| Malignant tumor is a major public health problem in China, laryngeal cancer is the most common malignant disease of head and neck. Due to the increasing incidence, laryngeal cancer has gradually caused extensive concern. The treatment strategies for laryngeal carcinoma include surgery, radiotherapy and chemotherapy. Recurrence and metastasis are common even after resection and the overall outcome is still unsatisfactory. Therefore, it is urgent to seek new cures for the treatment of laryngeal cancer. The melanoma-associated antigens family A(MAGE-A) belongs to Cancer/testis antigens(CTA), which are aberrantly expressed in a wide variety of malignant tumors, but not in normal adult tissues except for germ cells.In recent years, our group focused on the expression pattern of MAGE-A family in tumor tissues. However, the expression pattern of MAGE-A family in laryngeal squamous cell carcinoma(LSCC) patients is still unclear. In the present study, the expression of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 protein in LSCC tissues and corresponding adjacent non-neoplastic 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. The second part we detected the methylation levels of MAGE-A1 and MAGE-A3 in106 LSCC cases and 22 corresponding cancer adjacent normal tissues by methylation specific PCR(methylation specific polymerase chain reaction, MSP) method. The expression of DNA methyl transfer enzyme DNMT1, DNMT3 a and DNMT3 b were studied by immunohistochemistry. In addition, the m RNA expressions of MAGE-A genes in peripheral blood of LSCC were detected by multiple nested RT-PCR. These studies provided a theoretical basis for the prognosis and early detection of LSCC. The main research contents and results were shown as follows: Part I Expressions and significance of MAGE-A family in LSCCObjective:To investigate the expression of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 in LSCC and and corresponding adjacent non-neoplastic 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 The expressions of MAGE-As, MAGE-A9 and MAGE-A11 were assessed in the LSCC tissues and corresponding adjacent non-neoplastic tissues from 106 patients by immunohistochemistry(IHC), and the associations of MAGE-As, MAGE-A9 and MAGE-A11 expressions and the clinical parameters and the survival of LSCC patients were analyzed by chi-square test, Kaplan-Meier survival and Cox regression analysis.Results:1 MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein was detected in normal testis tissues and laryngeal squamous cell carcinoma. 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 laryngeal squamous cell carcinoma 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, 54.72%(58/106) laryngeal squamous cell carcinoma tissues showed MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein expression.In laryngeal squamous cell carcinoma tissues, the expression of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12) protein is positively related to clinical stages, tumor size and lymph node metastasis(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, gender, smoking histological grade, and pathological degree(P>0.05).2 MAGE-A9 protein was detected in normal testis tissues and laryngeal squamous cell carcinoma. 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, 46.22%(49/106) laryngeal squamous cell carcinoma tissues showed MAGE-A9 protein expression.In laryngeal squamous cell carcinoma tissues, the expression of MAGE-A9 protein is positively related to clinical stages, tumor size and lymph node metastasis(P<0.05). There were no significant differences between MAGE-A9 protein expression and patient’s age, gender, smoking histological grade, and pathological degree(P>0.05).3 MAGE-A11 protein was detected in normal testis tissues and laryngeal squamous cell carcinoma. 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, 51.89%(55/106)laryngeal squamous cell carcinoma tissues showed MAGE-A11 protein expression.In laryngeal squamous cell carcinoma tissues, the expression of MAGE-A11 protein is positively related to clinical stages, tumor size and lymph node metastasis(P<0.05). There were no significant differences between MAGE-A11 protein expression and patient’s age, gender, smoking histological grade, and pathological degree(P>0.05).4 In 106 laryngeal squamous cell carcinoma tissues, MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9 and-A11 showed uniform distribution. 