南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (04): 550-555.doi: 10.12122/j.issn.1673-4254.2020.04.16

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木犀草素通过逆转OPCML基因甲基化抑制乳腺癌MDA-MB-231细胞增殖

董欣敏，郑 媞，张子英，白喜玲，李 华，张 剑   

  • 出版日期:2020-04-30 发布日期:2020-04-20
  • 基金资助:

Luteolin reverses OPCML methylation to inhibit proliferation of breast cancer MDA-MB- 231 cells

  

  • Online:2020-04-30 Published:2020-04-20

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摘要： 目的 观察木犀草素对MDA-MB-231乳腺癌细胞增殖以及对OPCML基因表达的影响。方法 体外培养MDA-MB-231细 胞，加入终浓度为0（对照组）以及5、10和20 μmol/L的木犀草素处理孵育24 h或48 h后，MTT法检测细胞的增殖，流式细胞术检测细胞凋亡；并提取胞内总mRNA和蛋白，以实时定量PCR和Western blot检测OPCML的mRNA及蛋白变化；同时采用甲基化特异性PCR（MSP）检测OPCML启动子区域的甲基化水平，并分析细胞内甲基化酶的活性。结果 MTT结果显示，MDAMB-231细胞经5、10 以及20 μmol/L木犀草素孵育24 h后，随着浓度的递增，细胞活性逐渐降低（P<0.05）。流式细胞术结果也显示，不同浓度木犀草素可不同程度诱导MDA-MB-231细胞凋亡（P<0.05）。此外，木犀草素能以剂量依赖性方式诱导MDAMB-231细胞表达OPCML mRNA和蛋白（P<0.05）。MSP结果也显示，木犀草素可显著下调OPCML启动子的甲基化状态，上调非甲基化OPCML的水平，并能降低细胞内甲基化酶的活性（P<0.05）。结论 木犀草素可抑制MDA-MB-231乳腺癌细胞的增殖，其机制可能与影响OPCML基因的表达以及甲基化水平有关。

Abstract: Objective To observe the effect of luteolin on the proliferation and expression of OPCML in breast cancer cell line MDA-MB-231. Methods Cultured MDA-MB-231 cells were treated with luteolin at the concentrations of 5, 10 and 20 μmol/L for 24 or 48 h. MTT assay was used to detect cell proliferation and flow cytometry was used to detect the cell apoptosis. The expressions of OPCML mRNA and protein were detected using real-time quantitative PCR and Western blotting, respectively. OPCML gene methylation in the promoter region was detected using methylation-specific PCR (MSP), and the activity of methylase in the cells was analyzed. Results MTT assay showed that treatment with luteolin at 5, 10 and 20 μmol/L for 24 h concentration-dependently decreased the viability of MDA-MB-231 cells (P<0.05). Flow cytometry also showed that luteolin at different concentrations could induce apoptosis of MDA-MB-231 cells (P<0.05). Luteolin dose-dependently induced the expression of OPCML mRNA and protein in MDA-MB-231 cells (P<0.05), down-regulated the methylation status in the promoter region of OPCML gene, up-regulated the level of non-methylated OPCML, and reduced the activity of methylase in the cells (P<0.05). Conclusion Luteolin inhibits the proliferation of MDA-MB-231 breast cancer cells probably by upregulating OPCML expression and its demethylation.