常海平, 田原, 王敬芝, 徐杰, 勾晓娟, 程建新. siRNA特异性沉默TPX2基因对人宫颈腺癌Hela细胞体外生长的影响[J]. 中国肿瘤临床, 2012, 39(2): 80-84. DOI: 10.3969/j.issn.1000-8179.2012.02.006
引用本文: 常海平, 田原, 王敬芝, 徐杰, 勾晓娟, 程建新. siRNA特异性沉默TPX2基因对人宫颈腺癌Hela细胞体外生长的影响[J]. 中国肿瘤临床, 2012, 39(2): 80-84. DOI: 10.3969/j.issn.1000-8179.2012.02.006
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Haiping CHANG, Yuan TIAN, Jingzhi WANG, Jie XU, Xiaojuan GOU, Jianxin CHENG. Effect of Specific TPX2-siRNA on Growth of Human Cervical Adenocarcinoma Hela Cells in vitro[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2012, 39(2): 80-84. DOI: 10.3969/j.issn.1000-8179.2012.02.006
Citation: Haiping CHANG, Yuan TIAN, Jingzhi WANG, Jie XU, Xiaojuan GOU, Jianxin CHENG. Effect of Specific TPX2-siRNA on Growth of Human Cervical Adenocarcinoma Hela Cells in vitro[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2012, 39(2): 80-84. DOI: 10.3969/j.issn.1000-8179.2012.02.006
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siRNA特异性沉默TPX2基因对人宫颈腺癌Hela细胞体外生长的影响

Effect of Specific TPX2-siRNA on Growth of Human Cervical Adenocarcinoma Hela Cells in vitro

    摘要
  • 摘要:
      目的  应用TPX2-siRNA转染Hela细胞, 探讨TPX2基因沉默后对Hela细胞增殖和侵袭力及周期分布的影响。
      方法  合成TPX2特异性的siRNA 4对, 随机siRNA阴性对照1对。Lipofectamine 2000介导siRNA转染Hela细胞株。RT-PCR检测转染前后mRNA表达的变化, Western blot检测转染前后蛋白表达的变化, MTT、流式细胞术(FCM)、Transwell小室和Matrigel基质侵袭实验观察细胞增殖、周期、及侵袭能力的变化。
      结果  在600μL转染体积中, 在siRNA浓度为20μmoL/L时, siRNA和Lipoectamine2000以2.5:2比例搭配时, 转染效率最高, 24h为(98.33±1.53)%。TPX2mRNA沉默效果尤以TPX2-siRNA4最强达(82.5±0.43)%, 其蛋白表达水平明显降低, 抑制率达(63.4±1.05)%。TPX2-siRNA4转染组发生了明显的细胞周期S期停顿现象, 细胞增殖受到显著抑制。TPX2-siRNA4转染组穿膜细胞为13.33±1.53显著低于空白对照组54.33±2.52和阴性对照组53.00±4.00(P < 0.05)。
      结论  siRNA干扰TPX2后能抑制Hela细胞增殖和侵袭, 影响其周期分布, 因此TPX2可以作为人宫颈腺癌基因治疗的候选靶点。

     

    Abstract:
      Objective  To investigate the inhibitory action of TPX2 with RNA interference (RNAi) on cellular cycle, proliferation and invasiveness of Hela cells.
      Methods  Four pairs of TPX2-siRNA and a pair of randomly negative-controlled siRNA were synthesized. SiRNA was transfected into the Hela cells by Lipofectamine 2000. TPX2 mRNA was determined by RT-PCR. Western blot was used to detect the expression of TPX2 protein. Flow cytometry (FCM), MTT reduction assay, Transwell and matrigel invasion assay were used to observe the biological behaviors of the Hela cells.
      Results  In 600ul volume of the transfection liquid, when the concentration of siRNA was 20umol/L, the ratio between siRNA and lipo2000 was 2.5 vs 2, the transfection efficiency was the highest(98.33± 1.53) % after they were transfected for 24 hours. THE expression of TPX2 mRNA in Hela cells transfected with TPX2-siRNA4 was decreased(82.5 ± 0.43) % with the most silencing effect. Expression of TPX2 protein was reduced (63.4 ±1.05) %, and the proliferation of Hela cell was obviously inhibited in TPX2-siRNA4-transfected group. FCM analysis showed that the Hela cells transfected with TPX2-siRNA4 had S-phase cells cycle arrest. The penetrating cells were significantly fewer in the TPX2-siRNA4-transfected group (13.33± 1.53) than in the blank control (54.33 ± 2.52) and negative control group (53.00 ± 4.00) (P < 0.05).
      Conclusion  Down-regulating the expression of TPX2 gene by siRNA can inhibit the proliferation and invasion of Hela cells and can affect the cell cycle distribution. Therefore, TPX2 may become a therapy target for cervical adenocarcinoma.

     

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