WNT通路抑制因子的甲基化在大肠侧向发育型肿瘤（LST）中作用的实验研究
摘要
一、目的与意义
大肠侧向发育性肿瘤是一类主要沿粘膜表面呈侧向浅表扩散,具有与其大小不相称的高恶变潜能的表浅型大肠肿瘤。目前对该肿瘤的研究还停留在临床的内镜下诊断及治疗方面。
就形态上而言,早期大肠癌可以被分为两种,一种是隆起型,另一种是平坦型。但是到目前为止,有关于平坦型大肠肿瘤的准确的表观遗传方面的变化知之甚少。
越来越多的证据表明平坦型的大肠癌可以解释大约10%-20%的大肠癌。现在仍然存在有关平坦型与隆起型肿瘤的起源和进展的争论。
Wnt介导的信号途径可受胞外分泌蛋白的调节.有两类Wnt的拮抗物：第一包括分泌的Frizzled相关蛋白(sFRP)家族,Wnt抑制因子-1(WIF-1)和Cerberus,通过直接与Wnt分子结合而抑制Wnt信号传导；第二类包括DKK家族,通过与Wnt受体复合体LRP5/L和LRP6的结合而抑制Wnt信号传导。LRP5/6和Dkk低密度脂蛋白的受体相关蛋白(LRP, low-density lipoprotein-receptor-related protein)是一类在脂代谢中具有重要作用的蛋白家族,其中哺乳动物的LRP-5,6和果蝇中的Arrow具有高度的同源性,同属一个亚家族.新近的研究发现,LRP-5/6在WNT信号通路中具有重要的作用.他们的结构特点是,均含有三个保守的区域：(1)胞外区,具有表皮生长因子(EGF, epidermal growth factor)重复序列和LDLR (low-density lipoprotein-receptor)重复序列,(2)跨膜区,(3)胞内区.LRP-5/6胞外区可以结合WNT蛋白,和Frizzled受体相互作用将信号从胞外传入胞内。已有研究结果表明,胞内区并未见明显的催化活性的结构,在WNT信号存在时,LRP-5胞内区可以募集AXIN并使其降解,从而激活WNT通路。Dkkl (dikkopfl)作为WNT胞外的抑制子,正是通过结合其受体LRP6发挥作用的。
根据Wnt蛋白转导信号的方式,将Wnt信号途径分为经典途径(canonical Wnt signal pathway)和非经典的途径(noncanonical Wnt signal pathway)。经典Wnt/β-catenin信号途径。Wnt蛋白在胞膜上与一种卷曲蛋白(frizzled,Frz)的跨膜受体相结合,同时结合的还有低密度脂蛋白受体相关蛋白(lowdensity lipoprotein receptor related protein 5 and 6, LRP5/6),这是Wnt信号通路活化的重要起始信号。通过这些受体复合物将信号传至胞质内,激活一系列蛋白的活性,包括散乱蛋白(dishevelled, Dsh)、糖原合成酶激酶(GSK-3β)、APC(andenomatous polyposis coli)、轴蛋白(AXIN)和转录调节剂β-catenin。Wnt和Frz受体的结合被WIF1和分泌性Frz相关蛋白(secreted frizzled-related proteins, SFRPs)竞争性抑制,而Dickko家族(DKK-1,DKK-2)通过间接减少可利用的辅助受体LRP的数量来间接抑制Wnt与膜受体的结合。
在正常情况下胞质内β-catenin的水平被多蛋白降解复合体调控,这组复合体包括APC、AXIN、GSK-3β和酪蛋白激酶1α/ε(CK1α/ε),复合体的功能可使β-catenin磷酸化,而后被泛素-蛋白酶体系统降解。所以正常情况下,胞质内游离β-catenin处于极低的水平。一旦Wnt蛋白与受体Frz结合,可使胞质Dsh激活而被磷酸化,进一步与APC结合,抑制GSK-3β的活性,使β-catenin不能被磷酸化,导致β-catenin在胞质内积累继而入胞核,与核内转录因子TCF/LEF(T cell factor/lymphoid enhancer factor)形成复合体,激活一系列Wnt信号靶基因的转录,包括c-my、cyclinD1、MMP-7和免疫球蛋白转录因子2(ITF-2)。核内β-catenin的出现是Wnt信号通路激活的标志。β-catenin是Wnt信号通路的正向调节因子,而APC, AXIN, GSK-3β等则是负向调节因子。
非经典的Wnt信号途径Wnt蛋白与受体的结合也可以通过其他两种途径激活信号转导。其一,与某些Frz受体的结合可以使细胞内钙离子释放,激活蛋白激酶C(PKC)。其二,Dsh激活Jun-N末端激酶(JNK)通路,从而调控细胞支架重排以及细胞的极性。非经典的Wnt信号途径在肿瘤中的作用尚不明了。
Wnt信号途径的抑制因子在肿瘤的作用越来越受到重视,本研究即着重于Wnt信号途径的抑制因子的甲基化在侧向发育型肿瘤的研究。
二、方法与内容
材料和方法
Wnt信号通路在原发性结直肠肿瘤中的活性研究。
1、103例肿瘤标本(包括52例侧向发育型肿瘤和51例隆起型腺瘤)来自南方医院消化科2006年-2009年内镜切除的标本,所有标本经过福尔马林固定,石蜡包埋。103例腺瘤标本收集到新鲜肿瘤标本的有59例(包括32例侧向发育型肿瘤和27例隆起型腺瘤),59例肿瘤标本中,我们收集到肿瘤组织及肿瘤旁的正常组织,新鲜标本立即置入组织保存液(大连宝生物工程有限公司),-80℃低温冰箱保存。
2、采用半定量RT-PCR方法,检测结直肠肿瘤组织中wnt信号通路关键分子SFRP1、SFRP2、SFRP5mRNA的表达水平。所有标本经过反转录PCR(reverse transcriptase PCR RT-PCR)用Olympus-CRI (Creative Realities, Inc.)显微摄像系统拍摄图片,并用图像分析软件Image Pro plus 6.0分析各图片累积光密度(integrated optical density, IOD)和阳性表达面积(stained area, SA),计算平均光密度(Mean density, MD),即MD=IOD/SA。
3、通过甲基化特异性PCR (methylation specific PCR, MSP)检检测结直肠肿瘤组织中wnt信号通路关键分子SFRP1、SFRP2和SFRP5的的甲基化状态。亚硫酸氢盐修饰后的DNA作为MSP的模板,采用对甲基化或非甲基化序列特异的引物进行扩增。扩增产物在2%的琼脂糖凝胶上分离,EB染色,紫外灯下成像观察。
4、采用免疫组织化学方法,检测结直肠肿瘤组织中SFRP1蛋白质的表达,并采用半定量方法对组织化学染色进行评分。
5、去甲基化试验在结肠癌HCT116细胞株中进行去甲基化试验,10μM5-氮-2-脱氧胞苷(5-aza-dCyd)去甲基化处理HCT116细胞株5天,对照组仅给予PBS处理,同样条件下培养。每个细胞株均进行3次重复独立试验。观察去甲基化处理对HCT116细胞株SFRP1、SFRP2、和SFRP5mRNA表达的影响。
