Lu Yan-ming, Zhang Shu-lan, Meng Li-rong, Zhao Yan-yan
Department of Gynecology and Obstetrics, Second Affiliated Hospital, China Medical University, Shenyang 110004, China.
Zhonghua Yi Xue Za Zhi. 2006 Mar 7;86(9):619-23.
To investigate the effects of RNA interference (RNAi) targeting HER-2 gene on the biological traits of human ovarian carcinoma.
siRNA specific to HER-2 gene was synthesized according to the sequence in the GenBank. Human ovarian carcinoma cells of the line SKOV-3 were cultured and divided into 3 groups: control group; non-specific group, transfected with non-specific siRNA; and specific group, transfected with specific HER-2 siRNA. On the 5th day after transfection cisplatin was added into the culture fluid. The expression of HER-2 mRNA and the expression of protein both before and after transfection were detected by RT-PCR and Western blotting. The cell apoptosis was assessed by flow cytometry. The chemosensitivity of transfected cells to cisplatin was determined by MTT method.
Since the 3rd day after transfection the expression of HER-2 mRNA of the HER-2 siRNA group was suppressed till 10 days later. On the 7th day after transfection the expression rate of HER-2 protein of the HER-2 siRNA group was (25.5 +/- 0.8)%, significantly lower than those of the nonspecific siRNA group and control group, (95.7 +/- 0.8)% and (96.6 +/- 1.2)% respectively (both P < 0.001). On the 9th day after transfection no expression of HER-2 protein was found in the HER-2 siRNA group. The apoptosis rate of SKOV-3 cells increased time-dependently after transfection in the HER-2 siRNA group and reached the peak, (53.2 +/- 1.0)% on the 6th day, significantly higher than those of the non-specific siRNA group and control group, (4.1 +/- 0.3)% and (4.1 +/- 0.3)% respectively (both P < 0.001). After exposure to cisplatin for 24 hours, the survival rate of the HER-2 siRNA group was (58.4 +/- 0.8)%, significantly higher than those of the nonspecific siRNA group and control group, (68.0 +/- 0.6)% and (67.0 +/- 0.3)% respectively (both P < 0.001).
siRNA targeting HER-2 synthesized in vitro and transfected into human ovarian carcinoma cells effectively suppresses the HER-2 expression, induces cell apoptosis, and increases the sensitivity to cisplatin of the cells. The successful application of HER-2 siRNA extends the list of available therapeutic modalities in the treatment of human ovarian cancer.
探讨靶向HER-2基因的RNA干扰（RNAi）对人卵巢癌生物学特性的影响。
根据GenBank中的序列合成针对HER-2基因的小干扰RNA（siRNA）。培养人卵巢癌细胞系SKOV-3，并分为3组：对照组；非特异性组，转染非特异性siRNA；特异性组，转染特异性HER-2 siRNA。转染后第5天，向培养液中加入顺铂。采用逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测转染前后HER-2 mRNA及蛋白的表达。采用流式细胞术评估细胞凋亡情况。采用MTT法测定转染后细胞对顺铂的化学敏感性。
转染后第3天起，HER-2 siRNA组HER-2 mRNA的表达受到抑制，至10天后仍受抑制。转染后第7天，HER-2 siRNA组HER-2蛋白的表达率为（25.5±0.8）%，明显低于非特异性siRNA组和对照组，分别为（95.7±0.8）%和（96.6±1.2）%（均P<0.001）。转染后第9天，HER-2 siRNA组未检测到HER-2蛋白表达。HER-2 siRNA组转染后SKOV-3细胞凋亡率呈时间依赖性增加，第6天达到峰值（53.2±1.0）%，明显高于非特异性siRNA组和对照组，分别为（4.1±0.3）%和（4.1±0.3）%（均P<0.001）。顺铂作用24小时后，HER-2 siRNA组细胞存活率为（58.4±0.8）%，明显高于非特异性siRNA组和对照组，分别为（68.0±0.6）%和（67.0±0.3）%（均P<0.001）。
体外合成并转染人卵巢癌细胞的靶向HER-2的siRNA能有效抑制HER-2表达，诱导细胞凋亡，并增加细胞对顺铂的敏感性。HER-2 siRNA的成功应用拓宽了人卵巢癌治疗的可用治疗方式。
