SFRP1基因表达与大肠肿瘤发生的关系研究
摘要
目的通过检测正常大肠粘膜组织、大肠腺瘤组织、大肠腺癌组织中SFRP1、E-cad、β-cat、c-myc及cyclinD1的表达水平、SFRP1基因启动子甲基化状况,观察相关蛋白表达水平的变化和基因甲基化状况与大肠癌临床生物学行为之间的联系及基因甲基化状况与相应蛋白表达之间的关系,探讨大肠癌发生发展、侵袭转移中的有关分子机制,为临床治疗提供有意义的理论基础和治疗靶点。
方法随机收集80例大肠腺瘤、85例大肠腺癌、及其相应的切端处正常大肠粘膜组织165例新鲜标本。用免疫组化方法检测上述标本中β-cat、E-cad、c-myc及cyclinD1蛋白的表达情况,用半定量RT-PCR方法检测上述标本中SFRP1的mRNA表达水平,用甲基化特异性PCR检测SFRP1基因启动子甲基化状况。分析SFRP1基因的甲基化状况与其mRNA表达水平与相应蛋白表达水平之间的关系,及SFRP1基因甲基化状况和蛋白表达水平变化与大肠癌临床生物学行为之间的关系。
结果
1半定量RT-PCR SFRP1基因在大肠腺瘤、大肠腺癌组织中的表达水平均明显低于大肠正常组织的对照组,差异有统计学意义(P＜0.05),且大肠腺癌组织中SFRP1基因的表达水平低于大肠腺瘤(P＜0.05)
2甲基化PCR 165例正常大肠切端、80例腺瘤标本MSP产物均为阴性。85例大肠腺癌标本中有22例甲基化产物,甲基化阳性率为25.88%(22/85)。
3免疫组化正常大肠黏膜β-cat、E-cad定位于上皮细胞膜而c-myc、cyclinD1未见表达。大肠腺瘤、腺癌组织β-cat、E-cad胞膜表达不同程度缺失,β-cat呈胞质和胞核异位表达。腺瘤β-cat、E-cad的异常表达率(37.5%,28.9%),低于大肠腺癌(100.0%,93.4%,P＜0.05)。大肠腺瘤c-myc、cyclinD1蛋白异常表达率(36.4%,45.2%)明显低于大肠腺癌(73.6%,82.9%,P均＜0.05),且腺瘤染色程度明显较腺癌变淡。
结论
1 SFRP1基因在大肠腺瘤、大肠腺癌组织中的表达水平均明显低于大肠正常组织的对照组,且SFRP1mRNA水平的下调由正常肠黏膜、腺瘤、到大肠癌呈上升趋势。提示SFRP1作为Wnt通路的拮抗剂之一,可以对防止大肠肿瘤的发生发展起到一定的作用。SFRP1mRNA水平下调,导致其对Wnt-frizzled信号传导通路的拮抗功能降低、Wnt信号传导通路的持续存在。因此SFRP1水平的下调可能是大肠癌的启动及促进因素,它可能参与了早期大肠癌的发生,促进了腺瘤形成及其向早期大肠癌转变。
2 165例正常大肠切端、80例腺瘤标本MSP产物均为阴性。85例大肠腺癌标本中有22例甲基化产物,甲基化阳性率为25.88%(22/85)。这一结果提示我们可以通过对SFRP1基因启动子的高度甲基化区进行检测和进行一定的处理,将可能成为大肠肿瘤的早期诊断及大肠肿瘤的预防、甚至治疗的有效手段。故SFRP1可能为预防大肠肿瘤的发生、大肠肿瘤的早期诊断提供新思路,并为防治大肠肿瘤提供新靶标和实验依据。
3大肠腺瘤、腺癌组织β-cat、E-cad胞膜表达不同程度缺失,β-cat呈胞质和胞核异位表达。腺瘤β-cat、E-cad的异常表达率,低于大肠腺癌。大肠腺瘤c-myc、cyclinD1蛋白异常表达率明显低于大肠腺癌。且随着SFRP1基因mRNA及DNA的表达从腺瘤到癌过程的下调,β-cat、E-cad、c-myc、cyclinD1的异常表达上升。进一步提示SFRP1作为Wnt通路的拮抗剂,其水平的下调可能是大肠癌的启动及促进因素,它可能参与了早期大肠癌的发生,促进了腺瘤形成及其向早期大肠癌转变。
Objective To investigate the expressions of SFRP1 in matched normal large bowel mucosal samples, colorectal adenoma samples and colorectal cancer samples. To analyze the mechanism of gene silencing. To assess the functional change of SFRP1 ,in colorectal tumorigenesis, analyze the relationship between the aberrant expression and clinical pathologic characteristics.