6统计学处理
计量资料以X±SD表示,计数资料以百分比(%)表示。采用SPSS13.0对数据进行分析,连续变量两组间比较采用独立t检验或Mann-Whitney U检验。分类变量两组间比较采用x2检验或Fisher精确概率法,P<0.05认为差异有统计学显著性,所有检验均为双侧。
三、结果与分析
结果
早期CRC中存在Wnt信号通路的sFRP1过度甲基化。sFRP1、2、5在正常大肠黏膜甲基化率分别为13.6%(8/59)、28.8%(17/59)、27.1%(16/59)。sFRP1、2、5在LSTs甲基化率分别为61.5%(32/52)、65.4%(34/52)、55.8%(29/52)；sFRP1、2、5在PAs中分别为39.2%(20/51)、54.9%(28/51)、60.8%(31/51)；LSTs和Pas与正常黏膜比较sFRP1、2、5差异均有统计学意义(均P<0.05)。LSTs与PAs比较, sFRP2、5没有统计学意义(均P>0.05),sFRP1有统计学意义(P=0.030)。sFRP2、5 mRNA在LSTs组和PAs组中无显著差异；与PAs组相比,LSTs组sFRP1 mRNA水平(t=-3.239,P=0.002)显著降低。相应的,sFRP1蛋白(Mann-Whitney U检验,Z=-2.973,P=0.003)在PAs组的表达显著高于LSTs组；sFRP1蛋白在Pas组常常有阳性表达。
四、结论
甲基化作用在大肠侧向发育性肿瘤机制：LST的病因及病理机制仍然未完全明确,目前尚未阐明。
1.SFRP1表达缺失或下调可能是大肠侧向发育型肿瘤的一个重要特征。
2.SFRP1的甲基化在大肠侧向发育型肿瘤发生中起重要作用。
1. Objectives and significance
Laterally spreading tumors(LST) is a kind of flat colon tumor, which is charactered with laterally spread along the surface of colon membrana mucosa, and highly carcinogenesis potency. Till now, research about LST is limited in clinic endoscopy diagnosis and treatment.
Colorectal adenomas can be divided into two groups in morphology, protruded-type and flat-type. However, the accurate frequencies of genetic and epigenetic alterations in flat-type colorectal advanced adenomas (laterally spreading tumors) have remained largely unknown.
Growing evidence suggests that flat colorectal cancers (CRC) account for 10% to 20% of all CRCs and that these are frequently associated with more advanced pathologies. However, controversy exists as to the origin and progression of flat CRCs compared with the more common polypoid-type morphology.
Supporters of the adenoma-carcinoma sequence posit that flat carcinomas originate through an adenoma intermediate, either by ulceration of a polyp or through the formation of a flat adenoma. Others have asserted that the identification of flat carcinomas with no observable adenoma component suggests that flat carcinomas arise de novo without progressing through an adenomatous intermediate
The Wnt signaling pathway, which plays important roles in embryogenesis, development and homeostasis, is also closely linked to carcinogenesis. The Wnt ligands bind to Frizzled receptors and low-density lipoprotein receptor-related protein (Lrp) 5/6 co-receptors located at the plasma membrane and activate both canonical and non-canonical pathways. Activation of the canonical Wnt pathway leads to stabilization ofβ-catenin and its accumulation in the cytoplasm. This is followed by translocation to the nucleus, whereβ-catenin associates with T-cell factor (TCF)/lymphocyte enhancer factor family transcription factors to stimulate the expression of target genes. Constitutive activation of the canonical Wnt pathway resulting from mutation of APC, AXIN1 and CTNNB1 (β-catenin) has been observed in various human cancers.
