AXIN2基因在肝细胞癌中的表达及甲基化分析
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摘要
背景和目的
肝细胞癌(hepatocellular carcinoma, HCC),是世界上最常见的恶性肿瘤之,在我国,每年约有300,000人死于HCC,约占全世界HCC年死亡总数的50%。HCC的发生是一个涉及多基因改变的复杂过程,已有研究证实,在HCC发生过程中存在着多种抑癌基因启动子区CpG岛的高甲基化现象,干扰抑癌基因的转录,导致细胞异常增殖,引起细胞癌变。经典的Wnt/β-catenin信号转导通路的异常激活与包括HCC在内的多种肿瘤的形成相关。作为该途径的负性调控因子,AXIN2基因在结肠癌,肺癌等恶性肿瘤中存在启动子高甲基化所致表达缺失或下调,最终导致Wnt/(3-catenin信号通路异常激活并引起相应肿瘤的发生,但在HCC中是否存在AXIN2基因启动子甲基化异常尚不完全确定。本研究检测了AXIN2基因在肝癌细胞系和HCC组织中mRNA表达情况及启动子区甲基化状态,初步分析AXIN2基因甲基化水平对mRNA表达的影响,探讨AXIN2基因异常甲基化在HCC发生、发展中的意义。
材料与方法
收集53例河南省肿瘤医院2009年5月至2010年4月手术切除的HCC和相应的癌旁(距离癌组织大于2 cm)组织标本。所有患者均有肝硬化背景,多数(48/53)为乙肝病毒感染,其中男性48例,女性5例,年龄35-76岁,中位年龄52岁。参照2010巴塞罗那BCLC分期法：A期16例,B期4例,C期33例,D期0例。所有患者均签署知情同意书,均为第1次手术,术前均未实施化疗和其他治疗。正常对照17例,为因肝海绵状血管瘤手术切除的正常肝脏组织标本。所有标本均获得病理证实。
5株人肝癌细胞系HepG2、SMMC-7721、Skhep-1、Hep3B、PLC/PRF/5和1株正常肝细胞系L02,由北京大学医学部病原生物学系微生物学教研室提供。
用Trizol (Sigma公司)法提取组织细胞总RNA,分光光度计检测抽提的质量和浓度；再按照Fermentas逆转录试剂说明书操作步骤合成cDNA。荧光定量RT-PCR检测AXIN2基因mRNA在细胞系和40例配对肝癌组织中的表达。提取组织细胞DNA,荧光定量PCR检测细胞系和53例配对肝癌组织中AXIN2基因启动子区甲基化水平。数据经SPSS17.0进行Wilcoxon符号秩和检验、t检验、Fisher确切概率法和Spearman秩相关检验的统计学处理,以α=0.05为检验水准。
结果
1. AXIN2 mRNA在40例HCC和配对癌旁组织中均有表达。AXIN2基因在癌组织中的mRNA表达水平(0.1629±0.06791)低于癌旁组织(0.4155±0.23304),且具有统计学意义(Z=-2.567,P=0.010)。
2. AXIN2基因在HepG2、SMMC-7721、Skhep-1、Hep3B、PLC/PRF/5等5种人肝癌细胞系中的甲基化水平分别为：98.15%、98.38%、96.00%、93.14%、83.27%,均明显高于正常肝细胞系L02的甲基化水平7.38%。
3.53例HCC组织及配对癌旁组织的AXIN2基因甲基化水平均明显高于正常组织(t=-3.663, P=0.009; t=-4.591, P=0.007), HCC组织与配对癌旁组织之间甲基化水平无明显差异。
4. AXIN2 mRNA表达水平与甲基化状态呈负相关(r=-0.458, P=0.032); AXIN2基因高甲基化率低于AXIN2 mRNA低表达率。
5.AXIN2基因在BCLC C期HCC患者中的甲基化水平明显高于BCLC A/B期的HCC患者(t=-2.830,P=0.008)。与患者的年龄、性别、HBV是否感染、甲胎蛋白浓度和门静脉是否有癌栓均无关。
结论
1.肝细胞癌中,AXIN2基因mRNA表达下调,其表达下调的主要机制是该基因启动子区的异常高甲基化；
2.肝细胞癌中,AXIN2基因启动子区高甲基化与患者BCLC分期呈正相关。
Background and objective
Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide. Each year, at least 300,000 people die from HCC in China alone. In China, risk factors for HCC include chronic hepatitis B or C infection, liver cirrhosis, and nonalcoholic steatohepatitis, et al. As for many other cancers, HCC has been understood a set of disease that driven by progressive genetic abnormalities and epigenetic changes of suppressor genes. The CpG islands hypermethylation of tumor suppress genens has been recognized as an important and alternative mechanism in hepatocarcinogenesis. Aberrant activation of the canonical Wnt/p-catenin signaling pathway is frequently involved in a broad spectrum of human cancers including HCC. AXIN2 gene is a tumor suppressor, as a scaffold protein, it plays an important role in the canonical Wnt/β-catenin signaling pathway. Epigenetic silencing of AXIN2 by promoter methylation underlies an alternative mechanism in lung and colon cancers development. However, it is currently unknown whether AXIN2 hypermethylation is associated with the HCC carcinogenesis. We investigate the promoter methylation status and mRNA expression of AXIN2 gene in HCC, and analyze the correlation of HCC and AXIN2 methylation status.