19 of 60(31.67%) laryngeal squamous cell carcinoma 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 106 corresponding normal tissues(χ2=1.585, P=0.453>0.05).5 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 laryngeal squamous cell carcinoma. In univariate analysis, high protein expression levels of MAGE-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12), MAGE-A9, and MAGE-A11, high clinical stage, lymph node metastasis and large tumor size were correlated with poor prognosis of laryngeal squamous cell carcinoma patients. Further assessment with Cox’s multivariable analysis showed that only high MAGE-A9(P<0.001) expression level was an independent prognostic factor for poor overall survival of laryngeal squamous cell carcinoma patients.Conclusions: The melanoma-associated antigens-As(Including MAGE-A1,-A2,-A3,-A4,-A6,-A10 and-A12),-A9,-A11 are relative tumor-specific in laryngeal squamous cell carcinoma(LSCC), and could be ideal antigens for LSCC immunotherapy. In addition, MAGE-A9 probably is a poor prognostic marker for LSCC patients. Part Ⅱ Epigenetic mechanism of MAGE-A expression in laryngealsquamous cell carcinomaObjective:To investigate the methylation levels of MAGE-A1 and MAGE-A3 in LSCC and corresponding adjacent normal tissues, and to analyze the relationship between the methylation and the expression level of MAGE-A in the corresponding samples. At the same time, the expression of DNMT1, DNMT3 a and DNMT3 b in LSCC tissues was studied, and the relationship between the methylation status of MAGE-A1 and MAGE-A3 and the expression of MAGE-A family were analyzed.Methods:We detected the methylation levels of MAGE-A1 and MAGE-A3 in106 LSCC cases and 22 corresponding cancer adjacent normal tissues by MSP method. The expression of DNA methyl transfer enzyme DNMT1, DNMT3 a and DNMT3 b were studied by immunohistochemistry. The methylation of MAGE-A1 and MAGE-A3 were analyzed by chi square test or the Fisher ’s test statistics. The relationship between the expression of DNMT1, Dnmt3 a and DNMT3 b expression of MAGE-A methylatiom state and MAGE-A were analyzed by chi square test or the Fisher ’s test statistics and correlation using the Spearman rank correlation test.Results:1 In LSCC tissues,the rate of non-methylation of MAGE-A1 gene was 39.6%(42/106).MAGE-A1 gene of 22 normal tissues was methylation status.2 In LSCC tissues,the rate of non-methylation of MAGE-A3 gene was 46.2%(49/106).MAGE-A3 gene of 22 normal tissues was methylation status.3 There was a significant positive correlation between methylation status within MAGE-A1 and MAGE-A3 promoter and protein expression level in the whole MAGE-A family(r=0.388, P=0.000)(r=0.387, P=0.000).4 In LSCC tissues, DNMT1,DNMT3 a and DNMT3 b protein were mainly expressed in cell nuclear. 34.91%(37/106) LSCC tissues showed DNMT1 protein expression. 37.74 %(40/106) LSCC tissues showed DNMT3 a protein expression. 33.02%(35/106) LSCC tissues showed DNMT3 b protein expression.In LSCC tissues, the level of demethylation of MAGE-A1 is negative related to the expression of DNMT1, DNMT3 a and DNMT3 b protein(χ2=13.017, r=-0.350, P=0.000)(χ2=19.755, r=-0.432, P=0.000)(χ2=11.038, r=-0.323,P=0.001). The level of demethylation of MAGE-A3 is also negative related to the expression of DNMT1,DNMT3 a and DNMT3 b protein(χ2=22.490, r=-0.480, P=0.000)(χ2=21.327, r=-0.449,P=0.000)(χ2=14.459,r=-0.369,P=0.000). The expression of MAGE-As is negative related to the expression of DNMT1,DNMT3 a and DNMT3 b protein(χ2=25.127, r=-0.487, P=0.000)(χ2=19.206, r=-0.426,P=0.000)(χ2=21.406,r=-0.449,P=0.000).5 MAGE-As expression was positive in Eca109 cells and negative in Hep-2.6 The results of RT-PCR showed that the expression of m RNA MAGE-As was not found in Hep-2 cells without drug. The expression of MAGE-As gene was re-expressed after 72 h by 2.5μmol/L 、 5μmol/L 5-aza-Cd R. The expression of MAGE-As became more obvious when concentration was increased.Conclusions: The DNA methyl transferase DNMT gene family regulates the expression of MAGE-A gene by participating in the methylation process of MAGE-A family, which may play an important role in the occurrence and development of LSCC. Part Ⅲ Expression of MAGE-A gene in peripheral blood circulatingtumor cells(CTCs) of laryngeal squamous cell carcinomapatientsObjective:To detect the expression of MAGE-A gene in peripheral blood