OVCAR细胞转染miR-130b下调多梳基因-1蛋白表达增加对卡铂敏感性

目的观察miR-130b对卡铂抗卵巢癌的增敏作用,探讨其相关的作用机制。方法 OVCAR细胞Lipofectiamine 2000转染miR-130b模拟物和无miR-130b基因功能的miR-130b阴性对照,荧光定量PCR检测转染效果。转染细胞给予对倍稀释卡铂100,50,25,12.5,6.25,3.12,1.56和0.78μmo.lL-1处理68 h。MTT法检测OVCAR细胞存活;Western印迹法检测多梳基因-1(Bmi-1)蛋白表达。结果 荧光定量PCR结果显示,正常对照组、miR-130b模拟物转染组和阴性对照组miR-130b表达的相对值分别为0.22±0.08,0.46±0.12和0.23±0.10,miR-130b模拟物转染组显著高于正常对照组和阴性对照组(P<0.01),正常对照组和阴性对照组之间无统计学差异。卡铂对正常对照组、miR-130b模拟物转染组和阴性对照组卵巢癌OVCAR细胞的IC50值分别为32.4±4.6,14.7±2.1和(31.4±4.2)μmo.lL-1,miR-130b模拟物转染组IC50值显著低于正常对照组和阴性对照组(P<0.01)。正常对照组、miR-130b模拟物转染组和阴性对照组细胞多梳基因-1蛋白的相对表达量分别为0.75±0.16,0.21±0.12和0.77±0.18,miR-130b模拟物转染组显著降低(P<0.01)。结论 OVCAR细胞转染miR-130b可以显著增加卡铂对其敏感性,下调多梳基因-1蛋白表达可能是miR-130b增敏的作用机制之一。

Transfection of miR-130b to OVCAR cells upragulates Bmi-1 expression and increases its sensitivity to carboplatin

OBJECTIVE To observe the effect of miR-130b on cytotoxic activity of carboplatin against human ovarian carcinoma cells and explore its mechanisms. METHODS Mimics/miR-130b and negative/miR-130b were transfected with Lipofectiamine 2000, respectively. The expression of miR-130b was assessed by qRT-PCR. MTT assay was used to detect the cytotoxic activity of carboplatin(100, 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 μmol·L^-1) against ovarian carcinoma OVCAR cells. The protein expression was detected by Western blotting. RESULTS The miR-130b expression in control, mimics/miR-130b and negative/miR-130b group was 0.22±0.08, 0.46±0.12 and 0.23±0.10, respectively, which was higher in mimics/miR-130b group than that in control and negative/miR-130b groups(P<0.01). The IC₅₀ value of carboplatin against OVCAR cells in control, mimics/miR-130b and negative/miR-130b groups were 32.4±4.6, 14.7±2.1 and (31.4±4.2)μmol·L^-1, respectively. Significant difference was found between mimics/miR-130b group and control and negative/miR-130b groups(P<0.01). The protein expression of Bmi-1 was 0.75±0.16, 0.21±0.12 and 0.77±0.18 in control, mimics/miR-130b and negative/miR-130b groups, respectively, which was lower in mimics/miR-130b group than that in control and negative/miR-130b groups(P<0.01). CONCLUSION miR-130b Transfection enhances antitumor activity of carboplatin against human ovarian carcinoma. The downregulation of Bmi-1 plays an important role in enhancing the carboplatin-induced cytotoxic activity by miR-130b.