The non-canonical Wnt pathways are known to transduce signals independent ofβ-catenin and to include the planar cell polarity, Wnt/Ca2+ and c-jun N-terminal kinase pathways, yet much about their roles in cancer remains unknown.
Today more and more attention is payed to Wnt pathways.In our study,we investigate effect of Wnt antagonist genes methylation on Laterally spreading tumors(LSTs).
2. Methods and project
1. A total of 103 tumors (consisted of 52 LSTs and 51 PAs) were collected from individuals who underwent endoscopic resection under total colonoscopy at Nanfang Hospital from 2006 to 2009. All of 103 tumor tissues were fixed in 10% formalin and embedded in paraffin.There are 59 tumors (consisted of 32 LSTs and 27 PAs) have-collected fesh-specimens.We have collected tumor and adjacent normal tissues in fresh ones. All of the fresh tissues were frozen immediately in liquid nitrogen and stored at -80℃until required.
2. We investigated the mRNA levels of key molecules (SFRPs) in the wnt signaling by semi-quantitative RT-PCR in tumor specimens. CRI micro color system was applied to observe and take photos. The professional image analysis software, Image Pro plus 6.0 was used to analyse the difference of integrated optical density (IOD) and stained area (SA) in the two groups, mean density(MD)= IOD/SA, and the mean concentration of the two groups was compared and analyzed.
3. We also examined the methylation status of SFRPs family in primary colorectal tumors by methylation specific PCR (MSP). DNA of all samples were extracted and modified by sodium bisulfite treatment converting unmethylated, but not methylated. This modified DNA was used as a template for PCR. Methylation Specific PCR (MSP) was performed to observe the methylation of sFRP1, sFRP2,sFRP5 in LST,pAs and their normal tissues of patients. Frequencies of Methylation of Individual Genes in LST,PAs,and Normal Colon Samples were compared and analyzed.Bisulfite modified DNA was used as a template for MSP using primers specific for either the methylated or unmethylated sequences. The amplification products were separated on a 2% agarose gel and visualized by ethidium bromide staining and UV transillumination.
4. We also examined the protein expression of SFRP1 by immunostaining in neoplastic colonic tissues. Immunoreactivity was estimated semiquantitatively.
5. Fisher exact tests. Differences were considered significant if P<0.05. All significance.tests are two-tailed. All statistical tests were performed using the software SPSS
3. Results
1. The sFRP1 is inactivated by hypermethylation in primary CRCs.
The methylation of sFRP1, sFRP2, sFRP5 in colon tissue was significantly higher in LSTs and PAs than that in normal control samples (P<0.05). There was no significant difference in the methylation of sFRP2, sFRP5 between LSTs group and Pas group, while the methylation of sFRP1 in colon samples, LSTs group was significantly higher than that in PAs group (P=0.030). Meanwhile, the expression of sFRPl mRNA in LST group was significantly lower than that in PAs group (P=0.002).. However, Protein expressions of SFRP1 were decreased significantly in LSTs compared with those in protruded adenomas (PAs) (Mann-Whitney U test, Z=-2.973, P=0.003;). SFRP1 was expressed at a very low level in LSTs. In addition, positive staining for SFRP1 was frequently observed in the adjacent morphologically normal tissue of LSTs.
2. SFRPs mRNA showed a complete absence of expression in HCT116 cells. After treatment with 5-aza-dCyd for 5 days, induction of SFRPs mRNA expression occurred in HCT116 cells.
4. Conclusions
1. Absence or downregulation of SFRP1 expression may be a characteristic in LSTs.
2. Hypermethylation of SFRP1 plays an important role in LSTs.
大肠侧向发育性肿瘤是一类主要沿粘膜表面呈侧向浅表扩散,具有与其大小不相称的高恶变潜能的表浅型大肠肿瘤。目前对该肿瘤的研究还停留在临床的内镜下诊断及治疗方面。
就形态上而言,早期大肠癌可以被分为两种,一种是隆起型,另一种是平坦型。但是到目前为止,有关于平坦型大肠肿瘤的准确的表观遗传方面的变化知之甚少。
越来越多的证据表明平坦型的大肠癌可以解释大约10%-20%的大肠癌。现在仍然存在有关平坦型与隆起型肿瘤的起源和进展的争论。
