天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (4): 340-344.doi: 10.11958/j.issn.0253-9896.2015.04.002

• 细胞与分子生物学 • 上一篇    下一篇

甲基化调节miR-200b 的表达及其对胃癌MGC-803 细胞增殖侵袭能力的影响

郭蓉，宁向红，姜葵，张庆瑜△   

  1. 天津医科大学总医院消化内科（邮编300052）
  • 收稿日期:2014-11-10 修回日期:2015-01-20 出版日期:2015-04-15 发布日期:2015-04-13
  • 通讯作者: 张庆瑜 E-mail:1014852869@qq.com
  • 作者简介:郭蓉（1987），女，硕士在读，主要从事胃癌发病机制研究
  • 基金资助:
    国家自然科学基金资助项目（81172356）；天津市自然科学基金重点项目（10JCZDJC18500）

Methylation of miR-200b and its effects on proliferation，invasion of gastric cancer cell line MGC-803

GUO Rong，NING Xianghong，JIANG Kui，ZHANG Qingyu△   

  1. Department of Gastroenterology，Tianjin Medical University General Hospital，Tianjin 300052，China
  • Received:2014-11-10 Revised:2015-01-20 Published:2015-04-15 Online:2015-04-13
  • Contact: ZHANG Qingyu E-mail:1014852869@qq.com

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摘要： 摘要：目的体外研究基因miR-200b 发生甲基化的不同程度及其表达量的差异对胃癌细胞MGC-803 增殖、侵袭、凋亡及对上皮间质转化的影响。方法以正常胃黏膜上皮细胞GES-1、胃癌细胞MGC-803 为研究对象，提取细胞总RNA，逆转录后进行实时荧光定量PCR 检测2 种细胞系中miR-200b 的表达量；亚硫酸氢钠修饰PCR（BSP）法检测miR-200b 启动子区甲基化水平。用不同浓度5-氮杂胞苷（5′-Aza-CdR）药物处理MGC-803 细胞72 h 后，同法检测miR-200b 的表达量及启动子区甲基化水平变化。根据上述实验结果选择合适的药物作用浓度和时间即（10 μmol/L，72 h）作为处理组。通过Transwell 侵袭实验观测细胞侵袭能力；流式细胞技术检测细胞周期及凋亡的分布情况；Western-blot 检测上皮间质转化（EMT）相关指标E-cadherin、N-cadherin、ZEB1、Slug、基质金属蛋白酶（MMP）9 等的表达水平。结果miR-200b 在GES-1 中表达量较MGC-803 高（P=0.022）。启动子区甲基化水平则与之相反（P=0.034）。药物不同浓度作用后的MGC-803 细胞系miR-200b 表达量有随浓度升高而升高的趋势，作用浓度为10 μmol/L 时miR-200b 启动子区甲基化水平较对照组低（P=0.043）。与对照组相比，处理组细胞晚期凋亡数增多；G0/G1 期细胞数显著增多，S 期显著减少；穿过Transwell 小室细胞数显著减少；Western-blot 实验表明E-cad⁃ herin 表达量升高，N-cadherin、ZEB1、Slug、MMP9 表达量降低。结论miR-200b 启动子区甲基化程度的不同可以影响其表达量，而miR-200b 表达量的异常又与胃癌MGC-803 细胞增殖、侵袭能力密切相关。

关键词: 胃肿瘤, 腺癌, 细胞凋亡, 细胞增殖, DNA 甲基化, 微RNA-200b

Abstract: Abstract: Objective To investigate the effect of expression level and methylation level of miR-200b on proliferation, invasion and apoptosis of gastric cancer cell line MGC-803 in vitro. Methods Normal human gastric epithelium cell line GES-1, and gastric cancer cell line MGC-803 cells were cultured and harvested to extract total RNA. Then miR-200b ex⁃ pression level was examined via q-PCR；Methylation in promoter of miR-200b was revealed by Bisulphite PCR. MGC-803 cells were treated with different concentrations of 5′-Aza-CdR to test its effect on miR-200b expression and methylation of its promotor. The effect of its treatment at 10 μmol/L for 72 h on invasion，proliferation and apoptosis of both cell lines were detected by Transwell assay, Flow cytometry and apoptosis assay respectively. The gene related to EMT (epithelial-mesen⁃ chymal transition ) such as E-cadherin，N-cadherin，ZEB1，Slug were checked by Western blot. Results The expression of miR-200b in GES-1 is higher than that in MGC-803（P=0.022）. The methylation level in promoter region of miR-200b in GES-1 is lower than that in MGC-803（P=0.034）. After treated with 5′-Aza-CdR, the expression of miR-200b in MGC- 803 cell was up-regulated in a timely dependent manner. On the contrary, the methylation in promoter of mirR-200b were down-regulated when 5′-Aza-CdR were added at 10 μmol/L（P=0.043）. What’s more, 5′-Aza-CdR administration increas⁃ es cell apoptosis especially in aged cells and delays cell cycle at G0/G1 which in turn decrease ratio of cells in S phages. 5′- Aza-CdR treatment also decreased invasiveness and induced expression of E-cadherin, as well as down regulated the ex⁃ pressions of N-cadhrin, ZEB1, Slug and MMP9 in MGC-803 cells. Conclusion Expression of miR-200b could be affected by methylation of promoter of miR-200b and in turn regulate proliferation and invasion of gastric cells in vitro.

Key words: stomach neoplasms, adenocarcinoma, cell apoptosis, cell proliferation, DNA methylation, microRNA-200b