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目的:研究微小RNA-16(miR-16)在人上皮性卵巢癌顺铂耐药细胞中的表达及其与顺铂耐药的关系。方法:脂质体lipofectamine2000介导miR-16成熟体模拟物(mimics)及阴性对照片段(NC)分别转染人卵巢癌顺铂耐药细胞株SKOV3/DDP。实时荧光定量PCR检测各组细胞中miR-16的表达;蛋白印迹法检测细胞中Bcl-2蛋白、c-myc蛋白的表达;四甲基偶氮唑蓝(MTT)比色法检测细胞的顺铂半数抑制浓度(IC50);caspase-3活性检测试剂盒检测细胞内caspase-3酶的活性;流式细胞仪检测细胞的凋亡率。结果:(1)SKOV3/DDP细胞顺铂耐药指数(RI)为5.33;(2)SKOV3/DDP细胞中miR-16表达水平明显低于SKOV3细胞,约为后者的1/10(P=0.000),转染miR-16 mimics后SKOV3/DDP细胞中miR-16水平升高约660倍(P=0.001);(3)SKOV3/DDP细胞Bcl-2、c-myc蛋白的相对表达量较SKOV3细胞明显升高,转染miR-16 mimics后SKOV3/DDP细胞Bcl-2、c-myc蛋白的相对表达量明显降低(P<0.01);(4)SKOV3/DDP细胞转染miR-16 mim-ics后,顺铂IC50(14.19±0.06)较转染NC组(22.52±0.60)明显降低(P=0.002),且顺铂作用后caspase-3的酶活力单位也明显升高(P=0.000);(5)顺铂(20μg/ml)作用24h、48h后,SKOV3组凋亡率明显高于SKOV3/DDP组,转染miR-16 mimics的SKOV3/DDP细胞凋亡率约为转染NC细胞的1.5倍(P<0.01)。结论:miR-16可能通过靶向下调上皮性卵巢癌细胞Bcl-2蛋白,并协同调节c-myc蛋白的表达,增强卵巢癌顺铂耐药细胞对顺铂诱导凋亡的敏感性,发挥逆转耐药的作用。
Objective: To study the expression of miR-16 in cisplatin-resistant human epithelial ovarian cancer cells and its relationship with cisplatin resistance. Methods: Mimics of miR-16 mature mimics and negative control (NC) were transfected into human ovarian cancer cell line SKOV3 / DDP with lipofectamine 2000 respectively. The expression of miR-16 in each group was detected by real-time fluorescence quantitative PCR. The expression of Bcl-2 and c-myc in the cells was detected by Western blotting. The cisplatin was detected by MTT assay Platinum-half inhibitory concentration (IC50); caspase-3 activity detection kit detection of intracellular caspase-3 enzyme activity; flow cytometry cell apoptosis rate. Results: (1) The cisplatin resistant index (RI) of SKOV3 / DDP cells was 5.33. (2) The expression of miR-16 in SKOV3 / DDP cells was significantly lower than that in SKOV3 cells, 0.000). The miR-16 level in SKOV3 / DDP cells increased about 660-fold (P = 0.001) after transfected with miR-16 mimics. (3) The relative expression of Bcl-2 and c-myc proteins in SKOV3 / The expression of Bcl-2 and c-myc in SKOV3 / DDP cells was significantly decreased after transfected with miR-16 mimics (P <0.01). (4) The miR-16 mim -ics, the IC50 of cisplatin (14.19 ± 0.06) was significantly lower than that of NC group (22.52 ± 0.60) (P = 0.002), and the activity of caspase-3 was significantly increased after cisplatin treatment (5) The apoptosis rates of SKOV3 / SKOV3 / DDP cells treated with cisplatin (20μg / ml) for 24h and 48h were significantly higher than that of SKOV3 / DDP cells transfected with miR-16 mimics 1.5 times more cells (P <0.01). Conclusion: miR-16 may be targeted to downregulate Bcl-2 protein in epithelial ovarian cancer cells and coordinate the expression of c-myc protein to enhance the sensitivity of cisplatin-induced apoptosis in cisplatin-resistant ovarian cancer cells The role of resistance.