Wnt介导的信号途径可受胞外分泌蛋白的调节.有两类Wnt的拮抗物：第一包括分泌的Frizzled相关蛋白(sFRP)家族,Wnt抑制因子-1(WIF-1)和Cerberus,通过直接与Wnt分子结合而抑制Wnt信号传导；第二类包括DKK家族,通过与Wnt受体复合体LRP5/L和LRP6的结合而抑制Wnt信号传导。LRP5/6和Dkk低密度脂蛋白的受体相关蛋白(LRP, low-density lipoprotein-receptor-related protein)是一类在脂代谢中具有重要作用的蛋白家族,其中哺乳动物的LRP-5,6和果蝇中的Arrow具有高度的同源性,同属一个亚家族.新近的研究发现,LRP-5/6在WNT信号通路中具有重要的作用.他们的结构特点是,均含有三个保守的区域：(1)胞外区,具有表皮生长因子(EGF, epidermal growth factor)重复序列和LDLR (low-density lipoprotein-receptor)重复序列,(2)跨膜区,(3)胞内区.LRP-5/6胞外区可以结合WNT蛋白,和Frizzled受体相互作用将信号从胞外传入胞内。已有研究结果表明,胞内区并未见明显的催化活性的结构,在WNT信号存在时,LRP-5胞内区可以募集AXIN并使其降解,从而激活WNT通路。Dkkl (dikkopfl)作为WNT胞外的抑制子,正是通过结合其受体LRP6发挥作用的。
根据Wnt蛋白转导信号的方式,将Wnt信号途径分为经典途径(canonical Wnt signal pathway)和非经典的途径(noncanonical Wnt signal pathway)。经典Wnt/β-catenin信号途径。Wnt蛋白在胞膜上与一种卷曲蛋白(frizzled,Frz)的跨膜受体相结合,同时结合的还有低密度脂蛋白受体相关蛋白(lowdensity lipoprotein receptor related protein 5 and 6, LRP5/6),这是Wnt信号通路活化的重要起始信号。通过这些受体复合物将信号传至胞质内,激活一系列蛋白的活性,包括散乱蛋白(dishevelled, Dsh)、糖原合成酶激酶(GSK-3β)、APC(andenomatous polyposis coli)、轴蛋白(AXIN)和转录调节剂β-catenin。Wnt和Frz受体的结合被WIF1和分泌性Frz相关蛋白(secreted frizzled-related proteins, SFRPs)竞争性抑制,而Dickko家族(DKK-1,DKK-2)通过间接减少可利用的辅助受体LRP的数量来间接抑制Wnt与膜受体的结合。
在正常情况下胞质内β-catenin的水平被多蛋白降解复合体调控,这组复合体包括APC、AXIN、GSK-3β和酪蛋白激酶1α/ε(CK1α/ε),复合体的功能可使β-catenin磷酸化,而后被泛素-蛋白酶体系统降解。所以正常情况下,胞质内游离β-catenin处于极低的水平。一旦Wnt蛋白与受体Frz结合,可使胞质Dsh激活而被磷酸化,进一步与APC结合,抑制GSK-3β的活性,使β-catenin不能被磷酸化,导致β-catenin在胞质内积累继而入胞核,与核内转录因子TCF/LEF(T cell factor/lymphoid enhancer factor)形成复合体,激活一系列Wnt信号靶基因的转录,包括c-my、cyclinD1、MMP-7和免疫球蛋白转录因子2(ITF-2)。核内β-catenin的出现是Wnt信号通路激活的标志。β-catenin是Wnt信号通路的正向调节因子,而APC, AXIN, GSK-3β等则是负向调节因子。
非经典的Wnt信号途径Wnt蛋白与受体的结合也可以通过其他两种途径激活信号转导。其一,与某些Frz受体的结合可以使细胞内钙离子释放,激活蛋白激酶C(PKC)。其二,Dsh激活Jun-N末端激酶(JNK)通路,从而调控细胞支架重排以及细胞的极性。非经典的Wnt信号途径在肿瘤中的作用尚不明了。
Wnt信号途径的抑制因子在肿瘤的作用越来越受到重视,本研究即着重于Wnt信号途径的抑制因子的甲基化在侧向发育型肿瘤的研究。
二、方法与内容
材料和方法
Wnt信号通路在原发性结直肠肿瘤中的活性研究。
1、103例肿瘤标本(包括52例侧向发育型肿瘤和51例隆起型腺瘤)来自南方医院消化科2006年-2009年内镜切除的标本,所有标本经过福尔马林固定,石蜡包埋。103例腺瘤标本收集到新鲜肿瘤标本的有59例(包括32例侧向发育型肿瘤和27例隆起型腺瘤),59例肿瘤标本中,我们收集到肿瘤组织及肿瘤旁的正常组织,新鲜标本立即置入组织保存液(大连宝生物工程有限公司),-80℃低温冰箱保存。
2、采用半定量RT-PCR方法,检测结直肠肿瘤组织中wnt信号通路关键分子SFRP1、SFRP2、SFRP5mRNA的表达水平。所有标本经过反转录PCR(reverse transcriptase PCR RT-PCR)用Olympus-CRI (Creative Realities, Inc.)显微摄像系统拍摄图片,并用图像分析软件Image Pro plus 6.0分析各图片累积光密度(integrated optical density, IOD)和阳性表达面积(stained area, SA),计算平均光密度(Mean density, MD),即MD=IOD/SA。
3、通过甲基化特异性PCR (methylation specific PCR, MSP)检检测结直肠肿瘤组织中wnt信号通路关键分子SFRP1、SFRP2和SFRP5的的甲基化状态。亚硫酸氢盐修饰后的DNA作为MSP的模板,采用对甲基化或非甲基化序列特异的引物进行扩增。扩增产物在2%的琼脂糖凝胶上分离,EB染色,紫外灯下成像观察。
4、采用免疫组织化学方法,检测结直肠肿瘤组织中SFRP1蛋白质的表达,并采用半定量方法对组织化学染色进行评分。
5、去甲基化试验在结肠癌HCT116细胞株中进行去甲基化试验,10μM5-氮-2-脱氧胞苷(5-aza-dCyd)去甲基化处理HCT116细胞株5天,对照组仅给予PBS处理,同样条件下培养。每个细胞株均进行3次重复独立试验。观察去甲基化处理对HCT116细胞株SFRP1、SFRP2、和SFRP5mRNA表达的影响。
6统计学处理
计量资料以X±SD表示,计数资料以百分比(%)表示。采用SPSS13.0对数据进行分析,连续变量两组间比较采用独立t检验或Mann-Whitney U检验。分类变量两组间比较采用x2检验或Fisher精确概率法,P<0.05认为差异有统计学显著性,所有检验均为双侧。
三、结果与分析
结果
早期CRC中存在Wnt信号通路的sFRP1过度甲基化。sFRP1、2、5在正常大肠黏膜甲基化率分别为13.6%(8/59)、28.8%(17/59)、27.1%(16/59)。sFRP1、2、5在LSTs甲基化率分别为61.5%(32/52)、65.4%(34/52)、55.8%(29/52)；sFRP1、2、5在PAs中分别为39.2%(20/51)、54.9%(28/51)、60.8%(31/51)；LSTs和Pas与正常黏膜比较sFRP1、2、5差异均有统计学意义(均P<0.05)。LSTs与PAs比较, sFRP2、5没有统计学意义(均P>0.05),sFRP1有统计学意义(P=0.030)。sFRP2、5 mRNA在LSTs组和PAs组中无显著差异；与PAs组相比,LSTs组sFRP1 mRNA水平(t=-3.239,P=0.002)显著降低。相应的,sFRP1蛋白(Mann-Whitney U检验,Z=-2.973,P=0.003)在PAs组的表达显著高于LSTs组；sFRP1蛋白在Pas组常常有阳性表达。
四、结论
甲基化作用在大肠侧向发育性肿瘤机制：LST的病因及病理机制仍然未完全明确,目前尚未阐明。
1.SFRP1表达缺失或下调可能是大肠侧向发育型肿瘤的一个重要特征。
2.SFRP1的甲基化在大肠侧向发育型肿瘤发生中起重要作用。
1. Objectives and significance
Laterally spreading tumors(LST) is a kind of flat colon tumor, which is charactered with laterally spread along the surface of colon membrana mucosa, and highly carcinogenesis potency. Till now, research about LST is limited in clinic endoscopy diagnosis and treatment.
Colorectal adenomas can be divided into two groups in morphology, protruded-type and flat-type. However, the accurate frequencies of genetic and epigenetic alterations in flat-type colorectal advanced adenomas (laterally spreading tumors) have remained largely unknown.