Materials and Methods
53 HCC and adjacent non-cancerous tissues (> 2cm away from tumor) of cirrhotic liver,17 normal liver tissues were surgically obtained from the patients in Henan province tumor hospital between May 2009 and April 2010. Most of the patients (48/53) had chronic hepatitis B infection. The age of the 53 patients (48 male, 5 female) ranged from 35 to 76 years, with a median age of 52 years.16 patients are BCLC stage A,4 patients are BCLC stage B,33 patients are BCLC stage C, no patient is BCLC stage D. Informed consent was given by all patients for investigations and data anaylsis. All patients were not treated by preoperative radiotherapy or chemotherapy. As a control,7 samples of normal liver tissues were collected from the resection of hemangioma. All cases were confirmed by pathologic diagnosis.
Human HCC cell lines (HepG2、SMMC-7721、Skhep-1、Hep3B、PLC/PRF/5) were provided by Department of Microbiology, Peking University Health Science Center.
RNA was extracted from HCC tissues and cell lines. The purity of RNA was determined by OD260/OD280. cDNA synthesis was performed using Fermentas Reverse Transcription System. The expression of AXIN2 mRNA was determined by real-time quantitative RT-PCR. DNA was digested by proteinase K and later was extracted through a phenol/chloroform treatment. Real-time PCR was used to calculate the methylation status of AXIN2 gene. Statistical tests were done using SPSS17.0 statistical software. The level for a statistically significant difference was set at a=0.05 for all the tests.
Results
1. The expression of AXIN2 mRNA was lower in HCC tissues than that in adjacent non-cancerous tissues (Z=-2.567,P＝0.010)
2. In comparison with normal liver tissues, the methylation level of AXIN2 gene in HCC and adjacent non-cancerous tissues both increased significantly (t=-3.663, P=0.009; t=-4.591,P=0.007).
3. AXIN2 gene was also heavily methylated in all HCC cell lines. In contrast, the AXIN2 transcript was missing in all of the five HCC cell lines.
4. AXIN2 mRNA levels were inversely correlated with DNA methylation (r=-0.458, P=0.032).
5. Statistical analysis further indicated that AXIN2 hypermethylation was positively associated with BCLC stage.
Conclusions
In our experiment, decreased expression and hypermethylation of AXIN2 gene was found in HCC. AXIN2 mRNA levels were inversely correlated with DNA methylation. Methylation may be the main mechanism of AXIN2 gene down-regulation in HCC tissues, epigenetic silencing of AXIN2 may involve in the carcinogenesis of HCC.
