| Background and objectiveColorectal cancer(CRC)is one of the most common gastrointestinal malignancies that severely threatens the health of mankind.In western developed countries,CRC is the second cause of cancer-related death,only next to lung carcinoma.In China,the morbidity and mortality of CRC ranks from the 4thto 6thamong all the cancer-related death.Furthermore,the mortality is still increasing,expecially in the urban and developed rural area of China,due to the changs of environment,construction of food and drink and life style.Although the wide research on the pathogenesis of CRC had been achieved,and the technology of diagnosis and therapy had substantial improvement,breaking progresses were not achieved.Especially the 5-year survial of advanced CRC does not improve significantly.Since only 10%～15%of patients are diagnosed at an early stages,most hospital cases are in the advanced stages.The theraputic approaches are often limited to surgical treatment,radiation and chemotherapy. Therefore,it is of great realistic significance to study the carcinogenesis of CRC and search the specific and more efficaciously therapeutic methods including immunotherapy for its prevention and treatment.Since the 1990s of last century,with the elucidation of T cell recogniazed mechanism,development of recombinant DNA technology and extreme research of tumor antigen,the study on the tumor antigen properties,epitope structure and anti-tumor therapy with carcinoma antigen has a significant progress.It is one of most studying domains and hot-spots of tumor immunotherapy to develop and manufacture a new anti-tumor vaccine internationally in recent years.In the meantime,it is extraordinarily necessary to search a new biomarker in the earlier period of colorectal malignant transformation for the enhancement of earlier diagnosis and treament.The premise of immunotherapy is to identify the specific tumor antigen to aim directly at CRC.Among the numerous tumor associated antigen to date,Cancer/testis antigen(CTA)was found tumorassociated antigens encoded in various tumors of different origin,but not in normal tissues other than testis which lacks of human leuocyte antigen(HLA) molecule and is an immune-privilegd organ,cancer vaccination of CTA is not expected to cause damage to normal tissues due to autoimmune responses.Thus, CTA could be properly used as targets in activation of specific tumor immunotherapy.MAGE(melanoma antigen gene)is subset of CTA,widely expressed in the same manner in a varity of multiple tumor cells and human cancer of different histological origin,recognized as a group of highly interesting targets for polypeptide vaccine,and they encode tumor antigen recognized specifically by cytotoxic T leucocytes and induce specific immune kill to corresponding tumor cells in tumorous therapy.Therefore,MAGE is highly attractive targets mediated by CTL for specific tumor immunotherapy.There is a discrepant phenomenon of expression between mRNA and protein in the same tissues with the same gene occasionaly,that is to say that the transcriptional expression of mRNA is not always accompanied with the expression of protein.Since the key step of the feasibility of tumor vaccine based on MAGE genes is observed whether their protein are expressed in the meantime,the function of any gene is embodied at the level of protein. Therefore,it is necessary to observe the discrepancy at the expression level of transcription and protein.It is well known that gene expression is regulated by two main mechanism, genetic and epigenetics regulation.Genetic alterations,once regarded as the only important regulatory model in vivo,are characterized as gene structure alterations including mutations,gain or loss of gene copies,and chromosmal aberrations.Genetic mechanism only explains the part of caues resulted in the neoplasm.However,many functional genes participate the tumorigenesis through the epigenetic modifications which have attracted more attentions recently.Epigenetics alterations modulate gene expression via base modifications in the genome without changes in the primary DNA sequence, such as DNA methylation and demethylation,histone acetylation and deacetylation.Aberrant DNA methylation is the main modifications and most extensive and extreme modes of action,also the most common epigenetic mechanism of gene regulation.DNA 5'-cytosine methylation alteration, especially in the promoter CpG islands,which results in genome