Growing evidence suggests that flat colorectal cancers (CRC) account for 10% to 20% of all CRCs and that these are frequently associated with more advanced pathologies. However, controversy exists as to the origin and progression of flat CRCs compared with the more common polypoid-type morphology.
Supporters of the adenoma-carcinoma sequence posit that flat carcinomas originate through an adenoma intermediate, either by ulceration of a polyp or through the formation of a flat adenoma. Others have asserted that the identification of flat carcinomas with no observable adenoma component suggests that flat carcinomas arise de novo without progressing through an adenomatous intermediate
The Wnt signaling pathway, which plays important roles in embryogenesis, development and homeostasis, is also closely linked to carcinogenesis. The Wnt ligands bind to Frizzled receptors and low-density lipoprotein receptor-related protein (Lrp) 5/6 co-receptors located at the plasma membrane and activate both canonical and non-canonical pathways. Activation of the canonical Wnt pathway leads to stabilization ofβ-catenin and its accumulation in the cytoplasm. This is followed by translocation to the nucleus, whereβ-catenin associates with T-cell factor (TCF)/lymphocyte enhancer factor family transcription factors to stimulate the expression of target genes. Constitutive activation of the canonical Wnt pathway resulting from mutation of APC, AXIN1 and CTNNB1 (β-catenin) has been observed in various human cancers.
The non-canonical Wnt pathways are known to transduce signals independent ofβ-catenin and to include the planar cell polarity, Wnt/Ca2+ and c-jun N-terminal kinase pathways, yet much about their roles in cancer remains unknown.
Today more and more attention is payed to Wnt pathways.In our study,we investigate effect of Wnt antagonist genes methylation on Laterally spreading tumors(LSTs).
2. Methods and project
1. A total of 103 tumors (consisted of 52 LSTs and 51 PAs) were collected from individuals who underwent endoscopic resection under total colonoscopy at Nanfang Hospital from 2006 to 2009. All of 103 tumor tissues were fixed in 10% formalin and embedded in paraffin.There are 59 tumors (consisted of 32 LSTs and 27 PAs) have-collected fesh-specimens.We have collected tumor and adjacent normal tissues in fresh ones. All of the fresh tissues were frozen immediately in liquid nitrogen and stored at -80℃until required.
2. We investigated the mRNA levels of key molecules (SFRPs) in the wnt signaling by semi-quantitative RT-PCR in tumor specimens. CRI micro color system was applied to observe and take photos. The professional image analysis software, Image Pro plus 6.0 was used to analyse the difference of integrated optical density (IOD) and stained area (SA) in the two groups, mean density(MD)= IOD/SA, and the mean concentration of the two groups was compared and analyzed.
3. We also examined the methylation status of SFRPs family in primary colorectal tumors by methylation specific PCR (MSP). DNA of all samples were extracted and modified by sodium bisulfite treatment converting unmethylated, but not methylated. This modified DNA was used as a template for PCR. Methylation Specific PCR (MSP) was performed to observe the methylation of sFRP1, sFRP2,sFRP5 in LST,pAs and their normal tissues of patients. Frequencies of Methylation of Individual Genes in LST,PAs,and Normal Colon Samples were compared and analyzed.Bisulfite modified DNA was used as a template for MSP using primers specific for either the methylated or unmethylated sequences. The amplification products were separated on a 2% agarose gel and visualized by ethidium bromide staining and UV transillumination.
4. We also examined the protein expression of SFRP1 by immunostaining in neoplastic colonic tissues. Immunoreactivity was estimated semiquantitatively.
