结直肠癌中黑色素瘤抗原基因表达及启动子去甲基化状态的研究
摘要
背景和目的
结直肠癌(coloretal carcinoma,CRC)是世界上常见的消化道恶性肿瘤。在西方经济发达的国家,结直肠癌的死亡率是仅次于肺癌,居第二位;近二十年来,结直肠癌在我国的发病率总体上呈上升趋势,并随着人类环境、饮食结构和生活方式的改变,结直肠癌的发病率还会继续上升,在中国恶性肿瘤中发病率和死亡率居第4～6位。尽管相对于其它肿瘤来说,结直肠癌的发病机制研究的比较深入,诊治技术和方法也有了很大提高,但仍未取得突破性进展,尤其是进展期结直肠癌的五年生存率仍未得到根本性提高。只有10%～15%的病人能获得早期诊断,大部分住院病人已处于进展期,目前治疗方法仍局限于外科手术和放化疗,总的临床疗效不佳。积极开展结直肠癌发生机制的研究,寻找新的包括肿瘤免疫治疗在内的多种高效特异的治疗手段对于结直肠癌的防治具有重要的现实意义。
自上世纪90年代以来,随着T细胞识别机制的阐明,重组DNA技术的发展和肿瘤基因研究的深入,有关肿瘤抗原的性质、表位结构以及利用肿瘤抗原进行抗肿瘤治疗等研究都取得了很大的进展。研制和开发新型抗肿瘤疫苗已成为近年来国际上肿瘤免疫治疗的热点之一。在细胞癌变和肿瘤发生发展的早期非常有必要寻找一个新的分子生物学标记,以提高结直肠癌的早期诊断和治疗技术水平。同时,开展结直肠癌免疫治疗的前提是寻找针对结直肠肿瘤的特异性肿瘤抗原。在众多肿瘤相关抗原中,癌睾丸抗原(cancer/testis antigen,CTA)因其特异的表达模式,即在各种肿瘤组织中均有不同频率的表达,而在正常组织中仅限于睾丸的生殖细胞。因睾丸组织缺乏人类白细胞抗原(HLA)分子,是免疫豁免器官,不会因自体免疫反应对其它组织造成损害。因此,CTA适于作为特异性肿瘤免疫治疗的靶抗原。黑色素瘤抗原基因(melanoma antigen gene,MAGE)是CTA亚家族成员,因其在多种肿瘤细胞的特异性表达及在多种组织类型肿瘤细胞的广泛表达成为多肽疫苗研究的首选,其编码的抗原能被自体细胞毒性T淋巴细胞识别,诱导出对相应肿瘤细胞的特异性杀伤而达到肿瘤治疗的目的。因此,MAGE抗原肽是一种CTL(cytoxic T lymphocytes,CTL)介导的肿瘤特异性免疫治疗的理想靶分子。
MAGE基因在转录水平与蛋白表达水平表达有时出现分离的现象,即表达mRNA的组织不一定同时表达蛋白。因此,了解同一组织中基因转录和蛋白表达水平上的分离现象十分必要,因判断以MAGE抗原为基础的肿瘤疫苗可行性的关键步骤是了解这种抗原能否在蛋白水平上表达。
研究已表明,基因表达的调控主要通过遗传学(genetics)和表遗传学(epigenetics)两种机制实现。遗传学机制涉及到基因结构的改变,但只能解释肿瘤发病机制中的一部分原因,如基因突变、基因复制时的获得或丢失和染色体异常。还有许多具有重要的功能基因是通过表遗传学机制参与肿瘤的发生、发展。表遗传学是一种不涉及核苷酸序列变化可遗传的基因表达方式的改变,包括DNA甲基化和去甲基化、组蛋白乙酰化和去乙酰化。DNA甲基化是表遗传学的主要修饰形式,是目前研究最深入、最广泛的作用方式。DNA 5-胞嘧啶甲基化改变特别是启动子区CpG岛甲基化是导致基因组不稳定而引发肿瘤的重要机制。许多研究显示启动子5′CpG岛的超甲基化可使原来表达的基因沉默而导致肿瘤的发生,如肿瘤抑制基因、细胞循环调节基因、DNA错配修复基因。可以说启动子区CpG岛异常甲基化是肿瘤发生的早期事件。MAGE基因启动子区CpG岛的甲基化/去甲基化也是调控其表达的重要机制,并已在胃癌、黑色素瘤等肿瘤细胞和组织中观察到,但结直肠癌组织中MAGE基因表达与启动子的去甲基化是否有关,还没有得到完全证实。
尽管MAGE基因的研究已有多年,但有关MAGE家族基因在结直肠癌中的表达研究不多,且多是针对欧美人群,针对中国人的研究很少,其表达频率的差异也很大。目前,MAGE基因在结直肠癌组织中的较为准确的表达还不是很清楚,其表达调控的机制也未得到完全证实,在从正常组织、腺瘤到癌组织的发展过程中,MAGE基因启动子去甲基化状态的变化尚未证实。为此,本课题就以下方面进行研究:
