Cloning, Expression Analysis And Functions Of Mouse Magebl8Gene In Regulating The Malignant Phenotypes Of Melanoma B16-F0Cells

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 Arg³–Asp82and Ile⁹¹–Ala²⁸⁹respectively. 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 NP₇76144.1） 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.