解剖学报

解剖学报 ›› 2018, Vol. 49 ›› Issue (5): 611-616.doi: 10.16098/j.issn.0529-1356.2018.05.008

• 细胞和分子生物学 • 上一篇    下一篇

4株乳腺癌细胞中内皮素受体B基因的甲基化状态及对MCF-7细胞增殖的影响

刘立琨1 朱文斌1 刘得水1 李新2 林悦铭3 张微1 周丽1 岳丽玲1*   

  1. 1. 齐齐哈尔医学院医药科学研究院; 2. 齐齐哈尔医学院附属第三医院乳腺外科; 3. 齐齐哈尔医学院医学技术学院医学影像与生物医学工程实验教学中心，黑龙江 齐齐哈尔 161006
  • 收稿日期:2017-12-08 修回日期:2018-02-10 出版日期:2018-10-06 发布日期:2018-10-06
  • 通讯作者: 岳丽玲 E-mail:yuell1025@126.com
  • 基金资助:
    黑龙江省自然科学基金资助项目;黑龙江省教育厅科学技术研究项目

Methylation of endothelin receptor B gene in four breast cancer cell lines and its effect on MCF-7 cell proliferation

 LIU Li-kun1 ZHU Wen-bin1 LIU De-shui1 LI Xin2 LIN Yue-ming3 ZHANG Wei1 ZHOU Li1 YUE Li-ling 1*   

  1. 1. Research Institute of Medicine and Pharmacy, Qiqihar Medical University; 2. Breast Surgery, the Third Affiliated Hospital of Qiqihar Medical University; 3. the Medical Imaging and Biomedical Engineering Experimental Teaching Center, School of Medical Science and Technology, Qiqihar Medical University,Heilongjian Qiqihar 161006, China
  • Received:2017-12-08 Revised:2018-02-10 Online:2018-10-06 Published:2018-10-06
  • Contact: YUE Li-ling E-mail:yuell1025@126.com

PDF

摘要：

目的 探讨内皮素受体B（EDNRB）基因在4株乳腺癌细胞中的表达、甲基化状态以及恢复EDNRB表达对MCF-7细胞增殖的影响。 方法 采用甲基化特异性PCR（MS-PCR）和亚硫酸盐测序法（BSP）分析4株乳腺癌细胞中EDNRB的甲基化状态；反转录聚合酶链反应（RT-PCR）检测EDNRB mRNA的表达水平；采用四甲基偶氮唑蓝（MTT）法和集落形成实验检测EDNRB表达恢复对MCF-7细胞增殖的影响。 结果 EDNRB在乳腺癌细胞MCF-7和ZR-75-1中表达缺失，并呈高甲基化状态；而在EDNRB表达最高的MDA-MB-231细胞中其启动子呈低甲基化，表明乳腺癌细胞中EDNRB基因启动子甲基化状态与其表达成负相关。5-氮杂胞苷（5-Aza-CR）能够反转EDNRB基因的表达，EDNRB表达恢复后MCF-7细胞的增殖受到抑制。 结论 EDNRB基因启动子区CpG岛频繁甲基化可能在乳腺癌发生发展中发挥重要作用，EDNRB有望成为乳腺癌早期诊断的新的分子标志物。

关键词: 乳腺癌, 甲基化, 内皮素受体B, 5-氮杂胞苷, 细胞增殖, 甲基化特异性聚合酶链反应

Abstract:

Objective To investigate the expression level and methylation status of endothelin receptor B (EDNRB) gene in four breast cancer cell lines and the effect of re-expression of methylated EDNRB on MCF-7 cell proliferation. Methods Methylation-specific PCR (MS-PCR) and bisulfite sequencing PCR (BSP) were used to detect methylation status of EDNRB in breast cancer cell lines. EDNRB gene expression level was measured by reverse transcription-PCR (RT-PCR). The colorimetric MTT assay and colony formation were used to investigate the effect of re-expression of methylated EDNRB on MCF-7 cell proliferation. Results No gene expression and promoter hypermethylation of EDNRB were detected in MCF-7 and ZR-75-1 cell lines. While the low frequency of methylation event happened in MDA-MB-231 cells which had the highest expression level of EDNRB. These results suggested that, in 4 breast cancer cell lines, the negative correlation existed between methylation status in the promoter region and expression of EDNRB gene. The expression of EDNRB gene in MCF-7 cells could be recovered by 5-azacytidine (5-Aza-CR) in a dose-dependent manner from 5 μmol/L to 10 μmol/L. Re-expression of EDNRB gene significantly inhibited cell proliferation. Conclusion Methylation of CpG islands in the promoter region of EDNRB gene may play important roles in the carcinogenesis and development of breast cancer. EDNRB gene may be a new molecular marker in the early diagnosis of breast cancer.

Key words: Breast cancer, Methylation, Endothelin receptor B, 5-azacytidine, Cell proliferation, Methylation-specific PCR