instability,is the important mechanisms which initiates carcinogenesis.Many previous studies suggest that hypermethylation of the 5'-CpG islands silences genes expression including those of tumor suppressor genes,cell cycle regulatory genes and DNA mismatch repair genes in carcinogenesis.Abnormal DNA methylation of CpG islands is an early event in human carcinogenesis.Methylation/demethylation of the promoter CpG islands of MAGE genes is also recognizedas an important mechanism of the genes expression regulations.A correlation between some MAGEs expression and genome-wide hypomethylation has been observed in some types of human carcinomas,such as cells and tissues of gastric cancer, hepatocellular carcinoma and melonoma.However,it is not completely confirmed if this relationship is present in colorectal carcinomas.Notwithstanding there were many years on the research of MAGE genes, there were few studies on expression frequency about MAGE family in CRC and most is and mostly aim at foreigner,few studies aim at Chinese.The most different frequencies exist in expression of MAGEs.It is not completely confirmed that the mechanism of regulations of MAGEs in colorectal normal, adenoma and carcinomas.It is well unknown to the expression of MAGE genes in CRC tissues,and regulatory mechanism of expression.The demethylation ststus was not completely confirmed from normal,adenoma to cancerous tissues. The specific aims in this study include:1.Detect the expression of MAGE-A1,-A3 and -A4 genes in human CRC cell lines and tissues by immunohistochemical and RT-PCR analysis,and investigate the feasibility of genes encoding proteins used as a target for immunotherapy and immunological molecular markers in CRC2.Detect the demethylation status of their promoter CpG islands in MAGE-A1,-A3 genes from normal,adenoma and to cancer tissues,and their correlation with the expression of mRNA and protein in human cancer tissues. Explore the correlation between the demethylation status and clinicopathological features.Search the potential molecular marker used as an early diagnosis and biomarker of CRC.Materals And MethodsThree CRC cell lines:HT-29,HCT116,SW116 were bought from Shanghai cell banks,Chinese Academy of Medical Sciences.Samples collecting:65 surgical CRC samples from patients undergoing surgery including cancer tissues and corresponding normal mucosa,respectively,and 28 resected samples of adenoma surgically or endoscopically,at department of colorectum,the first affiliated hospital of Guangxi Medical University from July to November in 2006 were investigated in this study.The diagnosis of all the cases was confirmed by pathological histology.(1)The expression of MAGE-A1,-A3 and -A4 genes was detected by RT-PCR.(2)The expression of MAGE-A1,-A3 and -A4 proteins was detected by immunohistochemistry in the same samples. (3)Demethylation status in promoter CpG islands of MAGE-A1,-A3 genes was performed by MSP.Statistical methods:The data were analyzed using statistical software SPSS version 13.0.Paired numerous data were done using Pearson x2 or Fisher's exact test,corralation analysis Spearman corralated test.Kappa test was also used to compare the agreement.Statistical.significance was accepted at P＜0.05.Results1.RT-PCR1.1 The expression frequency of MAGE-A1,-A3,-A4 genes mRNA in cell lines was detected.MAGE-A1:HT-29(-),HCT116(-),SW116(+);MAGE-A3, HT-29(+),HCT116(+),SW116(-);MAGE-A4(-)in three cell lines,respectively; and 18.5%(12/65),33.9%(22/65)and 20%(13/65)in CRC tissues,respectively, at least one MAGE gene expression was positive in 53.8%(35/65).Conversely, none of the corresponding adjacent non-tumorous tissues expressed any MAGE gene expression.1.2 The expression frequency of MAGE-1 and MAGE-3 was related to metastasis to lymph node(P=0.023;P=0.022),and MAGE-A1 related to age. The expression of the MAGE genes was not related to age(exception of MAGE-1),gender,CEA,tumor size and location,differentiated grade,the depth of invasion,and TNM stage(P＞0.05).2.IHC2.1 MAGE-A1,-A3 antigens were located in cytoplasm,whereas MAGE-A4 was prevailingly observed in cytoplasm and minorly in nucleus.2.2 The protein expression of MAGE-A1,-A3,-A4 was positive observed in 15.3%,29.2%,and 16.9%out of the CRC tissues,respectively,while not observed in normal colorectal mucosal tissues.The expression of the genes had no relationship with age,gender,CEA,tumor size and location,differentiated grade,the depth of invasion,and