5. Fisher exact tests. Differences were considered significant if P<0.05. All significance.tests are two-tailed. All statistical tests were performed using the software SPSS
3. Results
1. The sFRP1 is inactivated by hypermethylation in primary CRCs.
The methylation of sFRP1, sFRP2, sFRP5 in colon tissue was significantly higher in LSTs and PAs than that in normal control samples (P<0.05). There was no significant difference in the methylation of sFRP2, sFRP5 between LSTs group and Pas group, while the methylation of sFRP1 in colon samples, LSTs group was significantly higher than that in PAs group (P=0.030). Meanwhile, the expression of sFRPl mRNA in LST group was significantly lower than that in PAs group (P=0.002).. However, Protein expressions of SFRP1 were decreased significantly in LSTs compared with those in protruded adenomas (PAs) (Mann-Whitney U test, Z=-2.973, P=0.003;). SFRP1 was expressed at a very low level in LSTs. In addition, positive staining for SFRP1 was frequently observed in the adjacent morphologically normal tissue of LSTs.
2. SFRPs mRNA showed a complete absence of expression in HCT116 cells. After treatment with 5-aza-dCyd for 5 days, induction of SFRPs mRNA expression occurred in HCT116 cells.
4. Conclusions
1. Absence or downregulation of SFRP1 expression may be a characteristic in LSTs.
2. Hypermethylation of SFRP1 plays an important role in LSTs.
引文
[1]Bird A P. CpG islands as gene markers in the vertebrate nucleus.Trends Genet, 1987,3:342～347
[2]Reik W, Santos F, Dean W. Mammalian epigenomics:reprogramming the genome for development and therapy.Theriogenology,2003,59(1):21～32
[3]Bird A. DNA methylation patterns and epigenetic memory. GenesDev,2002, 16(1):6～21
[4]Bird A P, Wolffe A P. Methylation-induced repression belts,braces, and chromatin. Cell,1999,99(5):451～454
[5]Kim J, Kollhoff A, Bergmann A, et al. Methylation-sensitive binding of transcription factor YY1 to an insulator sequence within the paternally expressed imprinted gene, Peg3. Hum Mol Genet,2003,12(3):233～245
[6]Bradbury J. Human epigenome project up and running. PloS Biol,2003,1(3): E82
[7]Eckhardt F, Lewin J, Cortese R, et al. DNA methylation profiling ofhuman chromosomes 6,20 and 22. Nat Genet,2006,38(12):1378～1385
[8]Ehrlich M, Gama-Sosa M A, Huang L H, et al. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells. Nucleic Acids Res,1982, 10(8):2709～2721
[9]Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J Mol Biol,1987,196(2):261～282
[10]Takai D, Jones P A. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci USA,2002,99 (6):3740～3745
[11]Wang Y, Leung F C. An evaluation of new criteria for CpG islands in the human genome as gene markers. Bioinformatics,2004,20(7):1170～1177
[12]Larsen F, Gundersen G, Lopez R, et al. CpG islands as gene markers in the human genome. Genomics,1992,13(4):1095～1107
[13]Hansen R S, Stoger R, Wijmenga C, et al. Escape from gene silencing in ICF syndrome:evidence for advanced replication time as a major determinant. Hum Mol Genet,2000,9(18):2575～2587
[14]Yamada Y, Watanabe H, Miura F, et al. A comprehensive analysis of allelic methylation status of CpG islands on human chromosome 21q. Genome Res, 2004,14(2):247～266
[15]Hanahan D, Weinberg R A. The hallmarks of cancer. Cell,2000,100(1):57～ 70
[16]Weber M, Davies J J, Wittig D, et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet,2005,37(8):853～862
[17]Jones P A, Baylin S B. The fundamental role of epigenetic events in cancer. Nat Rev Genet,2002,3(6):415～428
[18]Ordway J M, Budiman M A, Korshunova Y, et al. Identification of novel high-frequency DNA methylation changes in breast cancer. PLoS ONE,2007, 2(12):e1314
[19]Piotrowski A, Benetkiewicz M, Menzel U, et al. Microarray-based survey of CpG islands identifies concurrent hyper-and hypomethylation patterns in tissues derived from patients with breast cancer. Genes Chromosomes Cancer, 2006,45(7):656～667
[20]Keshet I, Schlesinger Y, Farkash S, et al. Evidence for an instructive mechanism of de novo methylation in cancer cells. Nat Genet,2006,38(2): 149～153
[21]Gebhard C, Schwarzfischer L, Pham T H, et al. Genome-wide profiling of CpG methylation identifies novel targets of aberrant hypermethylation in myeloid leukemia. Cancer Res,2006,66(12):6118～6128
[22]Yu J, Zhang H Y, Ma Z Z, et al. Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis. Cell Res,2003,13(5):319～333
[23]薛京伦.表观遗传学-原理、技术与实践.上海科学技术出版社,2006.60
[24]Costello, JF, Plass, C. Methylation matters. British Medical Journal,2001, 38:285.
[25]Dahl C, Guldberg P. DNA methylation analysis techniques. Biogerontology, 2003,4:233-50.
[26]Fraga MF, Herranz M, Espada J, et al. A mouse skin multistage carcinogenesis model reflects the aberrant DNA methylation patterns of human tumors. Cancer Res,2004,64:5527-34.
[27]Eden A, Gaudet F, Waghmare A, et al. Chromosomal instability and tumors promoted by DNA hypomethylation. Science,2003,300:455.
