沉默二氢叶酸还原酶基因对卵巢癌细胞体外生物学行为的影响

Effect of down-regulation of dihydrofolate reductase on biological function of ovarian cancer cells in vitro

背景与目的:二氢叶酸还原酶(dihydrofolate reductase,DHFR)在卵巢上皮癌铂类耐药细胞中高表达。该研究旨在探讨DHFR基因沉默对卵巢癌细胞体外生物学行为的影响,为卵巢癌铂类耐药的治疗奠定基础。方法:设计DHFR基因靶向的发夹状si RNA,筛选出最佳si RNA沉默片段,构建携带有目的基因的慢病毒载体细胞即转染组(DHFR-p GCSIL-SKOV3细胞)及阴性对照组细胞(p GCSIL-SKOV3细胞)。采用流式细胞仪检测3组细胞在不同的顺铂质量浓度(2.5、5.0、10.0和20.0μg/m L)下不同时间段(24、48及72 h)的凋亡情况以及IC_50顺铂质量浓度(4.4μg/m L)的周期变化;采用高效液相色谱法检测不同顺铂质量浓度(2.5、5.0和7.5μg/m L)不同时间段(24和48 h)药物作用后3组细胞内的顺铂浓度。采用透射电镜观察IC_50顺铂质量浓度(4.4μg/m L)3种细胞的超微结构变化。结果:将退火后的双链寡核苷酸片段连接到p GCSIL/GFP载体,测序结果正确。转染SKOV3细胞后,蛋白[质]印迹法(Western blot)鉴定干扰效果。在给药24、48和72 h后,DHFR-p GCSIL-SKOV3、p GCSIL-SKOV3及SKOV3三组细胞的凋亡率随着顺铂质量浓度(2.5、5.0、10.0和20.0μg/m L)的上升而升高,DHFR-p GCSIL-SKOV3细胞给药48和72 h后的凋亡率明显高于p GCSIL-SKOV3及SKOV3细胞,差异有统计学意义(P<0.05)。3组细胞在IC_50顺铂质量浓度(4.4μg/m L)给药24和48 h后,转染组G_0/G_1期细胞比例较阴性及空白对照组明显增多,G_2/M、S期则反之;而给药72 h后,3组细胞以G_2/M及S期为主,转染组低于阴性及空白对照组。采用高效液相法检测细胞内顺铂质量浓度时,转染组在2.5和5.0μg/m L顺铂质量浓度给药24和48 h后,其细胞内顺铂浓度均明显高于对照组。转染组在7.5μg/m L顺铂质量浓度给药24 h后,其细胞内顺铂浓度均明显低于对照组细胞,在48 h后却又高于对照组,差异有统计学意义(P=0.034,P=0.014)。未给药时,转染组细胞的微丝明显多于对照组,线粒体亦改变明显。IC_50(4.4μg/m L)给药24和48 h后3组细胞均未见微丝;而给药72 h后微丝明显增多,且排列紊乱。结论:成功构建携带DHFR基因的p GCSIL干扰慢病毒载体的卵巢癌细胞。通过对DHFR下调后耐药性能研究得知,DHFR的下调与经过铂类药物作用的体外卵巢癌细胞的药物耐药性有一定的联系,卵巢癌细胞中下调DHFR基因可以考虑作为多药耐药的逆转机制。

Background and purpose： Dihydrofolate reductase （DHFR） is expressed highly in platinum-resistant ovarian cancer. This study aimed to explore the relationship between the silence ofDHFR gene and platinum drug resistance in ovarian cancer, and lay the foundation for the treatment of platinum-resistant ovarian cancer. Methods： To design targeting hairpin siRNA of DHFR gene, the optimal siRNA silent sequence was selected, and lentiviral vector carrying DHFR gene was constructed successfully, named DHFR-pGCSIL-SKOV3 cell. Flow cytometry was used to detect the cell apoptosis of DHFR-pGCSIL-SKOV3 cells, pGCSIL-SKOV3 cells and SKOV3 cells incubated in various concentrations of cisplatin （2.5, 5.0, 10.0 and 20.0 μg/mL） at different time points （24, 48 and 72 h）, and cell cycle changes of these cells at IC50 cisplatin concentration （4.4 μg/mL）. High performance liquid chromatography was used to test intracellular concentration of cisplatin at different induction concentration of cisplatin （2.5, 5.0 and 7.5 μg/mL） and various time points （24 and 48 h）. Ultrastructural changes of these ceils at concentration of cisplatin IC50 （4.4 μg/mL） were observed by transmission electron microscope. Results： After annealing double-strand nucleotide was connected to pGCSIL/GFP vector, sequencing result was correct. SKOV3 cell were transfected with virus particles followed by Western blot detection of interference effect. Flow cytometry was used to detect apoptosis in three groups of ceils, and increased apoptosis rate was found at the raised cisplatin concentration （2.5, 5.0, 10.0 and 20.0 μg/mL） at 24, 48 and 72 h in DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3 and SKOV3 cells. The apoptosis rate in DHFR-pGCSIL-SKOV3 was significantly higher than that in pGCSIL-SKOV3 and SKOV3 cells at 24 and 48 h （P〈0.05）. Flow cytometry was adopted to test cells cycle of 3 groups at different time period under IC50 cisplatin concentration （4.4 μg/mL）, the results indicated that G0/G1 phase cell rate of DHFR-pGCSIL-SKOV3 was much more than the others, of which GJM and S phase cell rates were on the contrary. While at 72 h, 3 groups were mainly GJM and S phase cell rates, DHFR-pGC- SIL-SKOV3 was lower than the others. High performance liquid chromatography method was used to detect intracellular cisplatin concentration at 24 and 48 h after the cells were incubated at various concentrations of cisplatin （2.5 and 5.0μg/mL）. The results showed the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell was significantly higher than that ofpGCS1L-SKOV3 and SKOV3 cells. However, after incubation at cisplatin concentration of 7.5 μg/mL, the intracellular cisplatin content of DHFR-pGCSIL-SKOV3 cell was significantly lower than that of pGCSIL-SKOV3 and SKOV3 cells at 24 h, while higher than pGCSIL-SKOV3 and SKOV3 cells at 48 h （P=0.034, P=-0.014）. We observed ultrastructural changes of three different cell lines induced by IC50 cisplatin concentration（4.4 μg/mL） at different time points by the electron microscope. We found that the microfilaments were increased and gathered together and mitochondrial structure was also changed obviously without the drug. However, there was rare microfilament in three groups of cells at 24 and 48 h, while at 72 h, obviously increased inordinate microfilaments were observed. Conclusion： We successfully constructed pGCSIL lentivirus interference carrier carrying DHFR gene. The research indicates that down-regulation ofDHFR gene is related to cisplatin drug resistance in ovarian cancer. The results laid the foundation for us to investigate the molecular mechanisms of multidrug-resistance in tumor.