1.应用RT-PCR和免疫组化技术研究MAGE-A1、-A3、-A4在结直肠癌细胞和组织中的表达,探讨其作为结直肠癌肿瘤免疫治疗的靶抗原以及作为免疫学检测的分子生物学标记物的可能性。
2.应用甲基化特异性PCR(MSP)研究MAGE-A1、-A3基因启动子区CpG岛从正常粘膜→腺瘤→癌组织的去甲基化状态,探讨其去甲基化状态与基因表达的关系与临床病理特征的关系,寻找可能有助于结直肠癌早期诊断和具有生物学特征的分子标记物。
材料和方法
细胞株:HT-29,HCT116、SW116,购自中国医学科学院上海细胞库。标本采集:2006年7月～11月于广西医科大学第一附属医院结直肠外科行手术治疗的65例结直肠癌组织及相应的正常粘膜标本及行手术或纤维结肠镜腺瘤切除治疗的28例腺瘤标本。所有标本均经病理组织学证实。
(1)提取结直肠正常粘膜和癌组织的总RNA,用RT-PCR法检测组织中MAGE-A1、-A3和-A4基因mRNA表达频率。
(2)应用免疫组化技术(IHC),检测同一组织标本MAGE-A1、-A3和-A4蛋白的表达。
(3)提取正常粘膜、腺瘤和癌组织的DNA,用甲基化特异性PCR(MSP)检测MAGE-A1、-A3基因启动子区CpG岛去甲基化的状态。
统计学方法:应用SPSS13.0统计学软件进行数据分析。行×列配对计数资料采用Pearson X~2检验或Fisher确切概率法检验,相关性分析采用Spearman相关检验,一致性分析采用Kappa分析。P＜0.05有统计学意义。
结果
1.RT-PCR结果:
1.1细胞株中MAGE-A1、-A3、-A4基因mRNA表达分别为:MAGE-A1,HT-29(-),HCT116(-),SW116(+);MAGE-A3,HT-29(+),HCT116(+),SW116(-);MAGE-A4均为(-)。65例结直肠癌组织中其表达率分别为18.5%(12/65)、33.9%(22/65)和20%(13/65),53.8%(35/65)的标本至少表达一种抗原基因,相应的正常粘膜组织均未检测到基因的表达。
1.2 CRC组织中MAGE-A1和-A3基因表达均与淋巴结转移有关(P=0.023,0.022),MAGE-A1表达与年龄有关(P=0.011),与性别、CEA、肿瘤大小和位置、组织分化程度、肿瘤浸润深度及TNM分期无关(P＞0.05)。
2.IHC结果:
2.1结直肠癌组织MAGE-A1、-A3抗原定位于细胞质,MAGE-A4抗原主要定位于胞质,少部分定位于胞核。
2.2 MAGE-A1、-A3、-A4蛋白的表达率分别为15.3%、29.2%、16.9%,正常组织未见表达。MAGE-A1、-A3和-A4蛋白表达均与患者的年龄、性别、CEA、肿瘤大小和位置、侵袭深度及TNM分期之间均无显著性差异(P＞0.05),但MAGE-A3表达与有无淋巴结转移之间差异有显著性(P=0.004),且呈正相关(rs=0.392,P=0.001)。
2.3 65例CRC组织中MAGE-A1基因mRNA阳性12例,蛋白表达9例(r=0.786,K=0.782＞0.7,P均＜0.01);MAGE-A3基因mRNA阳性22例,蛋白阳性18例(r=0.827,k=0.822＞0.7,P均＜0.01);MAGE-A4基因mRNA阳性13例,蛋白阳性11例(r=0.903,K=0.898＞0.7,P均＜0.01)。其中MAGE-A1和-A3各有1例mRNA阴性表达而蛋白呈阳性表达。三种基因的mRNA和蛋白表达均有很高的一致性,呈正相关。
3.MSP结果
3.1结肠癌细胞株MAGE-A1基因表达与甲基化:HT-29mRNA(-),M(+),U(-);HCT116 mRNA(-),M(+)、U(+);SW116 mRNA(+),M(-)、U(+)。MAGE-A3基因表达与甲基化:HT-29 mRNA(+),M(+),U(-);HCT116mRNA(+),M(-),U(+);SW116 mRNA(-),M(+),U(-)。
