miR-490-3p逆转MCF-7／ADM细胞对阿霉素耐药性的影响

【摘 要】目的 探讨miR-490-3p在阿霉素（ADM）耐药乳腺癌细胞系MCF-7／ADM中的表达情况及其对MCF-7／ADM细胞增殖、凋亡和ADM耐药的影响。方法 以野生型人乳腺癌细胞系MCF-7及其耐药型细胞系MCF-7／ADM为研究对象，用实时荧光定量PCR（qPCR）比较两种细胞中miR-490-3p的表达水平，将miR-490-3p的过表达载体pcDNA1（+）miR-490-3p瞬时转染至MCF-7／ADM细胞（过表达组）后采用qPCR检测转染效果，同时设pcDNA3.1（-）空载体对照组和空白对照组；采用MTT法测定转染pcDNA3.1（+）miR-490-3p对各组MCF-7/ADM细胞增殖能力的影响，测定ADM对MCF-7／ADM细胞的增殖抑制率并计算半数抑制浓度（IC50）及逆转倍数以评价对ADM敏感性的变化；分别于瞬时转染48 h后用Annexin V／FITC流式细胞术检测各组MCF-7/ADM细胞的凋亡率，Western blotting检测耐药蛋白P-糖蛋白（P-gp），乳腺癌耐药蛋白（BCRP）和多药耐药相关蛋白1（MRP1）的表达，荧光分光光度计检测胞内ADM药物浓度，流式细胞仪检测P-gp活性。结果 MCF-7／ADM细胞中miR-490-3p的表达水平为0.24±0.07，显著低于野生型MCF-7细胞的1.02±0.03（P＜0.05）；过表达组瞬时转染24～96 h的miR-490-3p水平持续升高，均高于空载体对照组和空白对照组（P＜0.05）；过表达组的增殖抑制率随转染时间的延长而增加，转染24、48、72和96 h的增殖抑制率依次为（17.52±1.87）％、（31.67±2.79）％、（45.09±1.88）％和（61.82±2.52）％，高于空载体对照组（P＜0.05）；ADM对过表达组的IC50值为（11.27±2.34）μg／ml，低于空白对照组的和空载体对照组（P＜0.05），且过表达组相对于空白对照组和空载体对照组的逆转倍数分别为3.35倍和3.39倍；与其余两组相比，过表达组的MCF-7/ADM细胞早、晚期凋亡率和细胞内药物浓度均升高，P-gp、BCRP和MRP1表达及P-gp活性均降低，差异均有统计学意义（P＜0.05）。结论 上调miR-490-3p可逆转MCF-7／ADM细胞对ADM的耐药性，可能通过降低耐药蛋白表达及抑制P-gp活性有关，且升高其水平可抑制细胞增殖并诱导凋亡。

Reversal effect of miR-490-3p on the resistance of MCF-7/ADM cells to adriamycin

【Abstract】Objective To investigate the expression of miR-490-3p in adriamycin（ADM）resistant human breast cancer cell line MCF-7／ADM and its effect on the cell proliferation，apoptosis and resistance to ADM of MCF-7／ADM cell line. Methods Cultured wild-type MCF-7 human breast cancer cells and its resistant cells MCF-7／ADM were selected as research objectives. The real time fluorescent quantitative PCR（qPCR）was used to compare the level of miR-490-3p in both cell lines. The recombinant plasmid pcDNA3.1（+）miR-490-3p to over-express miR-490-3p was transiently transfected into MCF-7／ADM cells（over expression group）and then the transfection effect was verified by qPCR. Meanwhile，the pcDNA3.1（-）empty control group and blank control group were set up. MTT method was used to determine the effect of pcDNA3.1（+）miR-490-3p on the proliferation of each group. The inhibitory rate of ADM on the proliferation of MCF-7／ADM cells was determined, and the inhibition concentration （IC50） and the reversal factor were calculated to evaluate the sensitivity to ADM. The cells in each group were collected at 48 h post-transfection for the detection of apoptotic rate by flow cytometry with Annexin FITC／PI double staining. Meanwhile, the expression of resistance proteins including P-glycoprotein（P-gp），breast cancer resistance protein （BCRP）and multidrug resistance associated protein 1（MRP1）were tested by Western blotting，intracellular ADM concentration was detected by fluorescence photometer and P gp activity by flow cytometry. Results The level of miR-490-3p in MCF-7／ADM cells was 0.24±0.07，significantly lower than 1.02±0.03 of wild type MCF-7 cells（P＜005）. The levels of miR-490-3p in the over expression group were higher than those in the other two groups during 24-96 h post-transfection（P＜005）. The proliferation inhibition rates were（17.52 ±1.87）％，（31.67±2.79）％，（45.09±1.88）％ and（61.82±2.52）％ at 48，24，72 and 96 h post-transfection， all higher than those of empty control group（P＜0.05）. IC50 value of ADM for MCF-7／ADM cells was（11.27±2.34）μg／ml，lower than that of blank control group and empty control group（P＜0.05）. Moreover，reversal index was 3.35 and 3.39 fold of blank control group and empty control group for the over expression group. Compared with the other two groups，there were increased apoptotic rates and intracellular drug concentration but decreased expression of P-gp，BCRP and MRP1 as well as P-gp activity in over-expression group （P＜0.05）.Conclusion Upregulation of miR-490-3p exhibits the reversal effect of resistance to ADM in MCF-7／ADM cell，possibly by reducing the resistance gene protein expression and inhibition of P-gp activity. Elevated level of miR-490-3p can inhibit proliferation and induce apoptosis.