中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (3): 191-194.

• 论著 • 上一篇    下一篇

短发夹RNA干扰STAT3基因对恶性黑素瘤细胞A375生物学行为的影响

陈静1,李家文2,陈宏翔3,王豫平4,李振鲁1   

  1. 1. 郑州市河南省人民医院皮肤科
    2. 武汉市华中科技大学同济医学院附属协和医院皮肤科，Affiliated Union Hospital, Tongji Medical College of Huazhong University of Science and Technology
    3. 华中科技大学同济医学院附属协和医院
    4. 河南省人民医院
  • 收稿日期:2011-04-12 修回日期:2011-10-28 出版日期:2012-03-15 发布日期:2012-02-29
  • 通讯作者: 李振鲁 E-mail:lizhenlu@sohu.com
  • 基金资助:

    负调控STAT3和Ror-γ探讨DC-IL-23-Th17轴在银屑病发病中的作用机制

Effects of a short hairpin RNA targeting STAT3 gene on the biological behavior of a human malignant melanoma cell line A375

  • Received:2011-04-12 Revised:2011-10-28 Online:2012-03-15 Published:2012-02-29

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摘要：

目的 研究短发夹RNA（shRNA）沉默STAT3基因表达对人恶性黑素瘤细胞A375增殖和凋亡的作用。 方法 设计和构建靶向STAT3基因有小发夹结构的正义和反义寡核苷酸，退火后克隆入psiRNA-hH1neo质粒，使用脂质体转染A375。实验分为对照组、空载体组和STAT3转染组。通过RT-PCR和Western印迹检测A375细胞中STAT3 基因mRNA和蛋白表达水平，MTT比色法检测细胞生长抑制率，流式细胞仪分析细胞周期分布，观察细胞凋亡。结果 STAT3转染组A375细胞STAT3 mRNA和蛋白表达水平（分别为0.2136 ± 0.0626、0.8214 ± 0.0430）明显低于对照组（0.7826 ± 0.0701、3.1693 ± 0.0846）和空载体组（0.8518 ± 0.0783、3.2180 ± 0.0780），P值均 < 0.01。STAT3转染24、48、72 h组的细胞增殖抑制率明显增高（分别为21.35% ± 2.12%、32.52% ± 2.64%、40.40% ± 3.08%），与正常对照组（1.39% ± 0.53%、3.05% ± 1.16%、4.41% ± 1.42%）、空载体组（2.63% ± 1.38%、5.84% ± 2.47%、10.32% ± 2.48%）比较，差异均有统计学意义（P < 0.01）。流式细胞仪检测显示，STAT3转染组细胞的凋亡率（81.06% ± 2.10%）较对照组（26.28% ± 0.47%）和空载体组（27.31% ± 1.05%）明显增高，差异有统计学意义（P < 0.01）。细胞周期分析显示，STAT3转染组G0 / G1期细胞比例（68.43% ± 4.00%）增高，S期细胞比例（17.40% ± 2.05%）降低，与对照组（分别为60.07% ± 2.47%、28.40% ± 2.09%）及空载体组（分别为60.29% ± 2.26%、27.34% ± 3.63%）相比，差异有统计学意义（P < 0.01或 < 0.05）。结论 针对STAT3基因的特异性小RNA干扰质粒能够下调STAT3 基因和蛋白表达，抑制A375的增殖能力，诱导细胞凋亡。

关键词: 信号转导与转录活化因子3

Abstract:

Objective To study the effects of short hairpin RNA （shRNA） targeting STAT3 gene on the proliferation and apoptosis of A375 human malignant melanoma cells. Methods Sense and antisense oligonucleotides with small hairpin structures targeting STAT3 gene were designed, synthesized and cloned into the plasmid vector psiRNA-hH1neo after annealing. Cultured A375 cells were divided into 3 groups: control group receiving no treatment, psiRNA-H1 group transfected with empty plasmid, and psiRNA-H1/STAT3 group transfected with the recombinant plasmid containing the shRNA. After additional culture for different durations, reverse transcription PCR and Western blot were performed to detect the expression of STAT3 mRNA and protein, methyl thiazolyl tetrazolium （MTT） assay to evaluate cell proliferation, flow cytometry to assess cell cycle and apoptosis. Results The expression level of STAT3 mRNA and protein in A375 cells in psiRNA-H1/STAT3 group （0.2136 ± 0.0626, 0.8214 ± 0.043, respectively） were significantly lower than that in the control group（0.7826 ± 0.0701, 3.1693 ± 0.0846, respectively, both P < 0.01） and psiRNA-H1 group （0.8518 ± 0.0783, 3.218 ± 0.078, respectively, both P < 0.01）. The inhibition rates of cell proliferation at 24, 48 and 72 hours were 21.35% ± 2.12%, 32.52% ± 2.64% and 40.4% ± 3.08% respectively in psiRNA-H1/STAT3 group, statistically different from those in the control group （1.39% ± 0.53%, 3.05% ± 1.16%, 4.41% ± 1.42%, respectively, all P < 0.01） and psiRNA-H1 group （2.63% ± 1.38%, 5.84% ± 2.47%, 10.32% ± 2.48%, respectively, all P < 0.01）. Flow cytometry showed a statistical increase in cell apoptosis rate in psiRNA-H1/STAT3 group compared with the control and psiRNA-H1 group （81.06% ± 2.10% vs. 26.28% ± 0.47% and 27.31% ± 1.05%, both P < 0.01）. The psiRNA-H1/STAT3 group exhibited a higher percentage of cells at G0/G1 phase （68.43% ± 4.00%） but a lower percentage of cells at S phase （17.4% ± 2.05%） compared with the control group （60.07% ± 2.47%, P < 0.05; 28.40% ± 2.09%, P < 0.01） and psiRNA-H1 group（60.29% ± 2.26%, 27.34% ± 3.63%, both P < 0.05 ）. Conclusions The small interference RNA targeting STAT3 gene can specifically down-regulate the expressions of STAT3 mRNA and proteins in, inhibit cellular proliferation of, and induce apoptosis in, A375 cells.

Key words: STAT3