| Tumor is seriously threatening the human life and health for a long time. The digestive tract cancers, such as gastric cancer, esophageal cancer and colorectal cancer, are the most significant threat. Because of the complexity of the mechanisms of tumor development, we are facing huge challenges in looking for a more efficient treatment. Immune therapy makes a new direction towards the treatment. The therapy can enhance the immunogenicity of tumor antigen to stimulate the specific immune response and attack tumor cells. Therefore, looking for tumor specific antigens becomes a key. Melanoma associated antigen(MAGE) is just the kind of tumor specific antigens.In recent years, our group focused on the expression pattern of MAGE-A family in tumor tissues. In the present study, the expression of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 protein in esophageal squamous cell carcinomas and cardia adenocarcinoma and their corresponding normal tissues was detected by immunohistochemisty.The relationship between the expression of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 protein and their clinical biological was analyzed, especially the prognostic factors.We use real-time quantitative PCR to verify the genes related to MAGE-A11 screened out by gene chips. In order to provide a theoretical basis for MAGE-A family to become the tumor specific antigens of esophageal squamous cell carcinoma and cardia adenocarcinoma, and try to explain the mechanisms.The main research contents and results were shown as follows: Part I Expressions and significance of MAGE-A family in esophagealsquamous cell carcinoma and cardia adenocarcinoma tissuesObjective: To investigate the expression of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 in esophageal squamous cell carcinomas and cardia adenocarcinoma and their corresponding normal tissues,and analyze the relationship between the expression of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 protein and their clinical biological indicators, especially the prognostic factors.Methods:1 Immunohistochemical staining was used to detect MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 specificity in normal testis tissue.2 Immunohistochemical staining was used to detect the protein expression of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 protein in esophageal squamous cell carcinomas and cardia adenocarcinoma and the corresponding normal tissue and the clinical/biological factors were analyzed.Results:1 MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein was detected in normal testis tissues and esophageal squamous cell carcinomas and cardia adenocarcinoma. In normal testis tissues, MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) was mainly expressed in cell nuclears of spermatogonium and spermatocyte, particularly.In esophageal squamous cell carcinomas and cardia adenocarcinoma tissues, MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein was mainly expressed in cytoplasm, and part of them in cell nuclear. MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein was not expressed in corresponding normal tissue, 68.33%(41/60) esophageal squamous cell carcinomas tissues showed MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein expression; 56.67%(17/30) cardia adenocarcinoma tissues showed MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein expression.In esophageal squamous cell carcinomas tissues, there were no significant differences between MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein expression and patient’s age, histological grades, clinical stages, tumor size, metastasis of axillary lymph nodes(P>0.05).In cardia adenocarcinoma tissues, the expression of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein is positively related to the expressions of histological grade(P<0.05).There were no significant differences between MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein expression and patient’s age, clinical stages, tumor size, metastasis of axillary lymph nodes(P>0.05).2 MAGE-A9 protein was detected in normal testis tissues and esophageal squamous cell carcinomas and cardia adenocarcinoma. In normal testis tissues, MAGE-A9 was mainly expressed in cell nuclears of spermatogonium and spermatocyte, particularly.In esophageal squamous cell carcinomas and cardia adenocarcinoma tissues, MAGE-A9 protein was mainly expressed in cytoplasm, and part of them in cell nuclear. MAGE-A9 protein was not expressed in corresponding normal tissue, 45%(27/60) esophageal squamous cell carcinomas tissues showed MAGE-A9 protein expression; 43.33%(13/30) cardia adenocarcinoma tissues showed MAGE-A9 protein expression.In esophageal squamous cell carcinomas tissues, the expression of MAGE-A9 protein is positively related to the expressions of histological grade(P<0.05).There were no significant differences between MAGE-A9 protein expression and patient’s age, clinical stages, tumor size, metastasis of axillary lymph nodes(P>0.05).In cardia adenocarcinoma tissues, the expression of MAGE-A9 protein is positively related to the expressions of histological grade(P<0.05).There were no significant differences between MAGE-A9 protein expression and patient’s age, clinical stages, tumor size, metastasis of axillary lymph nodes(P>0.05).3 MAGE-A11 protein was detected in normal testis tissues and esophageal squamous cell carcinomas and cardia adenocarcinoma. In