Dact1和Dact3基因在结直肠癌组织中的表达研究
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摘要
目的
结直肠癌是我国常见的几种恶性肿瘤之一,目前对结直肠癌发生的分子生物学机制的探讨主要集中在抑癌基因方面。而在结直肠癌发生发展中起重要作用的Wnt通路是一个研究的热点,其中Dact基因是Wnt通路中重要的调节位点。本实验的研究目的就是探讨结直肠癌中Dact1和Dact3的表达及其与结直肠癌发生的机制问题。
材料与方法
结直肠癌标本收集:于2008年1月至2009年7月,安徽医科大学杭州临床学院经病理诊断明确的结直肠癌手术切除标本及相应的癌旁组织各50例和正常人结直肠组织20例,其中结直肠癌组织,癌旁组织取自于结直肠癌手术切除之标本,癌旁组织为癌肿切缘5cm之结肠组织;正常人结直肠组织取自行PPH手术之标本。所有患者术前均末接受放、化疗及其他抗癌治疗,标本切除后立即于-80℃冰冻保存用于下一步实验。
RT-PCR:采用Trizol法抽提组织总RNA,实验步骤严格按照试剂说明书进行。紫外分光光度计检测RNA的浓度和纯度,A260/A280在1.8～2.0之间的RNA入选进行逆转录。以两步法行RT-PCR。取PCR产物在2%的琼脂糖凝胶中进行电泳,凝胶成像仪扫描、拍照,用软件分析各条带灰度值,以目的基因与内参β-actin比值表示其相对含量。
MSP(甲基化特异性PCR):酚-氯仿法提取基因DNA进行亚硫酸氢钠修饰,利用亚硫酸氢钠使DNA变性,并进行亚硫酸氢钠修饰。DNA纯化系统对修饰后的DNA标本进行纯化,再次碱变性后乙醇沉淀,溶解后用于PCR扩增模板。l0ul PCR产物加于2.0%琼脂糖凝胶中电泳,0.1%EB染色,凝胶成像系统照相分析。结果判定:只要甲基化引物扩增出目的条带,则可判定Dact1或Dact3基因启动子发生甲基化。统计学分析:采用SPSS11.5统计软件包处理数据,P<0.05有统计学意义。
结果
RT-PCR定性检测结直肠癌组织中Dact1 mRNA表达(阳性率为12%,6/50)明显低于相应的癌旁组织(阳性率为90%,45/50)和正常结直肠组织(阳性率为95%,19/20)。与正常结直肠组织和癌旁组织相比,Dact1 mRNA在结直肠癌组织中的表达有显著性差异(P<0.05)。结直肠癌组织中Dact3 mRNA表达(阳性率为10%,5/50)明显低于相应的癌旁组织(阳性率为86%,43/50)和正常结直肠组织(阳性率为90%,18/20)。与正常结直肠组织和癌旁组织相比,Dact3 mRNA在结直肠癌组织中的表达有显著性差异(P<0.05)。
通过MSP检测,结直肠癌组织的Dact1基因启动子甲基化阳性表达(阳性率为46%,23/50)明显高于癌旁组织(阳性率为12%,6/50)和正常结直肠组织(阳性率为5%,1/20)(P<0.05)。结直肠癌组织的Dact3基因启动子甲基化阳性表达(阳性率为12%,6/50)与癌旁组织(阳性率为10%,5/50)和正常结直肠组织(阳性率为10%,2/20)没有明显的差异(P>0.05)。
结论
本研究工作以结直肠癌组织、癌旁组织及对照组正常结直肠组织为研究对象,研究结直肠癌Dact1基因(Dact3基因)失活与Dact1基因(Dact3基因)启动子甲基化是否存在相关性,得出以下结论:
1、结直肠癌Dact1基因表达减少或缺失,正常组织及癌旁组织中的Dact1基因表达正常
2、结直肠癌Dact1基因启动子甲基化阳性率高于正常组织及癌旁组织。
3、结直肠癌Dact1基因mRNA的失表达(低表达)和其启动子甲基化状态可能存在相关性,Dact1基因启动子甲基化可能参与了结直肠癌的发生。
4、结直肠癌Dact3基因表达减少或缺失,正常组织及癌旁组织中的Dact3基因表达正常.
