[短发夹RNA对卵巢癌细胞中生存素mRNA表达及对紫杉醇化疗敏感性的影响]

[Influence of short hairpin RNA on survivin mRNA expression and chemosensitivity to paclitaxel in ovarian cancer cells].

作者信息

Yan Xiao-jian, Liang Li-zhi, Zeng Zong-yuan, Fu Li-wu, Shi Zhi

机构信息

Department of Gynecology, Cancer Center, Sun Yat-sen University, Guangzhou 510060, China.

出版信息

Zhonghua Fu Chan Ke Za Zhi. 2005 Sep;40(9):609-13.

PMID:16202317

Abstract

OBJECTIVE

To investigate the influence of short hairpin RNA (shRNA) expression plasmid against gene survivin (mU(6)/survivin) on survivin mRNA expression and chemosensitivity to paclitaxel in ovarian cancer cells OVCAR3.

METHODS

OVCAR3 cells were transfected with plasmid pEGFPC(2) formulated with lipofectamine 2000 at different concentrations. The transfection efficiency was examined by flow cytometry. The expression of survivin mRNA of OVCAR3 cells after transfection with the plasmid mU(6)/survivin with the high efficiency ratio was observed by RT-PCR. The effect of the plasmid on the cell cycle and apoptosis was analyzed by flow cytometry. The chemosensitivities of transfected cells to paclitaxel were determined by methyl thiazolyl tetrazolium (MTT).

RESULTS

The optimal transfection efficiency was obtained when pEGFPC(2): lipofectamine 2000 was 1: 2. Compared with the non-transfected groups, 0.81 +/- 0.05, the mRNA of survivin in OVCAR3 cells was reduced clearly after transfection with mU(6)/survivin, 0.26 +/- 0.04. shRNA reduced the expression level of survivin mRNA to 32% of non-transfected groups (P < 0.01). The apoptotic rate of survivin shRNA group reached (31.9 +/- 1.2)%, which was much higher than those of non-transfected (4.9 +/- 0.7)% and lip alone groups (5.6 +/- 0.5)% (P = 0.000). Cell cycle analysis showed that survivin shRNA induced accumulation of cells in G(0)/G(1) phase with a decrease of cells in G(2)/M phase after being cultured for 24 hours compared with non-transfected group (P < 0.01). MTT results showed that the 50% inhibiting concentration (IC(50)) of paclitaxel in non-transfected, lip alone and survivin shRNA transfected groups was (0.305 +/- 0.032), (0.157 +/- 0.031), and (0.019 +/- 0.001) micromol/L respectively. Compared with non-transfected group, shRNA increased the chemosensitivity of OVCAR3 cells to paclitaxel by 16 fold (P = 0.000).

CONCLUSION

Sequence specific shRNA targeting survivin can effectively suppress the expression of survivin mRNA and enhance the chemosensitivity to paclitaxel in ovarian cancer cells significantly.

摘要

目的

探讨针对生存素基因的短发夹RNA（shRNA）表达质粒（mU(6)/survivin）对卵巢癌细胞OVCAR3中生存素mRNA表达及对紫杉醇化疗敏感性的影响。

方法

用不同浓度的脂质体2000将质粒pEGFPC(2)转染OVCAR3细胞。通过流式细胞术检测转染效率。用RT-PCR观察高效转染质粒mU(6)/survivin后OVCAR3细胞中生存素mRNA的表达。通过流式细胞术分析该质粒对细胞周期和凋亡的影响。用噻唑蓝（MTT）法测定转染细胞对紫杉醇的化疗敏感性。

结果

当pEGFPC(2)与脂质体2000比例为1∶2时转染效率最佳。与未转染组（0.81±0.05）相比，转染mU(6)/survivin后OVCAR3细胞中生存素mRNA明显降低（0.26±0.04）。shRNA使生存素mRNA表达水平降至未转染组的32%（P<0.01）。生存素shRNA组凋亡率达（31.9±1.2）%，远高于未转染组（4.9±0.7）%和单纯脂质体组（5.6±0.5）%（P = 0.000）。细胞周期分析显示，与未转染组相比，培养24小时后生存素shRNA诱导细胞在G(0)/G(1)期积聚，G(2)/M期细胞减少（P<0.01）。MTT结果显示，未转染组、单纯脂质体组和生存素shRNA转染组中紫杉醇的50%抑制浓度（IC(50)）分别为（0.305±0.032）、（0.157±0.031）和（0.019±0.001）μmol/L。与未转染组相比，shRNA使OVCAR3细胞对紫杉醇的化疗敏感性提高了16倍（P = 0.000）。