circulating tumor cells of laryngeal squamous cell carcinoma patients, and investigate their relationship with the prognosis.Methods:Multiplex nested RT-PCR was used to detect the level of MAGE-A m RNA in peripheral blood circulating tumor cells of 65 laryngeal squamous cell carcinoma patients and 30 health donors. Restriction endonuclease treatment was used to detect the expression of each member of MAGE-A family, including MAGE-A1,-A2,-A3,-A4 and-A6 genes. RT-PCR was used to detect the level of MAGE-A9 and MAGE-A11 genes.Results:1 The expression rate of MAGE-A gene was 29.23%(19/65) in LSCC peripheral blood, which was detected by multi RT-nested PCR. Restriction endonuclease treatment was proceeded by Bcl I, Eco RI, Eco47 III, Sph I and Afl III matching with MAGE-A1,-A2,-A3,-A4 and-A6 genes, respectively. The frequency of MAGE-A expression in LSCC peripheral blood was the following order: A2>A3>A1>A4>A6. The expression rate of MAGE-A1 was 16.90%(11/65), MAGE-A2 21.54%(14/65), MAGE-A3 20%(13/65), MAGE-A4 15.38%(10/65), MAGE-A6 13.85%(9/65), MAGE-A9 18.46%(12/65), MAGE-A11 23.08%(15/65). 17 of 65 LSCC peripheral blood were positive for only one MAGE-A gene, 11 of 65 LSCC peripheral blood were positive for two genes, 8 of 65 LSCC peripheral blood were positive for three genes, 0 of 65 LSCC peripheral blood were positive for four genes, 5 of 65 LSCC peripheral blood were positive for five genes, 3 of 65 LSCC peripheral blood were positive for six genes. In two patients, all seven MAGE-A genes tested were expressed.2 MAGE-A gene expression was correlated with multiple clinical/biological factors. With increase of tumor size, the MAGE-A2 gene expression rate was also increased(χ2=4.916,P=0.027). With increase of tumor size, the MAGE-A9 gene expression rate was also increased(χ2=7.304,P=0.007). With increase of histological grade, the MAGE-A6 gene expression rate was also increased(χ2=4.746,P=0.029). The MAGE-A gene expression rate of LSCC patients with lymph node metastasis was significantly higher than patients without lymph node metastasis(χ2=17.868,P=0.000). The MAGE-A1 gene expression rate of LSCC patients with lymph node metastasis was significantly higher than patients without lymph node metastasis(χ2=10.943,P=0.001). The MAGE-A2 gene expression rate of LSCC patients with lymph node metastasis was significantly higher than patients without lymph node metastasis(χ2=13.848,P=0.000). The MAGE-A3 gene expression rate of LSCC patients with lymph node metastasis was significantly higher than patients without lymph node metastasis(χ2=16.250,P=0.000). The MAGE-A4 gene expression rate of LSCC patients with lymph node metastasis was significantly higher than patients without lymph node metastasis(χ2=13.446,P=0.000). The MAGE-A6 gene expression rate of LSCC patients with lymph node metastasis was significantly higher than patients without lymph node metastasis(χ2=16.565,P=0.000). The MAGE-A9 gene expression rate of LSCC patients with lymph node metastasis was significantly higher than patients without lymph node metastasis(χ2=18.092,P=0.000). The MAGE-A11 gene expression rate of LSCC patients with lymph node metastasis was significantly higher than patients without lymph node metastasis(χ2=21.262,P=0.000). With increase of clinical stage, the MAGE-A gene expression rate was increased(χ2=5.311,P=0.021). With increase of clinical stage, the MAGE-A1 gene expression rate was increased(χ2=6.237,P=0.013). With increase of clinical stage, the MAGE-A2 gene expression rate was increased(χ2=6.032,P=0.014). With increase of clinical stage, the MAGE-A11 gene expression rate was increased(χ2=10.543,P=0.001). With increase of Smoking, the MAGE-A gene expression rate was also increased(χ2=5.831, P=0.016). With increase of Smoking, the MAGE-A2 gene expression rate was also increased(χ2=4.406,P=0.036). With increase of Smoking, the MAGE-A3 gene expression rate was also increased(χ2=4.406,P=0.036). The above results suggest the m RNA levels of MAGE-A gene in peripheral blood can be used as an important maker for monitoring the prognosis of LSCC.Conclusions:1 MAGE-A gene expression in peripheral blood may be as a important maker for detection of LSCC CTCs.2 The expression of MAGE-A gene may be correlated with prognosis of LSCC, so as to do an important marker for monitoring the treatment and prognosis of LSCC. |
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