乳腺癌WIF-1基因启动子区域的甲基化状态分析及多态性研究
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摘要
目的：本研究探索乳腺癌WIF-1基因启动子区域的甲基化状态及启动子区rs58172684 A/G与rs2336433 C/T位点单核苷酸多态性与散发性乳腺癌发生的关系。
方法：应用RT-PCR及甲基化特异性PCR技术检测乳腺癌组织、相应癌旁组织和乳腺良性病变组织中WIF-1基因mRNA表达及其启动子甲基化状态；用5-Aza-CdR和TSA分别单独和联合作用MCF-7乳腺癌细胞株,通过MTT比色法检测细胞生长活性；采用RT-PCR检测细胞作用前后WIF-1 mRNA的表达；采用等位基因特异性扩增法(ASA)对rs58172684 A/G与rs2336433 C/T位点单核苷酸多态性进行分析。
结果：癌组织与癌旁组织、乳腺良性病变组织WIF-1基因表达差异显著(P<0.05)；WIF-1基因在癌组织中甲基化频率明显高于癌旁组织及乳腺良性病变组织(χ2=16.484,P<0.05)；5-Aza-CdR和TSA均可显著抑制MCF-7细胞的增殖,呈时间和剂量依赖性；与未处理组比较,5-Aza-CdR和TSA分别单独和联合作用MCF-7乳腺癌细胞后,MCF-7细胞中WIF-1基因mRNA表达增强；乳腺癌患者rs58172684A/G与rs2336433 C/T位点基因型频率与正常对照者之间比较具有显著性差异(χ2=117.475,P<0.001；χ2=38.798,P<0.001)；乳腺癌病例组rs58172684 G等位基因频率显著高于对照组(χ2=11.62,P<0.001)；rs58172684 A/G位点与rs2336433C/T位点的基因型在年龄、病理分级、组织分型以及淋巴结转移个数之间比较差异均无显著性(P>0.05)。
结论：WIF-1基因甲基化发生能够增加乳腺癌的患病风险；5-Aza-CdR和TSA均可通过恢复该基因的表达,抑制肿瘤细胞生长；WIF-1基因启动子区单核苷酸多态性可能与散发性乳腺癌的发生相关,G等位基因可能为散发性乳腺癌发生的遗传危险因素。
Objective:To investigate the promoter methylation status of WIF-1 gene in breast cancer and the relationship between the single nucleotide polymorphism of WIF-1 gene promoter rs58172684 A/G and rs2336433 C/T site and the development of sporadic breast cancer.
Methods:RT-PCR and sensitive methylation-specific-PCR(MSP)were used to detect the mRNA expression and the promoter methylation status of WIF-1 gene in breast cancer and tumor adjacent tissues and benign breast disease. Human breast cancer cell line MCF-7 was treated with either different concentrations of 5-Aza-CdR or different concentrations of TSA. The growth of MCF-7 cells was observed by MTT assay. The expression of WIF-1 mRNA was observed by RT-PCR before and after 5-Aza-CdR and/or TSA. Allele specific amplification (ASA) was used to analyze the single nucleotide polymorphism of WIF-1 gene promoter rs58172684 A/G and rs2336433 C/T site.
Results:Significant differences of the mRNA expression were observed among three tissues. Methylation of WIF-1 genesin breast cancer increased significantly compared with tumor adjacent tissues and benign breast disease tissues (χ2=16.484, P<0.05); 5-Aza-CdR and TSA can inhibit the growth of MCF-7 cell line, which was time and dose dependent. After treated with 5-Aza-CdR and/or TSA, the expression of WIF-1 mRNA in MCF-7 was increased. The frequency of WIF-1 gene promoter genotypes rs58172684 A/G and rs2336433 C/T in sporadic breast cancer patients were significantly different from those in the healthy individuals. The frequency of allele gene G of WIF-1 gene promoter rs58172684 site was significantly higher than that of normal controls. The frequency of allele containing genotypes had no significant correlation with the age, clinical stage, histological grades in sporadic breast cancer patients, as well as increased numbers of lymph node metastasis.
Conclusion:The promoter methylation status of WIF-1 gene was significantly related with the occurrence of breast carcinoma.5-Aza-CdR and TSA could recover its expression, thereby inhibiting the growth of tumor cells. The single nucleotide polymorphism of WIF-1 gene promoter might play a role in the development of sporadic breast cancer, and G allele may be a genetic risk factor of sporadic breast cancer.
