| ObjectiveThe aim of the study was to analyze the expression pattern of MAGE-D4 mRNA and protein in glioma and other tumor tissues, and to discuss the significance of MAGE-D4 autoantibody in sera so that the function of MAGE-D4 gene in tumor pathogenesis and progression would be understood in detail, which can provide informations to evaluate the feasibility of applying MAGE-D4 for tumor adjuvant diagnosis and target specific immunotherapy.Methods1. Predicition and analysis of MAGE-D4 protein homology, physical and chemical parameter, transmembrane region, secondary structure, domain, functional site, cellular orientation and antigenic epitope by bioinformatics were practised.2. RT-PCR technique was used to amplify MAGE-D4a full-length coding region from human glioma sample, which was cloned into prokaryotic expression plasmid pMAL-c2. Positive clones were identified, sequenced and transformed into E.coli. Roseetta. After induced by IPTG, expression products were purified with Amylose Resin affinity chromatography and identified by mass spectrometry.3. Rabbits were immunized with fusion protein to get anti-MAGE-D4 antibody, which was separated and purified by Protein A agarose column. The titer and specificity of antibody was examined by indirect ELISA and Western Blot respectively.4. Explore the gene expression characteristic of MAGE-D4 at mRNA and protein level by RT-PCR and immunohistochemistry staining. Investigate the relationship betwteen the expression status of MAGE-D4 and tumor clinicopathological features.5. With the purified MAGE-D4 fusion protein as coating antigen, MAGE-D4 autoantibody in sera of six kinds of tumor and healthy controls were detected by indirect ELISA assay. The clinical value of autoantibody was primarily discussed.Results1. MAGE-D4 is an unstable, weakly acidic, soluble protein. There are no transmembrane region and signal peptide. MAGE-D4's secondary structure is mainly composed ofα-helix(59.6%) and random coil(23.8%). It has two coiled coils at amino acid sites of 334-368 and 450-480. The subcellular location of MAGE-D4 is presumed in the nuclear(55.5%). The MAGE conserved domain was found in amino acid sequence of MAGE-D4. 2 N-glycosylation sites, 1 cAMP- and cGMP-dependent protein kinase phosphorylation site, 1 tyrosine kinase phosphorylation site, 9 casein knaseⅡphosphorylation sites, 14 protein kinase C kinase phosphorylation sites and some functional motifs were predicted of MAGE-D4 protein, which suggested that it might play a potential role on cell growth, adhesion and signal transduction. Moreover, a number of HLA-A2 T cell epitopes was found in MAGE-D4 protein.2. Recombinant plasmid of MAGE-D4 was successfully constructed. Expression level of fusion protein was as much as 47.7% when the inducing conditions were optimized, which is soluble in part. The expression protein which purified after affinity chromatography was really indentical to MAGE-D4 protein by mass spectrometry analysis.3. The titer of MAGE-D4 polyclonal antibody was higher than 1﹕64000, which could specifically recognize and combine MAGE-D4 fusion protein.4. The frequencies of MAGE-D4 mRNA expression were 85.48%(53/62) in glioma, 76.92%(20/26) in meningioma, 71.70%(38/53) in hepatocelluar carcinoma and 81.03%(47/58) in colon carcinoma. The positive rate of adjacent non-tumorous tissues were lower than corresponding hepatocelluar carcinoma and colon carcinoma tissues(P<0.05), which were 50.94%(27/53) and 41.38%(24/58). MAGE-D4 mRNA was positively expressed in HepG2, BEL-7404, CNE, SK-OV-3, TCA-8113 , and was negatively expressed in SGC-7901, MGC80-3, LOVO, SW620, MCF-7. MAGE-D4 protein was mainly located in cytoplasm, which was positive observed in 77.8%(21/27) golima, 69.6%(16/23) non-small cell lung cancer,66.7%(2/3) hepatocelluar carcinoma, 50%(1/2) gastric carcinoma , and 100%(2/2) colon carcinoma, while not observed in 3 normal brain tissues and 27 adjacent non-tumorous lung tissues. The expression level of MAGE-D4 of glioma and non-small cell lung cancer had no relationship with clinicopathological parameters.5. Antibodies against MAGE-D4 were detected in 19.34%(47/243)of sera samples from patients with a wide spectrum of tumors. The percentages of autoantibody response were observed in patients with 29% hepatocelluar carcinoma(10/35), 27% lung carcinoma (8/30), 19% colon carcinoma (12/64), 18% glioma(9/51), 17% gastric carcinoma(5/30), and 9% meningioma(3/33), whereas MAGE-D4 antibody was not found in sera of 72 healthy controls.ConclusionMAGE-D4 is probably a tumor associated antigen which has relatively lower or negatively expression in adjacent non-tumorous tissue and normal tissue compared with malignant tissue. Because of its immunogenicity in different types of tumor, MAGE-D4 should become a promising biomarker for cancer diagnosis and immunotherapy.
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