Hypermethylation of AXIN2 gene may affect the invasion and metastasis of HCC.
肝细胞癌(hepatocellular carcinoma, HCC),是世界上最常见的恶性肿瘤之,在我国,每年约有300,000人死于HCC,约占全世界HCC年死亡总数的50%。HCC的发生是一个涉及多基因改变的复杂过程,已有研究证实,在HCC发生过程中存在着多种抑癌基因启动子区CpG岛的高甲基化现象,干扰抑癌基因的转录,导致细胞异常增殖,引起细胞癌变。经典的Wnt/β-catenin信号转导通路的异常激活与包括HCC在内的多种肿瘤的形成相关。作为该途径的负性调控因子,AXIN2基因在结肠癌,肺癌等恶性肿瘤中存在启动子高甲基化所致表达缺失或下调,最终导致Wnt/(3-catenin信号通路异常激活并引起相应肿瘤的发生,但在HCC中是否存在AXIN2基因启动子甲基化异常尚不完全确定。本研究检测了AXIN2基因在肝癌细胞系和HCC组织中mRNA表达情况及启动子区甲基化状态,初步分析AXIN2基因甲基化水平对mRNA表达的影响,探讨AXIN2基因异常甲基化在HCC发生、发展中的意义。
材料与方法
收集53例河南省肿瘤医院2009年5月至2010年4月手术切除的HCC和相应的癌旁(距离癌组织大于2 cm)组织标本。所有患者均有肝硬化背景,多数(48/53)为乙肝病毒感染,其中男性48例,女性5例,年龄35-76岁,中位年龄52岁。参照2010巴塞罗那BCLC分期法：A期16例,B期4例,C期33例,D期0例。所有患者均签署知情同意书,均为第1次手术,术前均未实施化疗和其他治疗。正常对照17例,为因肝海绵状血管瘤手术切除的正常肝脏组织标本。所有标本均获得病理证实。
5株人肝癌细胞系HepG2、SMMC-7721、Skhep-1、Hep3B、PLC/PRF/5和1株正常肝细胞系L02,由北京大学医学部病原生物学系微生物学教研室提供。
用Trizol (Sigma公司)法提取组织细胞总RNA,分光光度计检测抽提的质量和浓度；再按照Fermentas逆转录试剂说明书操作步骤合成cDNA。荧光定量RT-PCR检测AXIN2基因mRNA在细胞系和40例配对肝癌组织中的表达。提取组织细胞DNA,荧光定量PCR检测细胞系和53例配对肝癌组织中AXIN2基因启动子区甲基化水平。数据经SPSS17.0进行Wilcoxon符号秩和检验、t检验、Fisher确切概率法和Spearman秩相关检验的统计学处理,以α=0.05为检验水准。
结果
1. AXIN2 mRNA在40例HCC和配对癌旁组织中均有表达。AXIN2基因在癌组织中的mRNA表达水平(0.1629±0.06791)低于癌旁组织(0.4155±0.23304),且具有统计学意义(Z=-2.567,P=0.010)。
2. AXIN2基因在HepG2、SMMC-7721、Skhep-1、Hep3B、PLC/PRF/5等5种人肝癌细胞系中的甲基化水平分别为：98.15%、98.38%、96.00%、93.14%、83.27%,均明显高于正常肝细胞系L02的甲基化水平7.38%。
3.53例HCC组织及配对癌旁组织的AXIN2基因甲基化水平均明显高于正常组织(t=-3.663, P=0.009; t=-4.591, P=0.007), HCC组织与配对癌旁组织之间甲基化水平无明显差异。
4. AXIN2 mRNA表达水平与甲基化状态呈负相关(r=-0.458, P=0.032); AXIN2基因高甲基化率低于AXIN2 mRNA低表达率。
5.AXIN2基因在BCLC C期HCC患者中的甲基化水平明显高于BCLC A/B期的HCC患者(t=-2.830,P=0.008)。与患者的年龄、性别、HBV是否感染、甲胎蛋白浓度和门静脉是否有癌栓均无关。
结论
1.肝细胞癌中,AXIN2基因mRNA表达下调,其表达下调的主要机制是该基因启动子区的异常高甲基化；
2.肝细胞癌中,AXIN2基因启动子区高甲基化与患者BCLC分期呈正相关。
Background and objective
Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide. Each year, at least 300,000 people die from HCC in China alone. In China, risk factors for HCC include chronic hepatitis B or C infection, liver cirrhosis, and nonalcoholic steatohepatitis, et al. As for many other cancers, HCC has been understood a set of disease that driven by progressive genetic abnormalities and epigenetic changes of suppressor genes. The CpG islands hypermethylation of tumor suppress genens has been recognized as an important and alternative mechanism in hepatocarcinogenesis. Aberrant activation of the canonical Wnt/p-catenin signaling pathway is frequently involved in a broad spectrum of human cancers including HCC. AXIN2 gene is a tumor suppressor, as a scaffold protein, it plays an important role in the canonical Wnt/β-catenin signaling pathway. Epigenetic silencing of AXIN2 by promoter methylation underlies an alternative mechanism in lung and colon cancers development. However, it is currently unknown whether AXIN2 hypermethylation is associated with the HCC carcinogenesis. We investigate the promoter methylation status and mRNA expression of AXIN2 gene in HCC, and analyze the correlation of HCC and AXIN2 methylation status.