5、结直肠癌Dact3基因和正常组织及癌旁组织中的启动子甲基化状态没有明显的差异,Dact3基因不是通过启动子甲基化参与结直肠癌的发生。
我们认为Dact1基因在结直肠癌的表达失常影响了正常蛋白质的表达水平,从而直接或间接的导致了结直肠癌的发生。Dact1基因的启动子甲基化可能是Dact1基因在结直肠癌中表达失常的主要因素。而同属一族的Dact3虽也在结直肠癌中表达失常,但其和启动子甲基化的关系不大。
Background
Colorectal carcinoma is one of the most common tumor types in our country. In present, scientists focus their studies on cancer surppressor genes to find out the moleculor mechanisms of tumor oncogenesis. Wnt pathway,which plays an important role in the development of colorectal cancer,is a hot spot of research. On the while, Dact is a key regulator site in the Wnt pathway.The purpose of this study is to observe the methylation of promoter of gene Dact1(Dact3) and the expression of Dact1(Dact3) mRNA in colorectal tumor and to analyze its relationship between them with carcinogenesis of colorectal tumor.
Materials and Methods
Colorectal cancer tissues 50 pairs of cancerous and corresponding paracancerous were surgically obtained from colorectal cancer patients ,and 20 of normal colorectal tissues were surgically obtained from PPH patients, all of which in the Hangzhou Hospital of anhui Medical University between 2008.1 and 2009.7. All the cancerous tissues had clear pathological diagnosis. The paracancerous were far away about 5cm from cancerous mucoses, and also had clear pathological- diagnosis: normal mucoses cases. All patients were preoperatively end up receiving chemotherapy and other anti-cancer therapy. All the tissues were stored at -80℃for reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific polymerase chain reaction (MSP).
RT-PCR:
Total RNA was isolated from tissue samples using Trizol Reagent according to the manufacturer's protocol. The concentration and purity of total RNA were determined from the A260/A280 ratio using a UV spectrophotometer .One microgram of total RNA from each specimen was subjected to RT-PCR. Theβ-actin and Dact1(Dact3) PCR products were electrophoresed on a 2% agarose gel and visualized by staining with ethidum bromide. The integrated density values of the bands representing amplified products were acquired and analyzed by a image analysis system.
MSP:
DNA was extracted from the 50 colorectal cancers and paracancerous and 20 of normal colorectal tissues using phenol-chloroform extraction, than treated with bisulfite. briefly, DNA was denatured with NaOH and modified with sodium bisulfite. DNA samples were then purified using Wizard DNA purification resin, treated again with NaOH, precipitated with ethanol, and resuspended in water for template to PCR. l 0ul samples of each PCR reaction products were loaded directly onto 2.0% agarose gels, stained with 0.1% ethidium bromide, and visualized under UV illumination. Results determination: Only when PCR products were amplified with methylated primer, then the promoter of Dact1(Dact3) gene could be regarded as methylation.
Statistical analysis: All the data were analyzed with SPSS11.5 statistics software. The statistical significance was defined as P<0.05.
Results
The detection of colorectal cancer Dact1 mRNA positive expression (12%, 6 / 50) by Qualitative RT-PCR was lower than in the paracancerous (90%, 45/50) and normal colorectal tissues (95%, 19/20). And there were significantly different between colorectal tumor tissues and paracancerous, normal colorectal tissues respectively(P<0.05). In the same way, colorectal cancer Dact3 mRNA positive expression (10%,5/50) were lower than in paracancerous (86%,43/50) and normal colorectal tissues (90%,18/20). And there were significantly different between colorectal tumor tissues and paracancerous, normal colorectal tissues respectively(P<0.05).
Bisulphite sequencing revealed that the methylation was unequally distributed within the Dact1 promoter and by MSP analysis a region close to the transcription start point was shown to be hypermethylated in the majority of colorectal carcinomas tissues (46% , 23/50) . In contrast, normal colorectal tissues displayed lower hypermethylation (5%,1/20) as well as in paracancerous (12%,6/50). On the contrary, the hypermethylation of Dact3 have no significant difference between colorectal tumor tissues (46%,23/50) and paracancerous(10%,5/50), as well as normal colorectal tissues(10%,2/20)(P>0.05).
Conclusion
Promoter hypermethylation of Dact1 were presented in the vast majority of colorectal tumor, and only rarely in paracancerous and normal colorectal tissues, which was maybe correlated with Epigenetic silencing and down-regulation of the Dact1 gene in colorectal tumor .On the other hand, promoter hypermethylation of Dact3 had no significant difference between colorectal carcinomas tissues and paracancerous, as well as normal colorectal tissues.It maybe had no relevance in the loss (low expression) of Dact3 mRNA expression with the state of promoter methylation of Dact3.
