白藜芦醇调控miR-519c逆转乳腺癌多柔比星耐药作用机制研究

Effect and mechanisms of resveratrol reversing breast cancer cells’ Doxorubicin resistance by regulating and controlling miR-519c

目的 白藜芦醇具有一定抗肿瘤作用,但其对多柔比星耐药细胞株MCF-7/ADR逆转耐药的作用及机制尚不清楚.miR-519c是一种内源性短链非编码小分子核糖核苷酸,在肿瘤细胞耐药过程中具有重要作用.本研究旨在探讨白藜芦醇能否通过调控miR-519c逆转乳腺癌多柔比星耐药.方法 MTS法和克隆形成实验检测并计算白藜芦醇细胞逆转耐药倍数;裸鼠移植瘤实验检测白藜芦醇对MCF-7/ADR细胞体内移植瘤的抑制作用;实时荧光定量多聚核苷酸链式反应(real-time quantitative polymerase chain reaction,qRT-PCR)检测裸鼠移植瘤中miR-519c表达水平;细胞转染miR-519c抑制剂后,克隆形成实验检测MCF-7/ADR乳腺癌细胞增殖情况;蛋白质印迹法检测ATP结合盒转运蛋白G2(ATP binding cassette transporter G2,ABCG2)和HuR耐药蛋白表达.结果 10μmol/L白藜芦醇逆转MCF-7/ADR细胞对多柔比星的耐药性,多柔比星组和多柔比星+白藜芦醇组IC50值分别为(29.82±1.26)和(10.25±0.86)μmol/L,t=31.42,P<0.001,耐药逆转倍数约为2.9倍;多柔比星组和白藜芦醇组细胞克隆数分别为296±32和281±27,高于白藜芦醇+多柔比星组(81±11),差异有统计学意义,F=69.20,P<0.001;白藜芦醇体内逆转MCF-7/ADR细胞耐药,多柔比星组和白藜芦醇组裸鼠移植瘤组织质量分别为(0.71±0.12)和(0.66±0.23)g,高于白藜芦醇+多柔比星组(0.19±0.08)g,差异有统计学意义,F=6.25,P<0.001;白藜芦醇能够促进miR-519c表达,白藜芦醇组和白藜芦醇+多柔比星组miR-519c相对含量分别为2.913±0.793和3.526±0.827,高于多柔比星组(1.012±0.231),差异有统计学意义,F=10.72,P=0.010;转染miR-519c抑制剂后,多柔比星组和白藜芦醇+多柔比星+miR-519c抑制剂组克隆数分别为296±32和256±29,高于白藜芦醇+多柔比星组(81±9),差异有统计学意义,F=59.25,P<0.001;蛋白印迹结果显示,多柔比星组、白藜芦醇+多柔比星组和白藜芦醇+多柔比星+miR-519c抑制剂组ABCG2蛋白表达量分别为(1.056±0.079)、(0.432±0.051)和(0.895±0.084),F=59.41,P<0.001;HuR蛋白表达量分别为(1.138±0·093)、(0.495±0.073)和(0.937±0.098),F=41.30,P<0.001.多柔比星+白藜芦醇组耐药相关蛋白ABCG2和HuR表达低于多柔比星组,差异具有统计学意义,均P<0.001;多柔比星+白藜芦醇+miR-519c抑制剂组ABCG2和HuR蛋白表达高于多柔比星+白藜芦醇组,差异具有统计学意义,均P<0.001.结论 miR-519c可能参与MCF-7/ADR乳腺癌耐药,白藜芦醇可能通过升高MCF-7/ADR细胞中miR-519c水平,抑制细胞耐药蛋白表达,发挥逆转耐药作用.

OBJECTIVE Resveratrol has been proved to be effective in inhibiting tumor growth,but the effect and mechanism of resveratrol on doxorubicin-resistant MCF-7/ADR cell remain unclear. MicroRNA-519c is an endogenous short-chain non-coding small molecule ribonucleotide, which plays an important role in the process of drug resistance of cancer cells. This study aimed to investigate whether resveratrol can reverse doxorubicin resistance in breast cancer by regulating the expression of miR-519c. METHODS MTS and cloning formation assay were used to detect and calculate the resistance-reversing times of resveratrol. Real-time Quantitative polymerase chain reaction(qRT-PCR) was used to detect the expression of miR-519c in nude mice model with MCF-7/ADR breast cancer. After the cells were transfected with miR-519c inhibitor,clone formation assay was used to detect cell proliferation. Western Blot assay was used to detect the expression of ABCG2, HuR. RESULTS The sensitivity of cell to doxorubicin was increased after 10 μmol/L resveratrol treated ,the IC50 in doxorubicin group and resveratrol+doxorubicin group were (29. 82±1. 26) and (10. 25±0. 86)μmol/L(t= 31. 42,P<0. 001),and the reversal of drug resistance was approximately 2. 9 times. Compared with resveratrolH-doxorubicin group (81±11),the number of cell clones of doxorubicin group and resveratrol group were 296±32 and 281 i27, higher than resveratrol + doxorubicin group (81±11), the difference was statistically significant,(F= 69. 20, P<0. 001). The weight of transplanted tumor tissue of nude mice in doxorubicin group and resveratrol group was (0. 71±0. 12) and (0. 66±0. 23) g,respectively,higher than that in resveratrol4+doxorubicin group (0. 19±0. 08) g. The difference was statistically significant (F=6. 25,P<0.001). The expression of miR-519c in resveratrol group and resveratrol + doxorubicin group were (2. 913±0. 793) and (3. 526±0. 827), respectively, which were higher than doxorubicin group (1. 012±0. 231),the difference had highly statistical significance(F= 10. 72,P = 0. 010). After transfection with miR-519c inhibitor, the number of clones in the doxorubicin group and the resveratrol + doxorubicin+ miR-519c inhibitor group was 296±32 and 256±29 respectively,higher than that in the resveratrol + doxorubicin group (81±9),the difference was statistically significant(F= 59. 25,P<0.001). The western blot results showed that the protein expression levels of ABCG2 in the doxorubicin group,the resveratrol + doxorubicin group and the resveratrol + doxorubicin+miR-519c inhibitor group were (1. 056±0. 079),(0. 432±0. 051) and (0. 895±0. 084),respectively(F=59. 41 ,P<0. 001). The protein expression levels of HuR were (1. 138±0. 093),(0. 495±0. 073) and (0. 937±0. 098),respectively(F= 41. 30,P<0. 001). The expression of ABCG2 and HuR in the doverubicin+ resveratrol group was lower than that in the doverubicin group, the difference was statistically significant (all P<0.001). The expression of ABCG2 and HuR protein in the doxorubicin+ resveratrol + miR-519c inhibitor group was higher than that in the doxorubicin + resveratrol group, with statistically significant differences (all P<0. 001). CONCLUSIONS miR-519c may be involved in the drug resistance of breast cancer to MCF-7/ADR. Resveratrol may reverse the effect of drug resistance by increasing the level of miR-519c in MCF-7/ADR cells and inhibiting the expression of drug-resistant proteins in cells.