3.2正常组织、腺瘤及癌组织MAGE-A1基因启动子去甲基化阳性率分别为1.5%(1/65)、14.3%(4/28)、29.2%(19/65);MAGE-A3基因去甲基化阳性率分别为4.6%(3/65)、28.6%(8/28)、47.7%(31/65)。正常组织中两个基因启动子去甲基化率均显著低于腺瘤和癌组织的去甲基化率(P＜0.05),而腺瘤和癌组织之间比较则没有统计学意义(P＞0.05)。
3.3 MAGE-A1、-A3去甲基化状态分别与肿瘤分化程度、有无淋巴结转移有关(P=0.009,0.004;0.042,0.000),而与性别、肿瘤大小、位置、浸润深度和TNM分期无关(P＞0.05);MAGE-A1基因启动子去甲基化状态与年龄有关(P=0.001)。
3.4将癌组织标本按启动子去甲基化状态分为启动子去甲基化组和甲基化组。MAGE-A1启动子去甲基化组mRNA阳性表达率为57.9%(11/19),显著高于甲基化组的2.2%(1/46)(r=0.653,K=0.625＞0.4,P均＜0.01);MAGE-A3启动子去甲基化组的mRNA阳性表达率为64.5%(20/31)显著高于甲基化组的43%(2/46)(r=0.653,K=0.632＞0.4,P均＜0.01)。启动子去甲基化状态与基因表达均具有一致性,呈正相关。
3.5将癌组织标本按启动子去甲基化状态分为启动子去甲基化组和甲基化组。MAGE-A1启动子去甲基化组的蛋白表达阳性表达率为47.4%(9/19)显著高于甲基化组的2.2%(1/46)(r=0.570,K=0.525＞0.4,P均＜0.01);MAGE-A3启动子去甲基化组的蛋白阳性表达率为54.8%(17/31)显著高于甲基化组的5.9%(2/34)(r=0.538,k=0.498＞0.4,P均＜0.01)。两个基因去甲基化水平与其蛋白表达均具有一致性,呈正相关。
结论
1.结直肠癌细胞株和组织中MAGE-A1、-A3、-A4基因在mRNA和蛋白水平均有表达。MAGE-A3在结直肠癌细胞株和组织中表达较高,具有作为结直肠癌肿瘤免疫治疗靶基因的可能。
2.MAGE-A1、-A3基因mRNA表达与淋巴结转移有关,有淋巴结转移的结直肠癌可能伴随较高的表达,且MAGE-A3蛋白表达与有无淋巴结转移呈正相关。
3.MAGE-A1、-A3基因启动子去甲基化阳性率在正常组织、腺瘤和癌组织中显著增高,在腺瘤和癌组织中差异无显著性,正常组织很少呈去甲基化状态。CRC中去甲基化状态均与mRNA和蛋白表达呈正相关。
4.MAGE-A1、-A3启动子去甲基化状态与淋巴结转移、组织分化程度有关。MAGE-A3基因去甲基化阳性率较高,对预测淋巴结转移、判断组织分化可能有参考价值。
5.MAGE-A1、MAGE-A3基因启动子去甲基化在结直肠癌发病早期就存在,贯穿在结直肠癌发生进展的全过程,与基因的转录表达有关,在细胞癌变和肿瘤的发生发展过程中可能起到一个重要的作用。
Background and objective
Colorectal 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 4~(th)to 6~(th)among 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 CRC
2.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 Methods
Three 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 x~2 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.
Results
1.RT-PCR
1.1 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.IHC
2.1 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.MSP
3.1 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.