normal testis tissues, MAGE-A11 was mainly expressed in cell nuclears of spermatogonium and spermatocyte, particularly. In esophageal squamous cell carcinomas and cardia adenocarcinoma tissues, MAGE-A11 protein was mainly expressed in cytoplasm, and part of them in cell nuclear. MAGE-A11 protein was not expressed in corresponding normal tissue, 66.67%(40/60)esophageal squamous cell carcinomas tissues showed MAGE-A11 protein expression; 53.33%(16/30) cardia adenocarcinoma tissues showed MAGE-A11 protein expression.In esophageal squamous cell carcinomas tissues, the expression of MAGE-A11 protein is positively related to the expressions of clinical stages and tumor size(P<0.05).There were no significant differences between MAGE-A11 protein expression and patient’s age, histological grade, metastasis of axillary lymph nodes(P>0.05).In cardia adenocarcinoma tissues, the expression of MAGE-A11 protein is positively related to the expressions of clinical stages and histological grade(P<0.05).There were no significant differences between MAGE-A11 protein expression and patient’s age, tumor size, metastasis of axillary lymph nodes(P>0.05).4 In 60 esophageal squamous cell carcinomas tissues, MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 showed uniform distribution. 19 of 60(31.67%) esophageal squamous cell carcinomas tissues showed MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 expression. The expression rate of these three proteins was significantly different in 60 breast cancer tissues(χ2=8.472, P=0.014<0.05), and the expression rate of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) is the highest among these three proteins.5 In 30 cardia adenocarcinoma tissues, MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 showed uniform distribution. 11 of 30(36.67%) cardia adenocarcinoma tissues showed MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 expression. The expression rate of these three proteins was not significantly different in 30 cardia adenocarcinoma tissues(χ2=1.156, P=0.561>0.05).6 The survival time of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 expression is lower than those negative ones in patients with esophageal squamous cell carcinoma and cardia adenocarcinoma.Conclusions: MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 proteins are esophageal squamous cell carcinoma and cardia adenocarcinoma specific antigens. They may be important markers for poor prognosis of esophageal squamous cell carcinoma and cardia adenocarcinoma.Part Ⅱ Initial verification of screening with MAGE-A11 related gene in Gene chipObjective: In order to verify the genes screening by Gene chip related by TRIM33, CEP295, KIF23, FN1, CKAP5, PNN, MMP13, MMP26 and MMP28,the differences in the expression levels of the m RNA between transfection of MAGE-A11 group and the empty vectors group,to explore the possible mechanism of MAGE-A11 on occurrence and progression of esophageal carcinoma, to provide a preliminary theoretical basis for clarifying the role of MAGE-A11 in esophageal carcinoma.Methods: Real-time PCR confirmation: the design of the primers: logging in GENEBANK wetsite, design the pairs of the primers; SYBR® Green I real-time RT-PCR were conducted by ABI Prism 7500.(for details,please see the the SYBR®Green I real-time RT-PCR experimental instructions).Results:1 In Eca109 esophageal carcinoma cell line,the results of RT-PCR showed that the transfection of MAGE-A11 markedly increased the expression of CEP295, TRIM33, KIF23, FN1, MMP13, MMP26 and MMP28 m RNA, compared with the empty vectors group, transfection of MAGE-A11 increased the level of CEP295, TRIM33, KIF23, FN1, MMP13, MMP26 and MMP28 gene transcription; the transfection of MAGE-A11 markedly decrease the expression of CKAP5 and PNNm RNA, compared with the empty vectors group,transfection of MAGE-A11 decreased the level of CKAP5 and PNN gene transcription(P<0.05).2 In TE-1 esophageal carcinoma cell line,the results of RT-PCR showed that the transfection of MAGE-A11 markedly increased the expression of CEP295, TRIM33, KIF23, FN1, MMP13, MMP26 and MMP28 m RNA, compared with the empty vectors group, transfection of MAGE-A11 increased the level of CEP295, TRIM33, KIF23, FN1, MMP13, MMP26 and MMP28 gene transcription; the transfection of MAGE-A11 markedly decreased the expression of CKAP5 and PNN m RNA, compared with the empty vectors group,transfection of MAGE-A11 decreased the level of CKAP5 and PNN gene transcription(P<0.05).3 In TE-13 esophageal carcinoma cell line,the results of RT-PCR showed that the transfection of MAGE-A11 markedly increased the expression of CEP295, TRIM33, KIF23, FN1, MMP13, MMP26 and MMP28 m RNA, compared with the empty vectors group, transfection of MAGE-A11 increased the level of CEP295, TRIM33, KIF23, FN1, MMP13, MMP26 and MMP28 gene transcription; the transfection of MAGE-A11 markedly decreased the expression of CKAP5 and PNNm RNA, compared with the empty vectors group, transfection of MAGE-A11 decreased the level of CKAP5 and PNNgene transcription(P<0.05).Conclusion: By regulating ubiquitin related genes(TRIM33 upregulation), proliferation and apoptosis related genes(CEP295 and KIF23 upregulation), the related genes of tumor invasion and metastasis(MMP13, MMP26, MMP28 and FN1 upregulation; CKAP5 and PNN downregulation), MAGE-A11 plays an important role in esophageal cancer. |
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