| Part one Expression and prognostic significance of MAGE-A11 in esophageal squamous cell carcinomaObjective:To investigate the expression of MAGE-A11 in esophageal squamous cell carcinoma and adjacent normal esophageal epithelium and its relationship with clinical biological indicators and prognosis.Methods:The expression of MAGE-A11 protein in 106 cases of esophageal squamous cell carcinoma and adjacent normal esophageal epithelium was detected by protein tissue microarray and immunohistochemistry.Results:1.The positive rate of MAGE-A11 protein expression in 106 cases of esophageal squamous cell carcinoma was 56.6%(60/106),and MAGE-A11 was not expressed in normal esophageal epithelium.2.The expression of MAGE-A11 protein was not related to age,sex and tumor location in patients with squamous cell carcinoma of the esophagus(P>0.05).The expression of MAGE-A11 protein was positively correlated with histological grade,TNM stage,tumor invasion,lymph node metastasis,distant metastasis or recurrence in esophageal squamous cell carcinoma(P<0.05).3.The total 5 year survival rate of MAGE-A11 positive esophageal squamous cell carcinoma was significantly lower than that of the patients with negative expression(P<0.001).4.Cox regression analysis showed that the expression of MAGE-A11 protein,lymph node metastasis and TNM staging were independent factors affecting the poor prognosis of esophageal squamous cell carcinoma.Part two The apparent regulatory mechanism of the second part of MAGE-A11 in the progression of esophageal squamous cell carcinomaObjective:To detect the expression of MAGE-A11 in esophageal squamous cell carcinoma cell lines Eca109,Ec9706,TE1,TE13,KYSE30 and KYSE170,and to study the mechanism of epigenetic regulation of MAGE-A11 in esophageal squamous cell carcinoma.Methods:1 The expression of MAGE-A11 mRNA in 6 esophageal squamous cell carcinoma cell lines was detected by qRT-PCR.2 The effect of 5-Aza-CdR(DAC)treatment on the expression of MAGE-A11 mRNA in 6 esophageal squamous cell carcinoma cell lines was detected by qRT-PCR.3 The methylation level of Cp G island in the MAGE-A11 promoter region of esophageal squamous cell carcinoma cell lines and normal esophageal epithelial cells was analyzed by BSP clone sequencing.4 Western Blot,immunostaining,and CHIP were used to analyze the correlation between TFCP2 and ZEB1 and MAGE-A11 promoter.5 QRT-PCR and Western Blot were used to analyze the effect of TFCP2 and ZEB1 transfection on the expression of MAGE-A11.6 CCK8 assay was used to analyze the effects of TFCP2 and ZEB1 on cell proliferation.7 The qRT-PCR,Western Blot,and CHIP methods were used to analyze the regulation of histone acetylation on MAGE-A11 transcription.Results:1.QRT-PCR results showed that in the 6 cell lines of esophageal squamous cell carcinoma,the MAGE-A11 mRNA of the Eca109 and KYSE30 cell lines was relatively low,while the TE13 cells showed a relatively high expression(P < 0.05).2.QRT-PCR results showed that there were different expression levels of MAGE-A11 mRNA in 6 esophageal squamous cell carcinoma cell lines treated with DAC.The cells with low expression in MAGE-A11 could be induced to increase by DAC treatment,while those with high MAGE-A11 expression were only slightly induced(P < 0.05).3.BSP showed that the MAGE-A11 promoter –140bp to +1bp region consisted of 15 CpG loci.which contained 10 Cp G sites combined with transcription factor SP1 cluster,and 3 CpG sites combined with TFCP2/ZEB1.The methylation rate of the TFCP2/ZEB1 binding region was higher than that of the SP1 binding cluster.The MAGE-A11 promoter in ESCC cells showed hypermethylation status.After DAC treatment,these Cp G sites were significantly demethated.4.The results of Western Blot,immunostaining and CHIP showed that transcription factor TFCP2 and ZEB1 in ESCC was directly bound to CpG site of MAGE-A11 promoter in a methylation dependent manner(P<0.05).5.QRT-PCR and Western Blot results showed that over expression of TFCP2 and ZEB1 promoted MAGE-A11 gene transcription.DAC treatment increased transcription factor induced MAGE-A11 expression(P<0.05).6.CCK8 results showed that over expression of MAGE-A11 increased cell proliferation.TFCP2 and ZEB1 induced the proliferation of esophageal cancer cells by regulating MAGE-A11 transcription.After knocking down MAGE-A11,these transcription factors did not increase cell proliferation ability(P<0.05).7.QRT-PCR,Western,Blot,CHIP showed that MeCP2 binds to the MAGE-A11 promoter region hypermethylation,inhibited the expression of MAGE-A11 mRNA and protein,knockdown of MeCP2 can increase the expression of MAGE-A11 mRNA and DAC induced protein,histone deacetylase HDAC1 can bind to the MAGE-A11 promoter region of promoter hypermethylation,inhibition of histone deacetylase gene expression,enzyme inhibitor TSA can increase the expression of MAGE-A11 mRNA and protein induced by DAC.Conclusion:1.MAGE-A11 can be used as a biomarker for poor prognosis in patients with esophageal squamous cell carcinoma.2.The transcriptional expression of MAGE-A11 was regulated by DNA methylation and histone acetylation in esophageal squamous cell carcinoma cells. |
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