[28]Karpf AR, Matsui S. Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells. Cancer Res,2005,65:8635-9.
[29]Bestor TH. Transposons reanimated in mice. Cell,2005,122:322-5.
[30]Feinberg AP. Imprinting of a genomic domain of 11p15 and loss of imprinting in cancer:an introduction. Cancer Res,1999,59:1743s-1746s.
[31]Kaneda A, Feinberg AP. Loss of imprinting of IGF2:a common epigenetic modifier of intestinal tumor risk. Cancer Res,2005,65:11236-40.
[32]Sakatani T, Kaneda A, Iacobuzio-Donahue CA, et al. Loss of imprinting of Igf2 alters intestinal maturation and tumorigenesis in mice. Science,2005, 307:1976-8.
[33]Holm TM, Jackson-Grusby L, Brambrink T, et al. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell,2005,8:275-85.
[34]Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer,2004,4:143-53.
[35]Wu H, Chen Y, Liang J, et al. Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis. Nature,2005, 438:981-7.
[36]Brueckner B, Stresemann C, Kuner R, et al. The human let-7a-3 locus contains an epigenetically regulated microRNA gene with oncogenic function. Cancer Res,2007,67:1419-23.
[37]Laird PW, Jackson-Grusby L, Fazeli A, et al. Suppression of intestinal neoplasia by DNA hypomethylation. Cell,1995,81:197-205.
[38]Gaudet F, Hodgson JG, Eden A, et al. Induction of tumors in mice by genomic hypomethylation. Science,2003,300:489-92.
[39]Yarnada Y, Jackson-Grusby L, Linhart H, et al. Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis. Proc Natl Acad Sci U SA,2005,102:13580-5.
[40]Greger V, Passarge E, Hopping W, et al. Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet, 1989,83:155-8.
[41]Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med,2003,349:2042-54.
[42]Esteller M, Silva JM, Dominguez G, et al. Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst,2000,92:564-9.
[43]Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet, 2000,24:132-8.
[44]Esteller M, Fraga MF, Guo M, et al. DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis. Hum Mol Genet,2001, 10:3001-7.
[45]Esteller M. Cancer epigenomics:DNA methylomes and histone-modification maps. Nat Rev Genet,2007,8:286-98.
[46]Weber M, Davies JJ, Wittig D, et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet,2005,37:853-62.
[47]Kudo S. Endoscopic mucosal resection of flat and depressed types of early colorectal cancer. Endoscopy,1993,25:455-461.
[48]Sakiko H, Jun K, Masashi T, et al. Laterally Spreading Type of Colorectal Adenoma Exhibits a Unique Methylation Phenotype and K-ras Mutations. Gastroenterology,2006,131:379-389.
[49]Naoto Sakamoto, et al. Frequent hypermethylation of RASSF1A in early flat-type colorectal tumor。Oncogene (2004) 23,8900-8907
[50]Kazuo Hashimoto, et al.Hypermethylation Status of APC Inversely With the Presenceof Submucosal Invasion in Laterally Spreading Colorectal Tumors。 Molecular Carcinogenesis 47:1-8 (2008)
[51]LairdPW, Jackson-GrusbyL, FazeliA, etal.Suppression of intestinal neoplasia by DNA hypomethylation.Cell,1995,81:197-205.
[52]KantarjianHM, O'BrienS, CortesJ, etal.Result sofdecitabine(5-aza-20deoxycytidine) therapy in 130 patients with chronic myelogenous leukemia.Cancer,2003,98: 522-528.
[2]Reik W, Santos F, Dean W. Mammalian epigenomics:reprogramming the genome for development and therapy.Theriogenology,2003,59(1):21～32
[3]Bird A. DNA methylation patterns and epigenetic memory. GenesDev,2002, 16(1):6～21
[4]Bird A P, Wolffe A P. Methylation-induced repression belts,braces, and chromatin. Cell,1999,99(5):451～454
[5]Kim J, Kollhoff A, Bergmann A, et al. Methylation-sensitive binding of transcription factor YY1 to an insulator sequence within the paternally expressed imprinted gene, Peg3. Hum Mol Genet,2003,12(3):233～245
[6]Bradbury J. Human epigenome project up and running. PloS Biol,2003,1(3): E82
[7]Eckhardt F, Lewin J, Cortese R, et al. DNA methylation profiling ofhuman chromosomes 6,20 and 22. Nat Genet,2006,38(12):1378～1385
[8]Ehrlich M, Gama-Sosa M A, Huang L H, et al. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells. Nucleic Acids Res,1982, 10(8):2709～2721
[9]Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J Mol Biol,1987,196(2):261～282
[10]Takai D, Jones P A. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci USA,2002,99 (6):3740～3745
[11]Wang Y, Leung F C. An evaluation of new criteria for CpG islands in the human genome as gene markers. Bioinformatics,2004,20(7):1170～1177
[12]Larsen F, Gundersen G, Lopez R, et al. CpG islands as gene markers in the human genome. Genomics,1992,13(4):1095～1107
[13]Hansen R S, Stoger R, Wijmenga C, et al. Escape from gene silencing in ICF syndrome:evidence for advanced replication time as a major determinant. Hum Mol Genet,2000,9(18):2575～2587
[14]Yamada Y, Watanabe H, Miura F, et al. A comprehensive analysis of allelic methylation status of CpG islands on human chromosome 21q. Genome Res, 2004,14(2):247～266
[15]Hanahan D, Weinberg R A. The hallmarks of cancer. Cell,2000,100(1):57～ 70
[16]Weber M, Davies J J, Wittig D, et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet,2005,37(8):853～862
[17]Jones P A, Baylin S B. The fundamental role of epigenetic events in cancer. Nat Rev Genet,2002,3(6):415～428
[18]Ordway J M, Budiman M A, Korshunova Y, et al. Identification of novel high-frequency DNA methylation changes in breast cancer. PLoS ONE,2007, 2(12):e1314
[19]Piotrowski A, Benetkiewicz M, Menzel U, et al. Microarray-based survey of CpG islands identifies concurrent hyper-and hypomethylation patterns in tissues derived from patients with breast cancer. Genes Chromosomes Cancer, 2006,45(7):656～667
[20]Keshet I, Schlesinger Y, Farkash S, et al. Evidence for an instructive mechanism of de novo methylation in cancer cells. Nat Genet,2006,38(2): 149～153
[21]Gebhard C, Schwarzfischer L, Pham T H, et al. Genome-wide profiling of CpG methylation identifies novel targets of aberrant hypermethylation in myeloid leukemia. Cancer Res,2006,66(12):6118～6128
[22]Yu J, Zhang H Y, Ma Z Z, et al. Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis. Cell Res,2003,13(5):319～333
[23]薛京伦.表观遗传学-原理、技术与实践.上海科学技术出版社,2006.60
[24]Costello, JF, Plass, C. Methylation matters. British Medical Journal,2001, 38:285.