Conclusions
1.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.
结直肠癌(coloretal carcinoma,CRC)是世界上常见的消化道恶性肿瘤。在西方经济发达的国家,结直肠癌的死亡率是仅次于肺癌,居第二位;近二十年来,结直肠癌在我国的发病率总体上呈上升趋势,并随着人类环境、饮食结构和生活方式的改变,结直肠癌的发病率还会继续上升,在中国恶性肿瘤中发病率和死亡率居第4～6位。尽管相对于其它肿瘤来说,结直肠癌的发病机制研究的比较深入,诊治技术和方法也有了很大提高,但仍未取得突破性进展,尤其是进展期结直肠癌的五年生存率仍未得到根本性提高。只有10%～15%的病人能获得早期诊断,大部分住院病人已处于进展期,目前治疗方法仍局限于外科手术和放化疗,总的临床疗效不佳。积极开展结直肠癌发生机制的研究,寻找新的包括肿瘤免疫治疗在内的多种高效特异的治疗手段对于结直肠癌的防治具有重要的现实意义。
自上世纪90年代以来,随着T细胞识别机制的阐明,重组DNA技术的发展和肿瘤基因研究的深入,有关肿瘤抗原的性质、表位结构以及利用肿瘤抗原进行抗肿瘤治疗等研究都取得了很大的进展。研制和开发新型抗肿瘤疫苗已成为近年来国际上肿瘤免疫治疗的热点之一。在细胞癌变和肿瘤发生发展的早期非常有必要寻找一个新的分子生物学标记,以提高结直肠癌的早期诊断和治疗技术水平。同时,开展结直肠癌免疫治疗的前提是寻找针对结直肠肿瘤的特异性肿瘤抗原。在众多肿瘤相关抗原中,癌睾丸抗原(cancer/testis antigen,CTA)因其特异的表达模式,即在各种肿瘤组织中均有不同频率的表达,而在正常组织中仅限于睾丸的生殖细胞。因睾丸组织缺乏人类白细胞抗原(HLA)分子,是免疫豁免器官,不会因自体免疫反应对其它组织造成损害。因此,CTA适于作为特异性肿瘤免疫治疗的靶抗原。黑色素瘤抗原基因(melanoma antigen gene,MAGE)是CTA亚家族成员,因其在多种肿瘤细胞的特异性表达及在多种组织类型肿瘤细胞的广泛表达成为多肽疫苗研究的首选,其编码的抗原能被自体细胞毒性T淋巴细胞识别,诱导出对相应肿瘤细胞的特异性杀伤而达到肿瘤治疗的目的。因此,MAGE抗原肽是一种CTL(cytoxic T lymphocytes,CTL)介导的肿瘤特异性免疫治疗的理想靶分子。
MAGE基因在转录水平与蛋白表达水平表达有时出现分离的现象,即表达mRNA的组织不一定同时表达蛋白。因此,了解同一组织中基因转录和蛋白表达水平上的分离现象十分必要,因判断以MAGE抗原为基础的肿瘤疫苗可行性的关键步骤是了解这种抗原能否在蛋白水平上表达。
研究已表明,基因表达的调控主要通过遗传学(genetics)和表遗传学(epigenetics)两种机制实现。遗传学机制涉及到基因结构的改变,但只能解释肿瘤发病机制中的一部分原因,如基因突变、基因复制时的获得或丢失和染色体异常。还有许多具有重要的功能基因是通过表遗传学机制参与肿瘤的发生、发展。表遗传学是一种不涉及核苷酸序列变化可遗传的基因表达方式的改变,包括DNA甲基化和去甲基化、组蛋白乙酰化和去乙酰化。DNA甲基化是表遗传学的主要修饰形式,是目前研究最深入、最广泛的作用方式。DNA 5-胞嘧啶甲基化改变特别是启动子区CpG岛甲基化是导致基因组不稳定而引发肿瘤的重要机制。许多研究显示启动子5′CpG岛的超甲基化可使原来表达的基因沉默而导致肿瘤的发生,如肿瘤抑制基因、细胞循环调节基因、DNA错配修复基因。可以说启动子区CpG岛异常甲基化是肿瘤发生的早期事件。MAGE基因启动子区CpG岛的甲基化/去甲基化也是调控其表达的重要机制,并已在胃癌、黑色素瘤等肿瘤细胞和组织中观察到,但结直肠癌组织中MAGE基因表达与启动子的去甲基化是否有关,还没有得到完全证实。
尽管MAGE基因的研究已有多年,但有关MAGE家族基因在结直肠癌中的表达研究不多,且多是针对欧美人群,针对中国人的研究很少,其表达频率的差异也很大。目前,MAGE基因在结直肠癌组织中的较为准确的表达还不是很清楚,其表达调控的机制也未得到完全证实,在从正常组织、腺瘤到癌组织的发展过程中,MAGE基因启动子去甲基化状态的变化尚未证实。为此,本课题就以下方面进行研究:
