临床肿瘤学杂志

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siRNA干扰蛋白质精氨酸甲基转移酶5表达对人胃癌SGC7901细胞增殖和克隆形成能力的影响

吴晓斌,唐福婷,徐慧
  

  1. 210029 南京 江苏省中医院病理科
  • 收稿日期:2016-09-22 修回日期:2016-11-10 出版日期:2016-12-31 发布日期:2016-12-31

Effect of siRNA targeting protein arginine methyl transferase 5 on the proliferation and colony-formation in human gastric cancer SGC7901 cells

WU Xiaobin,TANG Futing,XU Hui   

  1. Department of Pathology,Jiangsu Provincial Hospital of Traditional Chinese Medicine,Nanjing210029,China
  • Received:2016-09-22 Revised:2016-11-10 Online:2016-12-31 Published:2016-12-31

摘要:

目的 探讨小干扰RNA(siRNA)靶向抑制蛋白质精氨酸甲基转移酶5(PRMT5)表达后对人胃癌SGC7901细胞增殖和克隆形成能力的影响。方法 设计靶向抑制PRMT5表达的特异siRNA(siRNA-1、siRNA-2),瞬时转染胃癌SGC7901细胞(干扰组),同时设置转染无义序列的阴性对照组。在瞬时转染48 h后采用Western blotting实验评估siRNA对PRMT5的干扰效果;采用CCK-8法检测siRNA抑制PRMT5表达后胃癌SGC7901细胞的增殖情况;采用EdU法检测siRNA抑制PRMT5表达后胃癌SGC7901细胞的DNA复制情况;采用平板克隆形成实验检测siRNA抑制PRMT5表达后对胃癌SGC7901细胞克隆形成能力的影响。结果 在胃癌SGC7901细胞中,瞬时转染siRNA-1和siRNA-2后PRMT5蛋白的表达水平均显著低于阴性对照组(P<0.01),表明两个siRNA具有良好的干扰效果,可用于后续实验。与阴性对照组相比,PRMT5表达干扰后的胃癌SGC7901细胞增殖速率显著下降,差异有统计学意义(P<0.01)。转染siRNA序列后,PRMT5干扰组(siRNA-1和siRNA-2)的EdU阳性细胞百分比为(16.0±2.0)%和(19.5±3.0)%,远低于阴性对照组的(38.0±4.0)%,差异有统计学意义(P<0.01)。PRMT5干扰组(siRNA-1和siRNA-2)的克隆形成数分别为(50±6)个和(68±7)个,远低于阴性对照组的(120±11)个,差异均有统计学意义(P<0.01)。结论 通过siRNA抑制PRMT5表达后可显著抑制胃癌SGC7901细胞的增殖和克隆形成能力,为胃癌的临床治疗提供新的策略和理论依据。

Abstract:

Objective To explore the effect of small interfering RNA(siRNA)targeting protein arginine methyltransferase 5(PRMT5)on the proliferation and colony-formation in human gastric cancer SGC7901 cells. Methods siRNA-1 and siRNA-2 designed for specific targeting PRMT5 were transiently transfected into SGC7901 cells. Meanwhile,antisense sequence was used as negative control(NC). 48 h after transfection,Western blotting method was used to confirm the interfering efficacy to ensure that siRNA can be used for following experiments. CCK-8 assay was used to detect the proliferation of SGC7901 cells after gene-specific siRNA was transfected. EdU assay was used to examine the DNA replication of SGC7901 cells and plate colonyformation assay was used to evaluate the colony-formation of SGC7901 cells when PRMT5 was silenced by siRNA. Results The level of PRMT5 protein was significantly decreased in SGC7901 cells when siRNA-1 and siRNA-2 were transfected,suggesting that siRNA-1 and siRNA-2 could be used for further studies. The proliferation rate of SGC7901 cells was significantly reduced when PRMT5 was silenced(P<0.01)in comparison with NC. Compared with NC,the positive rates of EdU cells interfered by siRNA-1 and siRNA-2 in PRMT5 group were(16.0±2.0)% and(19.5±3.0)%,much lower than(38.0±4.0)% in NC with significance(P<0.01). Furthermore,the numbers of SGC7901 colonies in PRMT5 group interfered by siRNA-1 and siRNA-2 were 50±6 and 68±7,lower than 120±11 of NC with significance(P<0.01). Conclusion Knockdown of PRMT5 by gene-specific siRNA can inhibit the proliferation and colony-formation abilities in human gastric cancer SGC7901 cells,which provide new methods and theoretical basis for the treatment of gastric cancer.

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