Methods We conducted immunohistochemical investigation、Analysis of Promoter Methylation Status (MSP) and semiquantitatively determined with reverse transcription-polymerase chain reaction (RT-PCR)to SFRP1、E-cad、β-cat、c-myc、cyclinD1 in matched normal large bowel mucosal samples, colorectal adenoma samples and colorectal cancer samples. The relationship of the methylation status with the expression of relevant protein was analysed. The relationship of the methylation status and the expression of protein with the biological behavior of colorectal cancer was analysed.
Results
1 .RT-PCR Compared to control groups,the expression of SFRP_1 mRNA in colorectal adenomas and colorectal cancer are downregulated(P< 0.05),and the expression of SFRP_1 mRNA in colorectal adenomas is lower than in colorectal cancer(P< 0.05).
2.Immunohistochemistryβ-cat and E-cad were deleted in different degrees when they were observed in colorectal adenomas and colorectal cancer.β-cat had ectopic expression in cytoplasm and cellular nucleus, and its rate was lower in colorectal adenomas than in colorectal cancer (87.5%,100%, respectively, P< 0.05). The depletion rates ofβ-cat and E-cad in colorectal adenoma samples (37.5%、37.5 % respectively)were lower than in colorectal cancer samples (58.8%、76.4% respectively, P< 0.05) . Abnormal expression rates of c-myc and cyclinD1 in colorectal adenoma were lower samples than in colorectal cancer samples.
3. MSP The methylation status of SFRP1 promtor region in 165 matched normal large bowel mucosal samples and 80 colorectal adenoma samples all were 0.00%, and 25.88%(22/85) in colorectal cancer. Methylation of the SFRP1 promotor region was increased in colorectal cancer samples.
Conclusions
1 The inactivation and down-regulation of SFRP1 observed are consistent with its acting as a tumor suppressor gene in colorectal tumorigenesis, which lead to the aberrant expressions ofβ-cat、E-cad、c-myc、cyclinD1.Furthermore , investigation of the methylation status of the promotor region of SFRP1 by methylationspecific PCR revealed hypermethylation in 22 colorectal cancers.
2 These data demonstrate that hypermethylation of the SFRP1 promoter region is a frequent event in colorectal cancer and is increased significantly compared with normal mucosa from the same patient. But there was not an absolute inverse correlation between methylation and transcription. Our results show that methylation and transcriptional repression are common events in colorectal cancer and this may influence the role of SFRP1 in tumorigenesis. Our findings clearly demonstrate the function of SFRP1 in colorectal carcinogenesis,and we can participateif we treat with a demethylating agent for the colon cancers which have SFRP1 promter methylation, demethylation of the promoter and re-expression of SFRP1 could be observed.
3 So we can detect hypermethylation of the SFRP1 promoter region to prognose colon cancers. Perhaps taking this method to colon cancers which, we can prevent and cure colon cancers. Because SFRP1 regulates a wide spectrum of Wnt activities and pathways, these results suggest that regulating Wnt pathways plays a significant role in colorectal carcinogenesis.
方法随机收集80例大肠腺瘤、85例大肠腺癌、及其相应的切端处正常大肠粘膜组织165例新鲜标本。用免疫组化方法检测上述标本中β-cat、E-cad、c-myc及cyclinD1蛋白的表达情况,用半定量RT-PCR方法检测上述标本中SFRP1的mRNA表达水平,用甲基化特异性PCR检测SFRP1基因启动子甲基化状况。分析SFRP1基因的甲基化状况与其mRNA表达水平与相应蛋白表达水平之间的关系,及SFRP1基因甲基化状况和蛋白表达水平变化与大肠癌临床生物学行为之间的关系。
结果
1半定量RT-PCR SFRP1基因在大肠腺瘤、大肠腺癌组织中的表达水平均明显低于大肠正常组织的对照组,差异有统计学意义(P＜0.05),且大肠腺癌组织中SFRP1基因的表达水平低于大肠腺瘤(P＜0.05)
2甲基化PCR 165例正常大肠切端、80例腺瘤标本MSP产物均为阴性。85例大肠腺癌标本中有22例甲基化产物,甲基化阳性率为25.88%(22/85)。
3免疫组化正常大肠黏膜β-cat、E-cad定位于上皮细胞膜而c-myc、cyclinD1未见表达。大肠腺瘤、腺癌组织β-cat、E-cad胞膜表达不同程度缺失,β-cat呈胞质和胞核异位表达。腺瘤β-cat、E-cad的异常表达率(37.5%,28.9%),低于大肠腺癌(100.0%,93.4%,P＜0.05)。大肠腺瘤c-myc、cyclinD1蛋白异常表达率(36.4%,45.2%)明显低于大肠腺癌(73.6%,82.9%,P均＜0.05),且腺瘤染色程度明显较腺癌变淡。
结论
1 SFRP1基因在大肠腺瘤、大肠腺癌组织中的表达水平均明显低于大肠正常组织的对照组,且SFRP1mRNA水平的下调由正常肠黏膜、腺瘤、到大肠癌呈上升趋势。提示SFRP1作为Wnt通路的拮抗剂之一,可以对防止大肠肿瘤的发生发展起到一定的作用。SFRP1mRNA水平下调,导致其对Wnt-frizzled信号传导通路的拮抗功能降低、Wnt信号传导通路的持续存在。因此SFRP1水平的下调可能是大肠癌的启动及促进因素,它可能参与了早期大肠癌的发生,促进了腺瘤形成及其向早期大肠癌转变。