结直肠癌是我国常见的几种恶性肿瘤之一,目前对结直肠癌发生的分子生物学机制的探讨主要集中在抑癌基因方面。而在结直肠癌发生发展中起重要作用的Wnt通路是一个研究的热点,其中Dact基因是Wnt通路中重要的调节位点。本实验的研究目的就是探讨结直肠癌中Dact1和Dact3的表达及其与结直肠癌发生的机制问题。
材料与方法
结直肠癌标本收集:于2008年1月至2009年7月,安徽医科大学杭州临床学院经病理诊断明确的结直肠癌手术切除标本及相应的癌旁组织各50例和正常人结直肠组织20例,其中结直肠癌组织,癌旁组织取自于结直肠癌手术切除之标本,癌旁组织为癌肿切缘5cm之结肠组织;正常人结直肠组织取自行PPH手术之标本。所有患者术前均末接受放、化疗及其他抗癌治疗,标本切除后立即于-80℃冰冻保存用于下一步实验。
RT-PCR:采用Trizol法抽提组织总RNA,实验步骤严格按照试剂说明书进行。紫外分光光度计检测RNA的浓度和纯度,A260/A280在1.8～2.0之间的RNA入选进行逆转录。以两步法行RT-PCR。取PCR产物在2%的琼脂糖凝胶中进行电泳,凝胶成像仪扫描、拍照,用软件分析各条带灰度值,以目的基因与内参β-actin比值表示其相对含量。
MSP(甲基化特异性PCR):酚-氯仿法提取基因DNA进行亚硫酸氢钠修饰,利用亚硫酸氢钠使DNA变性,并进行亚硫酸氢钠修饰。DNA纯化系统对修饰后的DNA标本进行纯化,再次碱变性后乙醇沉淀,溶解后用于PCR扩增模板。l0ul PCR产物加于2.0%琼脂糖凝胶中电泳,0.1%EB染色,凝胶成像系统照相分析。结果判定:只要甲基化引物扩增出目的条带,则可判定Dact1或Dact3基因启动子发生甲基化。统计学分析:采用SPSS11.5统计软件包处理数据,P<0.05有统计学意义。
结果
RT-PCR定性检测结直肠癌组织中Dact1 mRNA表达(阳性率为12%,6/50)明显低于相应的癌旁组织(阳性率为90%,45/50)和正常结直肠组织(阳性率为95%,19/20)。与正常结直肠组织和癌旁组织相比,Dact1 mRNA在结直肠癌组织中的表达有显著性差异(P<0.05)。结直肠癌组织中Dact3 mRNA表达(阳性率为10%,5/50)明显低于相应的癌旁组织(阳性率为86%,43/50)和正常结直肠组织(阳性率为90%,18/20)。与正常结直肠组织和癌旁组织相比,Dact3 mRNA在结直肠癌组织中的表达有显著性差异(P<0.05)。
通过MSP检测,结直肠癌组织的Dact1基因启动子甲基化阳性表达(阳性率为46%,23/50)明显高于癌旁组织(阳性率为12%,6/50)和正常结直肠组织(阳性率为5%,1/20)(P<0.05)。结直肠癌组织的Dact3基因启动子甲基化阳性表达(阳性率为12%,6/50)与癌旁组织(阳性率为10%,5/50)和正常结直肠组织(阳性率为10%,2/20)没有明显的差异(P>0.05)。
结论
本研究工作以结直肠癌组织、癌旁组织及对照组正常结直肠组织为研究对象,研究结直肠癌Dact1基因(Dact3基因)失活与Dact1基因(Dact3基因)启动子甲基化是否存在相关性,得出以下结论:
1、结直肠癌Dact1基因表达减少或缺失,正常组织及癌旁组织中的Dact1基因表达正常
2、结直肠癌Dact1基因启动子甲基化阳性率高于正常组织及癌旁组织。
3、结直肠癌Dact1基因mRNA的失表达(低表达)和其启动子甲基化状态可能存在相关性,Dact1基因启动子甲基化可能参与了结直肠癌的发生。
4、结直肠癌Dact3基因表达减少或缺失,正常组织及癌旁组织中的Dact3基因表达正常.