[25]Dahl C, Guldberg P. DNA methylation analysis techniques. Biogerontology, 2003,4:233-50.
[26]Fraga MF, Herranz M, Espada J, et al. A mouse skin multistage carcinogenesis model reflects the aberrant DNA methylation patterns of human tumors. Cancer Res,2004,64:5527-34.
[27]Eden A, Gaudet F, Waghmare A, et al. Chromosomal instability and tumors promoted by DNA hypomethylation. Science,2003,300:455.
[28]Karpf AR, Matsui S. Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells. Cancer Res,2005,65:8635-9.
[29]Bestor TH. Transposons reanimated in mice. Cell,2005,122:322-5.
[30]Feinberg AP. Imprinting of a genomic domain of 11p15 and loss of imprinting in cancer:an introduction. Cancer Res,1999,59:1743s-1746s.
[31]Kaneda A, Feinberg AP. Loss of imprinting of IGF2:a common epigenetic modifier of intestinal tumor risk. Cancer Res,2005,65:11236-40.
[32]Sakatani T, Kaneda A, Iacobuzio-Donahue CA, et al. Loss of imprinting of Igf2 alters intestinal maturation and tumorigenesis in mice. Science,2005, 307:1976-8.
[33]Holm TM, Jackson-Grusby L, Brambrink T, et al. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell,2005,8:275-85.
[34]Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer,2004,4:143-53.
[35]Wu H, Chen Y, Liang J, et al. Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis. Nature,2005, 438:981-7.
[36]Brueckner B, Stresemann C, Kuner R, et al. The human let-7a-3 locus contains an epigenetically regulated microRNA gene with oncogenic function. Cancer Res,2007,67:1419-23.
[37]Laird PW, Jackson-Grusby L, Fazeli A, et al. Suppression of intestinal neoplasia by DNA hypomethylation. Cell,1995,81:197-205.
[38]Gaudet F, Hodgson JG, Eden A, et al. Induction of tumors in mice by genomic hypomethylation. Science,2003,300:489-92.
[39]Yarnada Y, Jackson-Grusby L, Linhart H, et al. Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis. Proc Natl Acad Sci U SA,2005,102:13580-5.
[40]Greger V, Passarge E, Hopping W, et al. Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet, 1989,83:155-8.
[41]Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med,2003,349:2042-54.
[42]Esteller M, Silva JM, Dominguez G, et al. Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst,2000,92:564-9.
[43]Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet, 2000,24:132-8.
[44]Esteller M, Fraga MF, Guo M, et al. DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis. Hum Mol Genet,2001, 10:3001-7.
[45]Esteller M. Cancer epigenomics:DNA methylomes and histone-modification maps. Nat Rev Genet,2007,8:286-98.
[46]Weber M, Davies JJ, Wittig D, et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet,2005,37:853-62.
[47]Kudo S. Endoscopic mucosal resection of flat and depressed types of early colorectal cancer. Endoscopy,1993,25:455-461.
[48]Sakiko H, Jun K, Masashi T, et al. Laterally Spreading Type of Colorectal Adenoma Exhibits a Unique Methylation Phenotype and K-ras Mutations. Gastroenterology,2006,131:379-389.
[49]Naoto Sakamoto, et al. Frequent hypermethylation of RASSF1A in early flat-type colorectal tumor。Oncogene (2004) 23,8900-8907
[50]Kazuo Hashimoto, et al.Hypermethylation Status of APC Inversely With the Presenceof Submucosal Invasion in Laterally Spreading Colorectal Tumors。 Molecular Carcinogenesis 47:1-8 (2008)
[51]LairdPW, Jackson-GrusbyL, FazeliA, etal.Suppression of intestinal neoplasia by DNA hypomethylation.Cell,1995,81:197-205.
[52]KantarjianHM, O'BrienS, CortesJ, etal.Result sofdecitabine(5-aza-20deoxycytidine) therapy in 130 patients with chronic myelogenous leukemia.Cancer,2003,98: 522-528.