Materials and Methods
53 HCC and adjacent non-cancerous tissues (> 2cm away from tumor) of cirrhotic liver,17 normal liver tissues were surgically obtained from the patients in Henan province tumor hospital between May 2009 and April 2010. Most of the patients (48/53) had chronic hepatitis B infection. The age of the 53 patients (48 male, 5 female) ranged from 35 to 76 years, with a median age of 52 years.16 patients are BCLC stage A,4 patients are BCLC stage B,33 patients are BCLC stage C, no patient is BCLC stage D. Informed consent was given by all patients for investigations and data anaylsis. All patients were not treated by preoperative radiotherapy or chemotherapy. As a control,7 samples of normal liver tissues were collected from the resection of hemangioma. All cases were confirmed by pathologic diagnosis.
Human HCC cell lines (HepG2、SMMC-7721、Skhep-1、Hep3B、PLC/PRF/5) were provided by Department of Microbiology, Peking University Health Science Center.
RNA was extracted from HCC tissues and cell lines. The purity of RNA was determined by OD260/OD280. cDNA synthesis was performed using Fermentas Reverse Transcription System. The expression of AXIN2 mRNA was determined by real-time quantitative RT-PCR. DNA was digested by proteinase K and later was extracted through a phenol/chloroform treatment. Real-time PCR was used to calculate the methylation status of AXIN2 gene. Statistical tests were done using SPSS17.0 statistical software. The level for a statistically significant difference was set at a=0.05 for all the tests.
Results
1. The expression of AXIN2 mRNA was lower in HCC tissues than that in adjacent non-cancerous tissues (Z=-2.567,P＝0.010)
2. In comparison with normal liver tissues, the methylation level of AXIN2 gene in HCC and adjacent non-cancerous tissues both increased significantly (t=-3.663, P=0.009; t=-4.591,P=0.007).
3. AXIN2 gene was also heavily methylated in all HCC cell lines. In contrast, the AXIN2 transcript was missing in all of the five HCC cell lines.
4. AXIN2 mRNA levels were inversely correlated with DNA methylation (r=-0.458, P=0.032).
5. Statistical analysis further indicated that AXIN2 hypermethylation was positively associated with BCLC stage.
Conclusions
In our experiment, decreased expression and hypermethylation of AXIN2 gene was found in HCC. AXIN2 mRNA levels were inversely correlated with DNA methylation. Methylation may be the main mechanism of AXIN2 gene down-regulation in HCC tissues, epigenetic silencing of AXIN2 may involve in the carcinogenesis of HCC.
Hypermethylation of AXIN2 gene may affect the invasion and metastasis of HCC.
引文
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