5、结直肠癌Dact3基因和正常组织及癌旁组织中的启动子甲基化状态没有明显的差异,Dact3基因不是通过启动子甲基化参与结直肠癌的发生。
我们认为Dact1基因在结直肠癌的表达失常影响了正常蛋白质的表达水平,从而直接或间接的导致了结直肠癌的发生。Dact1基因的启动子甲基化可能是Dact1基因在结直肠癌中表达失常的主要因素。而同属一族的Dact3虽也在结直肠癌中表达失常,但其和启动子甲基化的关系不大。
Background
Colorectal carcinoma is one of the most common tumor types in our country. In present, scientists focus their studies on cancer surppressor genes to find out the moleculor mechanisms of tumor oncogenesis. Wnt pathway,which plays an important role in the development of colorectal cancer,is a hot spot of research. On the while, Dact is a key regulator site in the Wnt pathway.The purpose of this study is to observe the methylation of promoter of gene Dact1(Dact3) and the expression of Dact1(Dact3) mRNA in colorectal tumor and to analyze its relationship between them with carcinogenesis of colorectal tumor.
Materials and Methods
Colorectal cancer tissues 50 pairs of cancerous and corresponding paracancerous were surgically obtained from colorectal cancer patients ,and 20 of normal colorectal tissues were surgically obtained from PPH patients, all of which in the Hangzhou Hospital of anhui Medical University between 2008.1 and 2009.7. All the cancerous tissues had clear pathological diagnosis. The paracancerous were far away about 5cm from cancerous mucoses, and also had clear pathological- diagnosis: normal mucoses cases. All patients were preoperatively end up receiving chemotherapy and other anti-cancer therapy. All the tissues were stored at -80℃for reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific polymerase chain reaction (MSP).
RT-PCR:
Total RNA was isolated from tissue samples using Trizol Reagent according to the manufacturer's protocol. The concentration and purity of total RNA were determined from the A260/A280 ratio using a UV spectrophotometer .One microgram of total RNA from each specimen was subjected to RT-PCR. Theβ-actin and Dact1(Dact3) PCR products were electrophoresed on a 2% agarose gel and visualized by staining with ethidum bromide. The integrated density values of the bands representing amplified products were acquired and analyzed by a image analysis system.
MSP:
DNA was extracted from the 50 colorectal cancers and paracancerous and 20 of normal colorectal tissues using phenol-chloroform extraction, than treated with bisulfite. briefly, DNA was denatured with NaOH and modified with sodium bisulfite. DNA samples were then purified using Wizard DNA purification resin, treated again with NaOH, precipitated with ethanol, and resuspended in water for template to PCR. l 0ul samples of each PCR reaction products were loaded directly onto 2.0% agarose gels, stained with 0.1% ethidium bromide, and visualized under UV illumination. Results determination: Only when PCR products were amplified with methylated primer, then the promoter of Dact1(Dact3) gene could be regarded as methylation.
Statistical analysis: All the data were analyzed with SPSS11.5 statistics software. The statistical significance was defined as P<0.05.
Results
The detection of colorectal cancer Dact1 mRNA positive expression (12%, 6 / 50) by Qualitative RT-PCR was lower than in the paracancerous (90%, 45/50) and normal colorectal tissues (95%, 19/20). And there were significantly different between colorectal tumor tissues and paracancerous, normal colorectal tissues respectively(P<0.05). In the same way, colorectal cancer Dact3 mRNA positive expression (10%,5/50) were lower than in paracancerous (86%,43/50) and normal colorectal tissues (90%,18/20). And there were significantly different between colorectal tumor tissues and paracancerous, normal colorectal tissues respectively(P<0.05).
Bisulphite sequencing revealed that the methylation was unequally distributed within the Dact1 promoter and by MSP analysis a region close to the transcription start point was shown to be hypermethylated in the majority of colorectal carcinomas tissues (46% , 23/50) . In contrast, normal colorectal tissues displayed lower hypermethylation (5%,1/20) as well as in paracancerous (12%,6/50). On the contrary, the hypermethylation of Dact3 have no significant difference between colorectal tumor tissues (46%,23/50) and paracancerous(10%,5/50), as well as normal colorectal tissues(10%,2/20)(P>0.05).
Conclusion
Promoter hypermethylation of Dact1 were presented in the vast majority of colorectal tumor, and only rarely in paracancerous and normal colorectal tissues, which was maybe correlated with Epigenetic silencing and down-regulation of the Dact1 gene in colorectal tumor .On the other hand, promoter hypermethylation of Dact3 had no significant difference between colorectal carcinomas tissues and paracancerous, as well as normal colorectal tissues.It maybe had no relevance in the loss (low expression) of Dact3 mRNA expression with the state of promoter methylation of Dact3.
