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目的观察微小RNA-200c(miRNA-200c)过表达对卵巢癌A2780顺铂耐药细胞(A2780/DDP)的影响。方法建立A2780/DDP耐药细胞株,未经处理的对数生长期A2780细胞为对照组,将对数生长期A2780/DDP细胞分为空转染组和增强组,增强组使用miRNA-200c前体片段转染A2780/DDP细胞,实时荧光定量PCR检测各组细胞miRNA-200c表达水平,四甲基偶氮唑蓝法检测各组细胞对DDP药物敏感性,克隆形成实验检测各组细胞增殖能力,流式细胞术检测细胞凋亡变化,实时荧光定量PCR检测MDR1 mRNA表达水平,Western blot检测P-gp蛋白的表达。结果与对照组、空转染组比较,增强组miRNA-200c相对表达量增高为(4.65±1.23)、IC50明显降低为(11.20±1.62)μg/ml、克隆集落的数量较少、细胞凋亡率明显增高为(21.1±2.1)%、MDR1 mRNA相对表达量减少为(0.61±0.12)、P-gp蛋白相对表达量减少为(0.52±0.21),差异均有统计学意义(P<0.05)。结论 miRNA-200c可能通过抑制细胞增殖、诱导凋亡、降低MDR1 mRNA及P-gp蛋白的表达水平降低A2780/DDP细胞对DDP的耐药性。
Objective To investigate the effect of microRNA-200c (overexpressed miRNA-200c) on cisplatin-resistant cells (A2780 / DDP) in A2780 ovarian cancer. Methods The A2780 / DDP resistant cell line was established. The untreated logarithmic growth phase A2780 cells served as the control group, and the logarithmic growth phase A2780 / DDP cells were divided into empty transfected group and enhanced group. The enhancement group used miRNA-200c The expression of miRNA-200c in each group was detected by real-time fluorescence quantitative PCR. The sensitivity of cells to DDP was detected by MTT assay. The proliferation of cells in each group was detected by clone formation assay , The changes of apoptosis were detected by flow cytometry, the expression of MDR1 mRNA was detected by real-time fluorescence quantitative PCR, and the expression of P-gp protein was detected by Western blot. Results The expression of miRNA-200c in enhancement group was (4.65 ± 1.23) and IC50 was significantly decreased to (11.20 ± 1.62) μg / ml compared with the control and empty transfected groups, respectively. The number of cloned colonies was less and apoptosis (21.1 ± 2.1)%, the relative expression of MDR1 mRNA decreased (0.61 ± 0.12) and the relative expression of P-gp protein decreased (0.52 ± 0.21), the difference was statistically significant (P <0.05) . Conclusions miRNA-200c may reduce the resistance of A2780 / DDP cells to DDP by inhibiting cell proliferation, inducing apoptosis and decreasing the expression of MDR1 mRNA and P-gp protein.