天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (8): 984-987.doi: 10.11958/20150421

• 细胞与分子生物学 • 上一篇    下一篇

miR-200b 靶向DNMT3A 抑制非小细胞肺癌细胞增殖与诱导凋亡

罗卫民， 罗湘玉， 郭家龙， 林称意， 张军△   

  1. 湖北医药学院附属十堰市太和医院心胸外科 （邮编442000）
  • 收稿日期:2016-01-05 修回日期:2016-03-01 出版日期:2016-08-15 发布日期:2016-08-22
  • 通讯作者: 张军 E-mail： dengmin2006@163.com E-mail:2319218424@qq.com
  • 作者简介:罗卫民 （1978）， 男， 硕士研究生， 副主任医师， 主要从事食管癌、 肺癌临床研究
  • 基金资助:
    湖北省教育厅科学研究计划指导性项目 （B2015477）

miR-200b suppresses proliferation and induces apoptosis in non-small cell lung cancer cells by targeting DNMT3A

LUO Weimin, LUO Xiangyu, GUO Jialong, LIN Chengyi, ZHANG Jun△   

  1. Department of Cardiothoracic Surgery, the Affiliated Shiyan Taihe Hospital of Hubei College of Pharmacy, Shiyan 442000, China
  • Received:2016-01-05 Revised:2016-03-01 Published:2016-08-15 Online:2016-08-22
  • Contact: ZHANG Jun E-mail： dengmin2006@163.com E-mail:2319218424@qq.com

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摘要： 摘要： 目的 探讨miR-200b是否通过靶向调控DNMT3A抑制人非小细胞肺癌A549细胞增殖与诱导凋亡。方法 运用qRT-PCR检测miR-200b在不同非小细胞肺癌细胞株中的表达； 将miR-200b mimics、 scramble、 DNMT3A-siRNA、 control-siRNA分别转染于A549细胞， 其中scramble与control-siRNA分别作为miR-200b mimics与DNMT3A-siRNA 的阴性对照组。采用 Western blot 检测 A549 细胞中 DNMT3A 蛋白表达； 采用 MTT 与 AnnexinV-FITC/PI 染色法分别检测 A549 细胞的增殖与凋亡， 比较 miR-200b mimics 与 DNMT3A-siRNA 对 A549 细胞增殖与凋亡的影响。结果 qRT-PCR 结果显示， miR-200b 在非小细胞肺癌 A549、 H1299、 L78、 H460 细胞中的表达均明显低于正常人支气管上皮 16HBE 细胞， 其中以 A549 细胞下调最为明显 （P < 0.05）。Western blot 结果显示， 外源过表达 miR-200b 或沉默 DNMT3A 能明显下调 A549 细胞中 DNMT3A 蛋白的水平。MTT 结果显示， 转染 miR-200b mimics 或沉默 DN⁃ MT3A 48 h、 72 h、 96 h 后， 反映细胞增殖的光密度 （OD） 值与各自阴性对照组比较明显减小 （P < 0.05）。AnnexinV- FITC/PI 染色结果显示， 转染 miR-200b mimics 或沉默 DNMT3A 后， A549 细胞的凋亡率分别为 （23.33%±0.90%、 20.41%±0.70%） 均高于各自阴性对照组 （5.28%±0.55%、 5.68%±0.47%， P < 0.05）。结论 miR-200b通过下调DNMT3A 抑制人非小细胞肺癌细胞增殖与诱导凋亡。

关键词: 微 RNAs, 癌, 非小细胞肺, 细胞增殖, 细胞凋亡, miR-200b, DNA 甲基化转移酶 3A

Abstract: Abstract： Objective To investigate whether miR-200b suppresses proliferation and induces apoptosis of non-small cell lung cancer cells by targeting DNMT3A. Methods A qRT-PCR was employed for detecting the expression of miR-200b in different non-small cell lung cancer cells and human bronchial epithelial cells. A549 cells were transfected with miR-200b mimics, scramble, DNMT3A-siRNA and control-siRNA, respectively. The scramble and control-siRNA were served the negative control of miR-200b mimics and DNMT3A-siRNA, respectively. Western blot assay was conducted to detect the expression of DNMT3A protein in A549 cells. MTT and Annexin V/propidium iodide staining were employed to detect the proliferation ability and apoptosis rate of A549 cells. The effects of miR-200b mimics and DNMT3A-siRNA on the proliferation and apoptosis rate of A549 cells were compared between groups. Results Results of qRT-PCR showed that the expression of miR-200b was significantly down-regulated in A549, H1299, L78 and H460 cells than that of 16HBE cells. Among them, the most obviously reduction was found in A549 cells (P < 0.05). Western blot assay showed that the level of DNMT3A protein was inhibited by restored miR-200b or knock-down DNMT3A in A549 cells. After transfection of miR-200bmimics or knock-downDNMT3Afor 48 h, 72 h and 96 h,MTT showed that theODvalues, which reflected the optical density of cell proliferationwere significantly lower than those in the control group (P < 0.05). Annexin V/propidiumiodide staining showed that apoptosis rates of A549 cells after transfection of miR-200bmimics or knock-downDNMT3Awere (23.33%±0.90%and 20.41%±0.70%), comparedwith the control group (5.28%± 0.55%and 5.68%±0.47%, P < 0.01). Conclusion miR-200b suppresses cell proliferation and induces apoptosis by targeting DNMT3Ain non-small cell lung cancer.

Key words: microRNAs, carcinoma, non-small-cell lung, cell proliferation, apoptosis, miR-200b, DNMT3A