1.应用RT-PCR和免疫组化技术研究MAGE-A1、-A3、-A4在结直肠癌细胞和组织中的表达,探讨其作为结直肠癌肿瘤免疫治疗的靶抗原以及作为免疫学检测的分子生物学标记物的可能性。
2.应用甲基化特异性PCR(MSP)研究MAGE-A1、-A3基因启动子区CpG岛从正常粘膜→腺瘤→癌组织的去甲基化状态,探讨其去甲基化状态与基因表达的关系与临床病理特征的关系,寻找可能有助于结直肠癌早期诊断和具有生物学特征的分子标记物。
材料和方法
细胞株:HT-29,HCT116、SW116,购自中国医学科学院上海细胞库。标本采集:2006年7月～11月于广西医科大学第一附属医院结直肠外科行手术治疗的65例结直肠癌组织及相应的正常粘膜标本及行手术或纤维结肠镜腺瘤切除治疗的28例腺瘤标本。所有标本均经病理组织学证实。
(1)提取结直肠正常粘膜和癌组织的总RNA,用RT-PCR法检测组织中MAGE-A1、-A3和-A4基因mRNA表达频率。
(2)应用免疫组化技术(IHC),检测同一组织标本MAGE-A1、-A3和-A4蛋白的表达。
(3)提取正常粘膜、腺瘤和癌组织的DNA,用甲基化特异性PCR(MSP)检测MAGE-A1、-A3基因启动子区CpG岛去甲基化的状态。
统计学方法:应用SPSS13.0统计学软件进行数据分析。行×列配对计数资料采用Pearson X~2检验或Fisher确切概率法检验,相关性分析采用Spearman相关检验,一致性分析采用Kappa分析。P＜0.05有统计学意义。
结果
1.RT-PCR结果:
1.1细胞株中MAGE-A1、-A3、-A4基因mRNA表达分别为:MAGE-A1,HT-29(-),HCT116(-),SW116(+);MAGE-A3,HT-29(+),HCT116(+),SW116(-);MAGE-A4均为(-)。65例结直肠癌组织中其表达率分别为18.5%(12/65)、33.9%(22/65)和20%(13/65),53.8%(35/65)的标本至少表达一种抗原基因,相应的正常粘膜组织均未检测到基因的表达。
1.2 CRC组织中MAGE-A1和-A3基因表达均与淋巴结转移有关(P=0.023,0.022),MAGE-A1表达与年龄有关(P=0.011),与性别、CEA、肿瘤大小和位置、组织分化程度、肿瘤浸润深度及TNM分期无关(P＞0.05)。
2.IHC结果:
2.1结直肠癌组织MAGE-A1、-A3抗原定位于细胞质,MAGE-A4抗原主要定位于胞质,少部分定位于胞核。
2.2 MAGE-A1、-A3、-A4蛋白的表达率分别为15.3%、29.2%、16.9%,正常组织未见表达。MAGE-A1、-A3和-A4蛋白表达均与患者的年龄、性别、CEA、肿瘤大小和位置、侵袭深度及TNM分期之间均无显著性差异(P＞0.05),但MAGE-A3表达与有无淋巴结转移之间差异有显著性(P=0.004),且呈正相关(rs=0.392,P=0.001)。
2.3 65例CRC组织中MAGE-A1基因mRNA阳性12例,蛋白表达9例(r=0.786,K=0.782＞0.7,P均＜0.01);MAGE-A3基因mRNA阳性22例,蛋白阳性18例(r=0.827,k=0.822＞0.7,P均＜0.01);MAGE-A4基因mRNA阳性13例,蛋白阳性11例(r=0.903,K=0.898＞0.7,P均＜0.01)。其中MAGE-A1和-A3各有1例mRNA阴性表达而蛋白呈阳性表达。三种基因的mRNA和蛋白表达均有很高的一致性,呈正相关。
3.MSP结果
3.1结肠癌细胞株MAGE-A1基因表达与甲基化:HT-29mRNA(-),M(+),U(-);HCT116 mRNA(-),M(+)、U(+);SW116 mRNA(+),M(-)、U(+)。MAGE-A3基因表达与甲基化:HT-29 mRNA(+),M(+),U(-);HCT116mRNA(+),M(-),U(+);SW116 mRNA(-),M(+),U(-)。
3.2正常组织、腺瘤及癌组织MAGE-A1基因启动子去甲基化阳性率分别为1.5%(1/65)、14.3%(4/28)、29.2%(19/65);MAGE-A3基因去甲基化阳性率分别为4.6%(3/65)、28.6%(8/28)、47.7%(31/65)。正常组织中两个基因启动子去甲基化率均显著低于腺瘤和癌组织的去甲基化率(P＜0.05),而腺瘤和癌组织之间比较则没有统计学意义(P＞0.05)。
