Wang Aixin, Li Jian, Li Hongxia
Department of Obstetrics and Gynecology,Beijing Shijitan Hospital, Capital Medical University,Beijing 100038, China.
Department of Obstetrics and Gynecology,Beijing Shijitan Hospital, Capital Medical University,Beijing 100038, China. Email:
Zhonghua Fu Chan Ke Za Zhi. 2014 Mar;49(3):213-7.
To investigate the reversal effect of hMSH2 small interference RNA(siRNA) on chemo-resistance of ovarian carcinoma cell line OC3/TAX300, explore the clinical significance.
The specific hMSH2 siRNA (experimental group) and non-specific hMSH2 siRNA (negative control group) was designed, synthesized and transfected into ovarian carcinoma cell line OC3/TAX300. The expression mRNA and protein levels of hMSH2 were detected by real-time reverse transcription (RT)-PCR and western blot. The cell proliferation was detected by methyl thiazolyl tetrazolium(MTT) method after 12, 24, 48, 72 hours of 2 µg/ml taxol, the apoptosis rate after 24, 48 hours of 2 µg/ml taxol was analyzed by flow cytometry. Morphological changes and ultramicrostructure of cells after 48 hours of 2 µg/ml taxol were observed with transmission electron microscope.
(1) The mRNA levels of hMSH2 were 0.004 ± 0.000, 0.053 ± 0.006 and 0.057 ± 0.012 in experimental group, negative control group and non-infected group, respectively. The protein levels of hMSH2 were 0.19 ± 0.04, 1.00 ± 0.07 and 0.95 ± 0.03 in experimental group, negative control group and non-infected group, respectively. (2) Compared with the non-infected group and the negative control group. The cell proliferation was effectively inhibited after 12, 24, 48, 72 hours of 2 µg/ml taxol (P < 0.05). The cell cycle was arrested at G2/M phase, the apoptotic rate was significantly increased after 24, 48 hours of 2 µg/ml taxol (P < 0.05). The experimental group after 48 hours of 2 µg/ml taxol was found to have more visible cell shrinkage, more serious chromatin margination, nucleus condensation, fragmentation and apoptotic body formation, nucleolus disappeared, markedly swollen mitochondria, mitochondrial cristae disappeared and other signs of apoptosis. While the nucleus was located in the cells of the central and nucleolus is clear, only mild chromatin pyknosis and marginalized, mild swelling of mitochondria in the control group and blank group.
siRNA targeting hMSH2 may reverse the chemo-resistance of ovarian carcinoma cell line OC3/TAX300 and may become a treatment or a new direction in the adjuvant therapy of ovarian cancer.
探讨人错配修复蛋白2（hMSH2）小干扰RNA（siRNA）对卵巢癌细胞系OC3/TAX300化疗耐药的逆转作用，探讨其临床意义。
设计、合成特异性hMSH2 siRNA（实验组）和非特异性hMSH2 siRNA（阴性对照组），转染卵巢癌细胞系OC3/TAX300。采用实时逆转录（RT）-PCR和蛋白质印迹法检测hMSH2的mRNA和蛋白表达水平。用噻唑蓝（MTT）法检测2 μg/ml紫杉醇作用12、24、48、72小时后的细胞增殖情况，用流式细胞术分析2 μg/ml紫杉醇作用24、48小时后的细胞凋亡率。用透射电子显微镜观察2 μg/ml紫杉醇作用48小时后细胞的形态学变化和超微结构。
（1）实验组、阴性对照组和未感染组hMSH2的mRNA水平分别为0.004±0.000、0.053±0.006和0.057±0.012。实验组、阴性对照组和未感染组hMSH2的蛋白水平分别为0.19±0.04、1.00±0.07和0.95±0.03。（2）与未感染组和阴性对照组相比。2 μg/ml紫杉醇作用12、24、48、72小时后细胞增殖受到有效抑制（P<0.05）。细胞周期阻滞于G2/M期，2 μg/ml紫杉醇作用24、48小时后细胞凋亡率显著升高（P<0.05）。2 μg/ml紫杉醇作用48小时后，实验组细胞可见更明显的细胞皱缩、更严重的染色质边集、核浓缩、核碎裂和凋亡小体形成，核仁消失，线粒体明显肿胀，线粒体嵴消失等凋亡征象。而对照组和空白组细胞的细胞核位于中央，核仁清晰，仅见轻度染色质固缩和边缘化，线粒体轻度肿胀。
靶向hMSH2的siRNA可能逆转卵巢癌细胞系OC3/TAX300的化疗耐药，可能成为卵巢癌治疗或辅助治疗的新方向。
