基础医学与临床 ›› 2016, Vol. 36 ›› Issue (11): 1525-1530.

• 研究论文 • 上一篇    下一篇

siRNA干扰14-3-3ζ基因促进人胃癌SGC-7901细胞细胞凋亡并抑制其增殖

李小山1,李国利1,冯晓灵1,刘丹1,马强1,何雪莲2   

  1. 1. 重庆三峡医药高等专科学校
    2. 重庆三峡中心医院
  • 收稿日期:2016-01-08 修回日期:2016-05-03 出版日期:2016-11-05 发布日期:2016-10-24
  • 通讯作者: 何雪莲 E-mail:437089764@qq.com
  • 基金资助:
    熊果酸抗肿瘤作用靶点的表观遗传学研究

siRNA down-regulating 14-3-3ζ expression inhibits proliferation and induces apoptosis in SGC-7901cell line in vitro

  • Received:2016-01-08 Revised:2016-05-03 Online:2016-11-05 Published:2016-10-24

摘要: 目的 探讨siRNA干扰14-3-3ζ基因对人胃腺癌细胞系SGC-7901增殖凋亡的影响及其作用机制。方法 利用siRNA干扰技术降低14-3-3ζ基因的正常表达的SGC-7901细胞为实验组,siRNA-control的对照组,和未加处理的空白组。用实时荧光定量PCR 和Western blot检测14-3-3ζ基因mRNA 的表达水平和蛋白表达水平;CCK8实验检测各组SGC-7901细胞的增殖;流式细胞术检测细胞周期和凋亡;Western blot检测周期和凋亡相关蛋白p53、cyclinD1、PUMA、Bax和Bcl-2。结果 siRNA干扰组14-3-3ζ基因的mRNA和蛋白表达水平低于空白组和对照组(P<0.05);CCK8检测细胞增殖较对照组显著降低(P<0.05);siRNA干扰组细胞周期G0/G1的比率较对照组高,而G2/M和S期则显著降低(P<0.05);siRNA干扰组的细胞凋亡率也显著升高(P<0.05);相关通路蛋白在siRNA干扰组中cyclinD1和Bcl-2表达降低,p53、PUMA、Bax蛋白表达增高(P<0.05)。结论 siRNA干扰14-3-3ζ可以通过p53通路,下调cyclinD1抑制SGC-7901细胞增殖并阻滞周期,上调PUMA、Bax蛋白和下调Bcl-2蛋白促进细胞凋亡。

关键词: 14-3-3ζ, RNA干扰, 增殖, 细胞周期, 凋亡

Abstract: Objective To investigate the effects of siRNA targeting 14-3-3ζ gene on the proliferation and apoptosis in human gastric adenocarcinoma cell line SGC-7901 and the latent mechanisms. Methods Three groups were devided as blank, siRNA-control and siRNA-14-3-3ζ groups. The 14-3-3ζ-siRNA was transfected into SGC-7901 cells by LipofectamineTM 2000. The gene silencing effect of siRNA targeting 14-3-3ζ was determined by quantity real-time PCR (RT-qPCR) and western blot at mRNA and protein levels respectively. Cell proliferation was detected by CCK8 assay. The cell cycle and apoptosis were detected by flow cytometry (FCM). The protein levels of p53, cylinD1, PUMA, Bax and Bcl-2 were analyzed by western blot. Results After transfection of 14-3-3ζsiRNA into SGC-7901 cells, the mRNA and protein levels of 14-3-3ζ were both significantly reduced. Compared with the control groups, the down-regulation of 14-3-3ζ in SGC-7901 cells resulted in significant decrease of cell proliferation (P<0.05). FCM showed significant increase G0/G1 phase of cell cycle and apoptotic rate in siRNA-14-3-3ζ cells (P<0.05). Besides, down-regulation of 14-3-3ζ cells also increased the expression of p53, PUMA and Bax protein, reduced the expression of cyclinD1 and Bcl-2 protein (P<0.05). Conclusion Down-regulation of 14-3-3ζ in SGC-7901 cells can inhibit proliferation and induce apoptosis via p53 pathway by down-regulating CcyclinD1 and Bcl-2 proteins and up-regulating PUMA and Bax proteins.

Key words: 14-3-3ζ, siRNA, proliferation, cell cycle, apoptosis