引文
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1 Cheyette BN,Waxman JS,Miller JR,et a1.Dapper,a Dishevelled-associated antagonist ofβ-catenin and JNK signaling,is required for notochord formation.Dev Cell,2002,2(4):449-461.
2 Krieghof E,Behrens J,Mayr B.Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention.Celf Sc ,2006,119:1453-1463.
3 Boutros M,Mihaly J,Bouwmeester T,et a1.Signaling specificity by Frizzled receptors in Drosophila.Science,2000,288:1825-1828.
4 Kikuchi A.Regulation of beta-catenin signaling in the Wnt pathway.Biochem Biophys Res Commun,2000,268:243-248.
5 Behrens J. Biochemical interactions in the wnt pathway. Biochimical et biophysica acta,2000,1495(2):168-182.
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1 Cheyette BN,Waxman JS,Miller JR,et a1.Dapper,a Dishevelled-associated antagonist ofβ-catenin and JNK signaling,is required for notochord formation.Dev Cell,2002,2(4):449-461.
2 Krieghof E,Behrens J,Mayr B.Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention.Celf Sc ,2006,119:1453-1463.
3 Boutros M,Mihaly J,Bouwmeester T,et a1.Signaling specificity by Frizzled receptors in Drosophila.Science,2000,288:1825-1828.
4 Kikuchi A.Regulation of beta-catenin signaling in the Wnt pathway.Biochem Biophys Res Commun,2000,268:243-248.
5 Behrens J. Biochemical interactions in the wnt pathway. Biochimical et biophysica acta,2000,1495(2):168-182.
6 Friadrick A,Kullmann F.Familial adenomatous polyposis syndrome (FAP):pathogenesis and molecular mechanisms.Med Klim,2003,98(12):776-782.
7 Feng XH,Derynck R.Specificity and versatility in TGF-βsignaling through Smads.Annu Rev Cell Dev Biol,2005,21:659-693.
8 Gloy J,Hikasa H,Sokol SY.Frodo interacts with Dishevelled to transduce Wnt signals.Nat Cell Biol,2002,4(5):351-357.
9 Zhang LX,Zhou H,Su Y,et a1.Zebrafish Dpr2 inhibits mesoderm induction by promoting degradation of nodal receptors.Science,2004,306(5693):l14-l17.
10 Fisher DA, Kivimae S, Hoshino J, et a1.Three Dact gene family members are expressed during embryonic development and in the adult brains of mice. Dev Dyn , 2006,235:2620–2630.
11 Katoh M .Identification and characterization of human DAPPER1 and DAPPER2 genes in silico.Int J Oncol,2003,22(4):907-913.
12 Katoh M. Identification and characterization of rat Dact1 and Dact2 genes in silico[J]. Int J Mol Med,2005,15(6):1045-1049.
13高霞,陈旭煌,陈晔光,等. Dapper在胚胎发育和信号调控中的作用.生命科学,2007,19(5):471-476.
14 Waxman JS, Hocking AM, Stoick CL, et a1.Zebrafish Dapper1 and Dapper2 play distinct roles in Wnt-mediated developmental processes. Development,2004,131:5909–5921.
15 Yau TO, Chan CY, Chan KL, et a1. HDPR1,a novel inhibitor of the WNT/beta-catenin signaling, is frequently downregulated in hepatocellular carcinoma: involvement of methylation-mediated gene silencing. Oncogene,2004.24:1607–1614.
16 Brott BK, Sokol SY. A vertebrate homolog of the cell cycle regulator Dbf4 is an inhibitor of Wnt signaling required for heart development. Dev Cell,2005,8:703–715.
17 Zhang L, Gao X, Wen J, et a1. Dapper 1 antagonizes Wnt signaling by promoting dishevelled degradation.J Biol Chem,2006, 281:8607–8612.
18 Jiang X,Tan J,Li JS , et a1. DACT3 is an epigenetic regulator of Wnt/β-cateninsignaling in colorectal cancer and is a therapeutic target of histone modifications. Cancer Cell ,2008,13:529–541.
19 Su Y, Zhang L, Gao X, et a1. The evolutionally conserved activity of Dapper2 in antagonizing TGF-βsignaling . FASEB J ,2007,21: 682–690.