Lu Yan-Ming, Zhang Shu-Lan, Meng Li-Rong, Zhao Yan-Yan
Department of Gynecology and Obstetrics, Second Affiliated Hospital of China Medical University, Shenyang 110004, China.
Zhonghua Yi Xue Za Zhi. 2008 Apr 1;88(13):909-13.
To investigate the effects of RNA interference (RNAi) targeting human epidermal growth factor receptor 2 (HER-2) gene on the change of chemosensitivity to cisplatin of ovarian carcinoma cells. Methods Three kinds of HER-2 gene targeting siRNA, HER-2 siRNA I-III, were synthesized and the best one (HER-2 siRNA III) was screened. Human ovarian carcinoma cells of the line SKOV3 were cultured randomly divided into 3 groups: HER-2 siRNA III group, transfected with HER-2 siRNA III, non-specific siRNA group, transfected with non-specific siRNA III, and control group, without transfection. Cisplatin of the concentrations of 0, 0.05, 0.2. 0.4, 0.8, 1.0, 2.0, 10, and 20 microg/ml was added into the culture fluid for 24 h. MTT method was used to detect the proliferation rate of the SKOV3 cells. Other SKOV3 cells were divided into 3 groups: siRNA group, transfected with HER-2 siRNA III, cisplatin group, exposed to cisplatin, and HER-2 siRNA III and exposed to cisplatin. Annexin V method and flow cytometry were used to detect the apoptosis of the SKOV3 cells. The HER-2 gene expression was assessed by Western blotting. The chemosensitivity of transfected cells to cisplatin was measured by MTT. Western blotting was used to detect the protein expression of apoptosis related proteins: Bcl-2, surviving, XIAP, and Smac.
After exposed to cisplatin, the cell survival rate decreased as the dose of cisplatin increased. The proliferation rate of the SKOV3 cells transfected with HER-2 siRNA III and exposed to 1 microg/ml cisplatin was (58 +/- 5)%, significantly lower than those of the nonspecific siRNA III transfection group [(65 +/- 6)%] and the control group [(68 +/- 3)%, both P < 0.01]. No significant difference in the cell survival rate was found between the control and nonspecific groups (P > 0.05). The apoptosis rates at different time point of the HER-2 siRNA III + cisplatin group were all higher than those of the other 2 groups (all P < 0.01). The protein expression levels of the antiapoptotic proteins Bcl-2, survivin, and XIAP were of the HER-2 siRNA III + cisplatin group were significantly lower than those of the cisplatin group, and the protein expression level of the apoptotic protein Smac of the HER-2 siRNA III + cisplatin group significantly higher than that of the cisplatin group (all P < 0.05).
HER-2 siRNA III induces cell apoptosis significantly and increases the sensitivity of the human ovarian carcinoma cells to cisplatin. The mechanism of induction of cell apoptosis by HER-2 siRNA III + cisplatin may be related to the downregulation of Bcl-2, survivin and XIAP protein and the up-regulation of Smac protein.
探讨靶向人表皮生长因子受体2（HER-2）基因的RNA干扰（RNAi）对卵巢癌细胞顺铂化疗敏感性变化的影响。方法合成3种靶向HER-2基因的小干扰RNA（siRNA），即HER-2 siRNA I-III，筛选出最佳的一种（HER-2 siRNA III）。将人卵巢癌细胞系SKOV3培养后随机分为3组：HER-2 siRNA III组，转染HER-2 siRNA III；非特异性siRNA组，转染非特异性siRNA III；对照组，未转染。向培养液中加入浓度分别为0、0.05、0.2、0.4、0.8、1.0、2.0、10和20μg/ml的顺铂作用24 h。采用MTT法检测SKOV3细胞的增殖率。另将SKOV3细胞分为3组：siRNA组，转染HER-2 siRNA III；顺铂组，暴露于顺铂；HER-2 siRNA III +顺铂组，转染HER-2 siRNA III并暴露于顺铂。采用膜联蛋白V法和流式细胞术检测SKOV3细胞的凋亡情况。通过蛋白质印迹法评估HER-2基因表达。采用MTT法检测转染细胞对顺铂的化疗敏感性。用蛋白质印迹法检测凋亡相关蛋白Bcl-2、生存素、X连锁凋亡抑制蛋白（XIAP）和第二线粒体衍生激活剂（Smac）的蛋白表达。
暴露于顺铂后，细胞存活率随顺铂剂量增加而降低。转染HER-2 siRNA III并暴露于1μg/ml顺铂的SKOV3细胞增殖率为（58±5）%，显著低于非特异性siRNA III转染组[（65±6）%]和对照组[（68±3）%，P均<0.01]。对照组和非特异性组细胞存活率差异无统计学意义（P>0.05）。HER-2 siRNA III +顺铂组不同时间点的凋亡率均高于其他2组（P均<0.01）。HER-2 siRNA III +顺铂组抗凋亡蛋白Bcl-2、生存素和XIAP的蛋白表达水平显著低于顺铂组，HER-2 siRNA III +顺铂组凋亡蛋白Smac的蛋白表达水平显著高于顺铂组（P均<0.05）。
HER-2 siRNA III可显著诱导细胞凋亡并增加人卵巢癌细胞对顺铂的敏感性。HER-2 siRNA III +顺铂诱导细胞凋亡的机制可能与下调Bcl-2、生存素和XIAP蛋白以及上调Smac蛋白有关。