方法：应用RT-PCR及甲基化特异性PCR技术检测乳腺癌组织、相应癌旁组织和乳腺良性病变组织中WIF-1基因mRNA表达及其启动子甲基化状态；用5-Aza-CdR和TSA分别单独和联合作用MCF-7乳腺癌细胞株,通过MTT比色法检测细胞生长活性；采用RT-PCR检测细胞作用前后WIF-1 mRNA的表达；采用等位基因特异性扩增法(ASA)对rs58172684 A/G与rs2336433 C/T位点单核苷酸多态性进行分析。
结果：癌组织与癌旁组织、乳腺良性病变组织WIF-1基因表达差异显著(P<0.05)；WIF-1基因在癌组织中甲基化频率明显高于癌旁组织及乳腺良性病变组织(χ2=16.484,P<0.05)；5-Aza-CdR和TSA均可显著抑制MCF-7细胞的增殖,呈时间和剂量依赖性；与未处理组比较,5-Aza-CdR和TSA分别单独和联合作用MCF-7乳腺癌细胞后,MCF-7细胞中WIF-1基因mRNA表达增强；乳腺癌患者rs58172684A/G与rs2336433 C/T位点基因型频率与正常对照者之间比较具有显著性差异(χ2=117.475,P<0.001；χ2=38.798,P<0.001)；乳腺癌病例组rs58172684 G等位基因频率显著高于对照组(χ2=11.62,P<0.001)；rs58172684 A/G位点与rs2336433C/T位点的基因型在年龄、病理分级、组织分型以及淋巴结转移个数之间比较差异均无显著性(P>0.05)。
结论：WIF-1基因甲基化发生能够增加乳腺癌的患病风险；5-Aza-CdR和TSA均可通过恢复该基因的表达,抑制肿瘤细胞生长；WIF-1基因启动子区单核苷酸多态性可能与散发性乳腺癌的发生相关,G等位基因可能为散发性乳腺癌发生的遗传危险因素。
Objective:To investigate the promoter methylation status of WIF-1 gene in breast cancer and the relationship between the single nucleotide polymorphism of WIF-1 gene promoter rs58172684 A/G and rs2336433 C/T site and the development of sporadic breast cancer.
Methods:RT-PCR and sensitive methylation-specific-PCR(MSP)were used to detect the mRNA expression and the promoter methylation status of WIF-1 gene in breast cancer and tumor adjacent tissues and benign breast disease. Human breast cancer cell line MCF-7 was treated with either different concentrations of 5-Aza-CdR or different concentrations of TSA. The growth of MCF-7 cells was observed by MTT assay. The expression of WIF-1 mRNA was observed by RT-PCR before and after 5-Aza-CdR and/or TSA. Allele specific amplification (ASA) was used to analyze the single nucleotide polymorphism of WIF-1 gene promoter rs58172684 A/G and rs2336433 C/T site.
Results:Significant differences of the mRNA expression were observed among three tissues. Methylation of WIF-1 genesin breast cancer increased significantly compared with tumor adjacent tissues and benign breast disease tissues (χ2=16.484, P<0.05); 5-Aza-CdR and TSA can inhibit the growth of MCF-7 cell line, which was time and dose dependent. After treated with 5-Aza-CdR and/or TSA, the expression of WIF-1 mRNA in MCF-7 was increased. The frequency of WIF-1 gene promoter genotypes rs58172684 A/G and rs2336433 C/T in sporadic breast cancer patients were significantly different from those in the healthy individuals. The frequency of allele gene G of WIF-1 gene promoter rs58172684 site was significantly higher than that of normal controls. The frequency of allele containing genotypes had no significant correlation with the age, clinical stage, histological grades in sporadic breast cancer patients, as well as increased numbers of lymph node metastasis.
Conclusion:The promoter methylation status of WIF-1 gene was significantly related with the occurrence of breast carcinoma.5-Aza-CdR and TSA could recover its expression, thereby inhibiting the growth of tumor cells. The single nucleotide polymorphism of WIF-1 gene promoter might play a role in the development of sporadic breast cancer, and G allele may be a genetic risk factor of sporadic breast cancer.
引文
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