3.3 MAGE-A1、-A3去甲基化状态分别与肿瘤分化程度、有无淋巴结转移有关(P=0.009,0.004;0.042,0.000),而与性别、肿瘤大小、位置、浸润深度和TNM分期无关(P＞0.05);MAGE-A1基因启动子去甲基化状态与年龄有关(P=0.001)。
3.4将癌组织标本按启动子去甲基化状态分为启动子去甲基化组和甲基化组。MAGE-A1启动子去甲基化组mRNA阳性表达率为57.9%(11/19),显著高于甲基化组的2.2%(1/46)(r=0.653,K=0.625＞0.4,P均＜0.01);MAGE-A3启动子去甲基化组的mRNA阳性表达率为64.5%(20/31)显著高于甲基化组的43%(2/46)(r=0.653,K=0.632＞0.4,P均＜0.01)。启动子去甲基化状态与基因表达均具有一致性,呈正相关。
3.5将癌组织标本按启动子去甲基化状态分为启动子去甲基化组和甲基化组。MAGE-A1启动子去甲基化组的蛋白表达阳性表达率为47.4%(9/19)显著高于甲基化组的2.2%(1/46)(r=0.570,K=0.525＞0.4,P均＜0.01);MAGE-A3启动子去甲基化组的蛋白阳性表达率为54.8%(17/31)显著高于甲基化组的5.9%(2/34)(r=0.538,k=0.498＞0.4,P均＜0.01)。两个基因去甲基化水平与其蛋白表达均具有一致性,呈正相关。
结论
1.结直肠癌细胞株和组织中MAGE-A1、-A3、-A4基因在mRNA和蛋白水平均有表达。MAGE-A3在结直肠癌细胞株和组织中表达较高,具有作为结直肠癌肿瘤免疫治疗靶基因的可能。
2.MAGE-A1、-A3基因mRNA表达与淋巴结转移有关,有淋巴结转移的结直肠癌可能伴随较高的表达,且MAGE-A3蛋白表达与有无淋巴结转移呈正相关。
3.MAGE-A1、-A3基因启动子去甲基化阳性率在正常组织、腺瘤和癌组织中显著增高,在腺瘤和癌组织中差异无显著性,正常组织很少呈去甲基化状态。CRC中去甲基化状态均与mRNA和蛋白表达呈正相关。
4.MAGE-A1、-A3启动子去甲基化状态与淋巴结转移、组织分化程度有关。MAGE-A3基因去甲基化阳性率较高,对预测淋巴结转移、判断组织分化可能有参考价值。
5.MAGE-A1、MAGE-A3基因启动子去甲基化在结直肠癌发病早期就存在,贯穿在结直肠癌发生进展的全过程,与基因的转录表达有关,在细胞癌变和肿瘤的发生发展过程中可能起到一个重要的作用。
Background and objective
Colorectal 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 4~(th)to 6~(th)among 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 CRC
2.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 Methods
Three 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 x~2 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.
Results
1.RT-PCR
1.1 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.IHC
2.1 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.MSP
3.1 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.
Conclusions
1.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.
引文
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