in MAGEB18-knockdown cells.These results collectively suggested that knockdown of MAGEB18indeed inhibitedcell proliferation and induced cell apoptosis, and that TP53played an important rolein this process.
Further analyzing the expression of Mageb18in various types of mouse-derivedcell lines found that Mageb18is over-expressed not only in cancer cell lines withhighly tumorigenic and metastatic properties, but also in cancer stem cell line andprecancerous stem cell lines, which indicates that the Mageb18gene may alsomediate the migration phenotype as well as proliferation and apoptosis. Therefore, wecombined the wound healing assay, transwell invasion assay and in vivo lung metastasis mouse model to investigate the impact of Mageb18gene on the mobility ofB16-F0cells. Our results indicated that knockdown of MAGEB18not only inhibitedthe in vitro migration and invasion of B16-F0cells, but also suppressed theirmetastasis to lung in vivo. It is well-known that MMPs plays important roles inregulating the migration and metastasis of cancer cells. As revealed by RT-PCRanalysis, we showed that parental B16-F0cells expressed various types of MMPs;however, knockdown of MAGEB18resulted in specifically down-regulating theexpression of gelatinases MMPs2and9. These results collectively indicated thatMageb18indeed mediated migration, invasion and metastasis phenotype of B16-F0cells through MMPs2and9.
In summary, we have cloned and identified a member of MAGE gene family,Mageb18, and firstly characterized it as a non-testis-specific type I MAGE gene. Theresults of the present study thus reveal an important phenomenon that the expressionof some type I MAGE genes, at least for Mageb18, is not testis-specific, butubiquitous. Because the various type I MAGE genes are the most frequently usedtargets in tumor immunotherapy nowadays, our results therefore also suggest thenecessity to study further the expression pattern of these type I MAGE genes innormal tissues prior to using them to develop more effective and safer cancervaccines.
引文
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