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沉默Rock2对肝癌细胞增殖及凋亡作用的研究

刘天德1,袁荣发2,王庆诺1,蒋成行1,杨志强1,邵江华3   

  1. 1. 南昌大学
    2. 南昌大学第二附属医院肝胆外科
    3. 南昌大学第二附属医院
  • 收稿日期:2011-12-27 修回日期:2012-06-13 出版日期:2012-10-15 发布日期:2012-10-15
  • 通讯作者: 邵江华

Study of Rock2 on the Proliferation and Apoptosis of Hepatocellular Carcinoma Cells

  • Received:2011-12-27 Revised:2012-06-13 Published:2012-10-15 Online:2012-10-15

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摘要： 目的：探讨沉默Rock2基因对人肝癌细胞Huh-7和HepG2增殖和凋亡作用的影响。方法：实验分为空白对照组、干扰无意义组、转染PBS组及干扰Rock2组。将Rock2干扰质粒shRock2转染到人肝癌细胞Huh-7和HepG2中，通过实时荧光定量PCR检测Rock2 mRNA的表达水平；Western blot检测Rock2蛋白的表达的水平；MTT比色法检测沉默Rock2后对Huh-7和HepG2细胞增殖抑制的影响；流式细胞术检测沉默Rock2对Huh-7和HepG2细胞周期及早期凋亡的变化。结果：实时荧光定量PCR以及Western blot结果显示，将shRock2转染肝癌细胞系Huh-7和HepG2后，Rock2 mRNA以及Rock2蛋白的表达水平均明显下降。沉默Rock2表达后，MTT结果显示Huh-7和HepG2细胞较对照组细胞增殖能力明显减弱（P<0.01）；流式细胞术检测结果显示Huh-7和HepG2细胞G0/G1期细胞比例明显升高，而S期和G2/M期细胞比例明显降低，与对照组比较差异有统计学意义（P <0.01）；肝癌细胞的早期凋亡较对照组明显增加（P<0.01）。结论：沉默Rock2能明显抑制肝癌细胞系Huh-7和HepG2的增殖，并诱导其早期凋亡，提示Rock2可作为肝癌基因治疗的一个新的分子靶点。

关键词: 癌, 肝细胞, 细胞增殖, 细胞凋亡, 肝肿瘤, 蛋白质丝氨酸苏氨酸激酶, 转染, 基因沉默

Abstract: Objective: To investigate the effects of Rock2 on proliferation and apoptosis of hepatocellular carcinoma Huh-7 and HepG2 cell lines. Methods: The experiment was divided into untreated group, non-targeting group, PBS group and shRock2 group. Hepatocellular carcinoma Huh-7 and HepG2 cell lines were transfected with shRock2. Reverse transcriptase polymerase chain reaction and Western blot assays were used to detect the Rock2 mRNA and protein levels, respectively. Huh-7 and HepG2 cell proliferation was measured by MTT assay. Change of cell cycle was detected by PI staining method through flow cytometry and earlier apoptosis was demonstrated by Annexin V apoptosis kit. Results: The Rock2 mRNA and protein levels both noticeably decreased in shRock2 transfected Huh-7 and HepG2 cells. After the silence of Rock2, cell proliferation was blocked in the shRock2 cells compared to the control groups (P<0.01). Knockdown of Rock2 promoted cell-cycle arrest at the G0/G1 phase. But the percentage of cells in S phase and G2/M decreased compared to the control groups（P<0.01）. The earlier apoptosis rate of shRock2 Huh-7 and HepG2 cells increased. This value was significantly higher than those of the control groups (P<0.01). Conclusion: Knockdown of Rock2 led to decreased proliferation, increased apoptosis in HCC cells and Rock2 may be a new target for HCC gene therapy.

Key words: carcinoma, hepatocellular, cell proliferation, apoptosis, liver neoplasms, protein-serine-threonine kinases, transfection, 基因沉默