Deng Kai-xian, Zhong Ling, Jiang Mei-xian, Wang Ping-ling, Chen Ying
Department of Obstetrics and Gynecology, Second Affiliated Hospital, Chongqing University of Medical Science, Chongqing 400010, China.
Zhonghua Fu Chan Ke Za Zhi. 2005 Dec;40(12):836-9.
To examine expression of survivin gene in ovarian epithelial carcinoma drug resistant cell line SKOV3/ADM and its parental cell line SKOV3, and induction of cells apoptosis and reversal of drug resistance in SKOV3/ADM after RNA interference (RNAi) silencing survivin gene.
The transcription of survivin gene in cells was detected by semi-quantitative RT-PCR, the protein expression level of survivin gene was analyzed by immunofluorescence staining. SKOV3/ADM cells were treated with pshRNA-survivin and paclitaxel (Taxol), and acridine orange (AO)/ethidium bromide (EB) staining was performed to evaluate the apoptosis of cells.
Survivin gene mRNA expressed by 99.1% and 75.3% respectively in cell lines SKOV3/ADM and SKOV3, while fluorescent cells were 59 +/- 5 and 42 +/- 3 (P < 0.05). After the introduction of pshRNA-survivin into SKOV3/ADM, mRNA transcription level of survivin gene decreased distinctly from 99.1% to 7.9%. The apoptotic cells of control group detected by AO/EB staining was 3.6 +/- 0.6, of Taxol group 10.2 +/- 1.0, of RNAi group 48.5 +/- 4.9, of RNAi + Taxol group 71.5 +/- 6.8. Apoptosis ratio between RNAi + Taxol group and RNAi group had significant difference (P < 0.05), and that between RNAi + Taxol group and Taxol group also had significant difference (P < 0.05).
Both survivin gene mRNA and its protein are over-expressed in ovarian epithelial carcinoma cell lines SKOV3 and SKOV3/ADM, the level of survivin gene expressed in SKOV3/ADM is obviously different compared with that in its parental cell line SKOV3. RNA interference targeted against specific sequences of survivin in SKOV3/ADM cell could significantly reduce the level of survivin mRNA transcripts and protein, effectively induce the cells apoptosis and restore the sensitivity of cell to conventional chemotherapeutic agents Taxol.
检测生存素基因在卵巢上皮癌耐药细胞系SKOV3/ADM及其亲本细胞系SKOV3中的表达情况，以及RNA干扰（RNAi）沉默生存素基因后对SKOV3/ADM细胞凋亡及耐药逆转的影响。
采用半定量RT-PCR检测细胞中生存素基因的转录情况，免疫荧光染色分析生存素基因的蛋白表达水平。用pshRNA-生存素和紫杉醇（泰素）处理SKOV3/ADM细胞，采用吖啶橙（AO）/溴化乙锭（EB）染色评估细胞凋亡情况。
生存素基因mRNA在SKOV3/ADM和SKOV3细胞系中的表达率分别为99.1%和75.3%，荧光细胞数分别为59±5和42±3（P<0.05）。将pshRNA-生存素导入SKOV3/ADM后，生存素基因的mRNA转录水平从99.1%明显降至7.9%。AO/EB染色检测对照组凋亡细胞数为3.6±0.6，紫杉醇组为10.2±1.0，RNAi组为48.5±4.9，RNAi+紫杉醇组为71.5±6.8。RNAi+紫杉醇组与RNAi组凋亡率比较差异有统计学意义（P<0.05），RNAi+紫杉醇组与紫杉醇组比较差异也有统计学意义（P<0.05）。
生存素基因mRNA及其蛋白在卵巢上皮癌细胞系SKOV3和SKOV3/ADM中均过度表达，SKOV3/ADM中生存素基因的表达水平与其亲本细胞系SKOV3相比有明显差异。针对SKOV3/ADM细胞中生存素的特定序列进行RNA干扰可显著降低生存素mRNA转录本和蛋白水平，有效诱导细胞凋亡并恢复细胞对传统化疗药物紫杉醇的敏感性。