2 165例正常大肠切端、80例腺瘤标本MSP产物均为阴性。85例大肠腺癌标本中有22例甲基化产物,甲基化阳性率为25.88%(22/85)。这一结果提示我们可以通过对SFRP1基因启动子的高度甲基化区进行检测和进行一定的处理,将可能成为大肠肿瘤的早期诊断及大肠肿瘤的预防、甚至治疗的有效手段。故SFRP1可能为预防大肠肿瘤的发生、大肠肿瘤的早期诊断提供新思路,并为防治大肠肿瘤提供新靶标和实验依据。
3大肠腺瘤、腺癌组织β-cat、E-cad胞膜表达不同程度缺失,β-cat呈胞质和胞核异位表达。腺瘤β-cat、E-cad的异常表达率,低于大肠腺癌。大肠腺瘤c-myc、cyclinD1蛋白异常表达率明显低于大肠腺癌。且随着SFRP1基因mRNA及DNA的表达从腺瘤到癌过程的下调,β-cat、E-cad、c-myc、cyclinD1的异常表达上升。进一步提示SFRP1作为Wnt通路的拮抗剂,其水平的下调可能是大肠癌的启动及促进因素,它可能参与了早期大肠癌的发生,促进了腺瘤形成及其向早期大肠癌转变。
Objective To investigate the expressions of SFRP1 in matched normal large bowel mucosal samples, colorectal adenoma samples and colorectal cancer samples. To analyze the mechanism of gene silencing. To assess the functional change of SFRP1 ,in colorectal tumorigenesis, analyze the relationship between the aberrant expression and clinical pathologic characteristics.
Methods We conducted immunohistochemical investigation、Analysis of Promoter Methylation Status (MSP) and semiquantitatively determined with reverse transcription-polymerase chain reaction (RT-PCR)to SFRP1、E-cad、β-cat、c-myc、cyclinD1 in matched normal large bowel mucosal samples, colorectal adenoma samples and colorectal cancer samples. The relationship of the methylation status with the expression of relevant protein was analysed. The relationship of the methylation status and the expression of protein with the biological behavior of colorectal cancer was analysed.
Results
1 .RT-PCR Compared to control groups,the expression of SFRP_1 mRNA in colorectal adenomas and colorectal cancer are downregulated(P< 0.05),and the expression of SFRP_1 mRNA in colorectal adenomas is lower than in colorectal cancer(P< 0.05).
2.Immunohistochemistryβ-cat and E-cad were deleted in different degrees when they were observed in colorectal adenomas and colorectal cancer.β-cat had ectopic expression in cytoplasm and cellular nucleus, and its rate was lower in colorectal adenomas than in colorectal cancer (87.5%,100%, respectively, P< 0.05). The depletion rates ofβ-cat and E-cad in colorectal adenoma samples (37.5%、37.5 % respectively)were lower than in colorectal cancer samples (58.8%、76.4% respectively, P< 0.05) . Abnormal expression rates of c-myc and cyclinD1 in colorectal adenoma were lower samples than in colorectal cancer samples.
3. MSP The methylation status of SFRP1 promtor region in 165 matched normal large bowel mucosal samples and 80 colorectal adenoma samples all were 0.00%, and 25.88%(22/85) in colorectal cancer. Methylation of the SFRP1 promotor region was increased in colorectal cancer samples.
Conclusions
1 The inactivation and down-regulation of SFRP1 observed are consistent with its acting as a tumor suppressor gene in colorectal tumorigenesis, which lead to the aberrant expressions ofβ-cat、E-cad、c-myc、cyclinD1.Furthermore , investigation of the methylation status of the promotor region of SFRP1 by methylationspecific PCR revealed hypermethylation in 22 colorectal cancers.
2 These data demonstrate that hypermethylation of the SFRP1 promoter region is a frequent event in colorectal cancer and is increased significantly compared with normal mucosa from the same patient. But there was not an absolute inverse correlation between methylation and transcription. Our results show that methylation and transcriptional repression are common events in colorectal cancer and this may influence the role of SFRP1 in tumorigenesis. Our findings clearly demonstrate the function of SFRP1 in colorectal carcinogenesis,and we can participateif we treat with a demethylating agent for the colon cancers which have SFRP1 promter methylation, demethylation of the promoter and re-expression of SFRP1 could be observed.
3 So we can detect hypermethylation of the SFRP1 promoter region to prognose colon cancers. Perhaps taking this method to colon cancers which, we can prevent and cure colon cancers. Because SFRP1 regulates a wide spectrum of Wnt activities and pathways, these results suggest that regulating Wnt pathways plays a significant role in colorectal carcinogenesis.
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