Yan Xiao-Jian, Liang Li-Zhi, Zeng Zong-Yuan, Shi Zhi, Fu Li-Wu
State Key Laboratory of Oncology in South China, Guangzhou, Guangdong 510060, P. R. China.
Ai Zheng. 2006 Apr;25(4):398-403.
BACKGROUND & OBJECTIVE: Drug resistance is a major obstacle to the successful chemotherapy of ovarian cancer. Recent studies have shown overexpression of Survivin in ovarian cancer tissues and cell lines, which may play an important role in the drug resistance of ovarian cancer. This study was to explore the effects of Survivin short hairpin RNA (shRNA) on Survivin expression, apoptosis, and chemosensitivity of human ovarian cancer cell line OVCAR3.
OVCAR3 cells were transfected with Survivin shRNA. Untransfected, lip-transfected, and mU6-transfected cells were set as controls. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of Survivin mRNA. Flow cytometry was applied to examine the expression of Survivin protein and cell apoptosis. MTT assay was used to examine the effect of Survivin shRNA on chemosensitivity of OVCAR3 cells.
The mRNA and protein levels of Survivin were obviously lower in Survivin shRNA-transfected OVCAR3 cells than in untransfected cells, lip-transfected cells, and mU6-transfected cells 24 h after transfection. The apoptotic rates of OVCAR3 cells 12 h, 24 h, 36 h, 48 h after Survivin shRNA transfection were 20.7%, 31.9%, 39.0%, and 46.7%, respectively, that showed a time-dependent manner. The 50% inhibitory concentrations (IC50) of paclitaxel were (0.305+/-0.032) micromol/L for untransfected cells, (0.157+/-0.031) micromol/L for lip-transfected cells, (0.175+/-0.010) micromol/L for mU6-transfected cells, and (0.019+/-0.001) micromol/L for Survivin shRNA-transfected cells; and the IC50 of cisplatin were (9.410+/-0.796) micromol/L, (6.675+/-1.739) micromol/L, (6.930+/-1.273) micromol/L, and (7.862+/-0.081) micromol/L, respectively. Survivin shRNA increased the sensitivity of OVCAR3 cells to paclitaxel by 16 folds (P<0.01), but had no significant effect on the sensitivity to cisplatin (P>0.05).
Sequence-specific shRNA targeting Survivin can suppress the expression of Survivin gene effectively in OVCAR3 cells, and sensitize OVCAR3 cells to paclitaxel, but has no significant effect on the sensitivity to cisplatin.
耐药性是卵巢癌化疗成功的主要障碍。近期研究表明，Survivin在卵巢癌组织和细胞系中过表达，这可能在卵巢癌耐药中起重要作用。本研究旨在探讨Survivin短发夹RNA（shRNA）对人卵巢癌细胞系OVCAR3中Survivin表达、细胞凋亡及化疗敏感性的影响。
用Survivin shRNA转染OVCAR3细胞。将未转染、脂质体转染及mU6转染的细胞设为对照。采用逆转录-聚合酶链反应（RT-PCR）检测Survivin mRNA的表达。应用流式细胞术检测Survivin蛋白表达及细胞凋亡。采用MTT法检测Survivin shRNA对OVCAR3细胞化疗敏感性的影响。
转染Survivin shRNA的OVCAR3细胞在转染后24 h，Survivin的mRNA和蛋白水平明显低于未转染细胞、脂质体转染细胞及mU6转染细胞。Survivin shRNA转染后12 h、24 h、36 h、48 h，OVCAR3细胞的凋亡率分别为20.7%、31.9%、39.0%和46.7%，呈时间依赖性。未转染细胞紫杉醇的50%抑制浓度（IC50）为（0.305±0.032）μmol/L，脂质体转染细胞为（0.157±0.031）μmol/L，mU6转染细胞为（0.175±0.010）μmol/L，Survivin shRNA转染细胞为（0.019±0.001）μmol/L；顺铂的IC50分别为（9.410±0.796）μmol/L、（6.675±1.739）μmol/L、（6.930±1.273）μmol/L和（7.862±0.081）μmol/L。Survivin shRNA使OVCAR3细胞对紫杉醇的敏感性提高了16倍（P<0.01），但对顺铂敏感性无显著影响（P>0.05）。
靶向Survivin的序列特异性shRNA能有效抑制OVCAR3细胞中Survivin基因的表达，使OVCAR3细胞对紫杉醇敏感，但对顺铂敏感性无显著影响。
