中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (11): 772-775.

• 论著 • 上一篇    下一篇

siRNA干扰水通道蛋白3和磷脂酶D2表达对A431细胞株生长和凋亡的影响

王小勇1,陶承军1,袁丞达1,王敏磊1,应航宇2,任金平2   

  1. 1. 杭州市中医院
    2. 杭州市中医院皮肤科
  • 收稿日期:2013-12-11 修回日期:2014-06-20 出版日期:2014-11-15 发布日期:2014-11-01
  • 通讯作者: 王小勇 E-mail:wangxiaoyong1974@163.com
  • 基金资助:
    浙江省自然科学基金

Effects of interference with the expressions of aquaporin 3 and phospholipase D2 by small interfering RNAs on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431

  • Received:2013-12-11 Revised:2014-06-20 Online:2014-11-15 Published:2014-11-01

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摘要： 目的 探讨水通道蛋白3（AQP3）和磷脂酶D2（PLD2）在人皮肤鳞状细胞癌细胞株A431细胞增殖和凋亡中的作用。 方法 针对AQP3和PLD2各设计3条siRNA，用脂质体转染法将siRNA导入A431细胞。用荧光定量PCR方法筛选出其中干扰效果最佳的AQP3-siRNA和PLD2-siRNA，Western印迹法检测转染后AQP3和PLD2蛋白的表达水平。将A431细胞分为5组：正常培养组（不加任何处理），转染试剂组（加入转染试剂），阴性对照组（转染阴性对照siRNA），AQP3-siRNA组（转染AQP3-siRNA），PLD2-siRNA组（转染PLD2-siRNA）。CCK8法检测AQP3和PLD2表达下调对A431细胞增殖的影响，膜联蛋白-V-异硫氰酸荧光素/碘化丙锭双染色后用流式细胞仪检测细胞凋亡。 结果 用脂质体转染法将siRNA导入A431细胞后，AQP3和PLD2的mRNA和蛋白表达均较阴性对照组明显下降。与阴性对照组比较，转染AQP3-siRNA后A431细胞增殖在24、48、72 h均受到明显抑制（t值分别为24.10、11.00、9.54，均P < 0.01）；转染PLD2-siRNA后，A431细胞增殖同样在24、48、72 h均受到明显抑制（t值分别为30.47、7.02、8.73，均P < 0.01）。与阴性对照组比较，转染AQP3-siRNA后，A431细胞凋亡在48 h和72 h均明显增加（t值分别为11.36、20.91，均P < 0.01）；转染PLD2-siRNA后，A431细胞凋亡在72 h明显增加（t值为4.86，P < 0.05）。 结论 siRNA下调AQP3或PLD2表达可抑制A431细胞增殖，诱导A431细胞凋亡。

关键词: 水通道蛋白质3, 磷脂酶D, RNA，小分子干扰, 癌，鳞状细胞, 细胞系，肿瘤

Abstract: Wang Xiaoyong, Tao Chengjun, Yuan Chengda, Wang Minlei, Ying Hangyu, Ren Jinping. Department of Dermatology, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou 310007, China Corresponding author: Wang Xiaoyong, Email: wangxiaoyong1974@163.com 【Abstract】 Objective To investigate the effects of aquaporin 3 （AQP3） and phospholipase D2 （PLD2） on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431. Methods Three small interfering RNAs （siRNAs） were constructed targeting the AQP3 and PLD2 genes separately, and transfected into A431 cells using liposomes. Then, fluorescence quantitative PCR was performed to find the most efficient siRNAs. Western blot was conducted to detect the protein expression levels of AQP3 and PLD2 in A431 cells after transfection with the selected AQP3-siRNA and PLD2-siRNA. Some A431 cells were divided into five groups: normal control group without any treatment, transfection reagent group treated with the oligofectamine reagent only, negative control group transfected with the negative control siRNA, AQP3-siRNA group transfected with the selected AQP3-siRNA, PLD2-siRNA group transfected with the selected PLD2-siRNA. After additional culture, cell counting kit-8 assay was performed to evaluate the proliferation of A431 cells, flow cytometry to detect the apoptosis of A431 cells after annexin V-fluorescein isocyanate/propidium iodide double-staining. Statistical analysis was carried out by the paired t test. Results The transfection with AQP3-siRNA and PLD2-siRNA induced a significant decrease in the mRNA and protein expressions of AQP3 and PLD2 respectively in A431 cells when compared with the untransfected cells. Compared with the negative control group, the proliferation of A431 cells was significantly decelerated at 24, 48 and 72 hours after transfection in the AQP3-siRNA group （t = 24.10, 11.00, 9.54, respectively, all P < 0.01） and PLD2-siRNA group （t = 30.47, 7.02, 8.73, respectively, all P < 0.01）. A significant increase was observed in the apoptosis of A431 cells at 48 and 72 hours after transfection with AQP3-siRNA （t = 11.36, 20.91, respectively, both P < 0.01）, and at 72 hours after transfection with PLD2-siRNA （t = 4.86, P < 0.05）compared with the negative control group. Conclusion The down-regulation of AQP3 and PLD2 expressions by siRNA can inhibit the proliferation, but induce the apoptosis, of A431 cells.

Key words: Aquaporin 3, Phospholipase D, RNA，small interfering, Carcinoma, squamous cell, Cell line, tumor