TNM stage(P＞0.05)out of CRC tissues. However,the expression of MAGE-A3 genes was significantly correlated with regional lymph node metastasis in CRC(P=0.004),moreover positive correlation(rs=0.392,P=0.001).2.3 Out of 65 cases,the mRNA expression of MAGE-A1 was 12 cases, while protein expresssion was 9 cases(r=0.786,κ=0.782＞0.7,P＜0.01),the mRNA expression of MAGE-A3 22 cases,while protein expresssion 18 cases (r=0.827,κ=0.822＞0.7,P＜0.01),the mRNA expression of MAGE-A4 13 cases, while protein expresssion 11 cases(r=0.903,κ=0.898＞0.7,P＜0.01). Interstingly,mRNA of 1case was positive while protein negative in MAGE-A1 and MAGE-A3,respectively.The mRNA expression was highly consistent with protein expression,moreover positive correlation.3.MSP3.1 The mRNA expression and demethylation of MAGE-A1 in CRC cell lines:HT-29 mRNA(-),M(+),U(-);HCT116 mRNA(-),M(+),U(+); SW116 mRNA(+),M(-),U(+).MAGE-A3 in CRC cell lines:HT-29 mRNA (+),M(+),U(-);HCT116 mRNA(+),M(-),U(+);SW116 mRNA(-),M(-),U(+).3.2 The demethylation rate of MAGE-A1 promoter was observed 1.5%(1/65),14.3%(4/28),29.2%(19/65)in normal mucosal tissues,adenoma and CRC tissues,respectively.The demethylation rate of MAGE-A3 promoter was 4.6%(3/65),28.6%(8/28),47.7%(31/65)in normal mucosal tissues, adenoma and CRC tissues,respectively.The demthylation rate of the two genes in normal mucosa was significantly lower than that of the adenoma and CRC tissues(P＜0.05),but the difference between the adenoma tissues and the CRC tissues was not statistically significant P＞0.05).3.3 The demethylation status of MAGE-A1 and MAGE-A3 promoters had no relationship with gender,tumor size and location,the depth of invasion,and TNM stage(P＞0.05).However,The demethylation status of MAGE-1 and MAGE-3 promoters was related to differentiated grade,metastasis to lymph node(P=0.009,0.004;0.042,0.000).In additional,MAGE-A1 related to age(P=0.001).3.4 Sixty five samples were divided into two groups according to demethylation status:demethylation group and methylation group.The mRNA expression rate of MAGE-A1 in promoter demethylation group was significantly higher than that of methylation group(r=0.653,κ=0.625＞0.4, P＜0.01),while MAGE-A3 was also identical(r=0.653,κ=0.632＞0.4,P＜0.01). The demethylation,status of the two genes was consistent with mRNA expression,moreover positive correlation.3.5 Sixty five samples were divided into two groups according to demethylation status:demethylation group and methylation group.The protein expression of MAGE-A1 in promoter demethylation group was significantly higher than that of methylation group(r=0.570,κ=0.525＞0.4,P＜0.01),while MAGE-A3 was also identical(r=0.538,κ=0.498＞0.4,P＜0.01).The demethylation status of the two genes was consistent with protein expression, moreover positive correlation.Conclusions1.The expression of MAGE-A1,-A3,-A4 was all found positive expression in CRC cell lines and tissues at mRNA and protein levels.The higher expression of MAGE-A3 was detected in in CRC cell lines and tissues,perhaps used as candidate of targeted gene for CRC.2.The expression frequency of MAGE-1 and MAGE-3 genes mRNA was related to metastasis to lymph node,suggesting that metastasis to lymph node was accompanied with higher expression of MAGE-A1,-A3 mRNA in CRC. The expression of MAGE-3 protein was positively related to metastasis to lymph node.3.The demethylation level of promoter of MAGE-A1,-A3 was significantly increased from normal tissues,adenoma to cancerous tissues, whereas,demethylation of the genes is quite rare in normal tissues.However, there was no significant difference of demethylation level of the two genes between adenoma and CRC tissues,but the demethylation status of the two genes was positively correlated with the expression of their mRNA and protein.4.Demethylation of both MAGE-A1 and MAGE-A3 in CRC was significantly correlated with lymph node metastasis and histological differentiation.Furthermore,there was a possible application with detection of the demethylation of MAGE-A3 in predicting lymph node metastasis and judging histological differentiation for its higher demethylation.5.Demethylation occurs in the early stage of carcinogenesis,throughout the total developing process of tumorigenesis as well as upregulating transcription of both MAGE-A1 and MAGE-A3,and perhaps plays an crucial role in malignant transformation of cell and the development of carcinogenesis in CRC.
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