【摘要】 目的 探讨miR-152-3p对紫杉醇耐药人卵巢癌细胞A2780T对紫杉醇耐药性的影响及机制。方法(1)紫杉醇（1.875,3.75,7.5,17和23μmol·L-1）与人卵巢癌A2780和A2780T细胞作用48 h,MTT法检测细胞存活率，计算抑制细胞存活半数抑制浓度（IC50）值和耐药指数（RI）。Western印迹法检测A2780和A2780T细胞耐药蛋白P-糖蛋白（P-gp）、多药耐药相关蛋白1(MRP1)和三磷酸腺苷结合转运蛋白G超家族成员2(ABCG2)蛋白表达。(2)实时荧光定量PCR(RT-qPCR)检测A2780和A2780T细胞miR-152-3p表达水平。脂质体瞬时转染技术转染miR-152-3p抑制物降低A2780T细胞中miR-152-3p表达（miR-152-3p抑制物组），同时设转染miR-152-3p阴性对照组，RT-qPCR检测转染效率，MTT法、划痕实验和流式细胞术分别检测转染miR-152-3p抑制物对A2780T细胞存活、迁移和凋亡的影响；Western印迹法检测转染miR-152-3p抑制物对A2780T细胞Bax和Bcl-2蛋白表达的影响。(3)用miRDB,Targetscan,miRWalk和Starbase数据库预测miR-152-3p的靶基因，并用Western印迹法检测转染miR-152-3p抑制物后A2780T细胞磷酸酯酶张力蛋白同源物（PTEN）蛋白表达的变化及RT-qPCR检测A2780和A2780T细胞PTEN mRNA表达水平予以验证。随后，脂质体瞬时转染技术转染PTEN siRNA沉默A2780T细胞中PTEN表达，同时设转染siRNA阴性对照组，RT-qPCR检测转染效率，MTT法检测沉默PTEN表达后A2780T细胞存活率和IC50值，Western印迹法检测P-gp,MRP1和ABCG2蛋白表达。结果 (1)紫杉醇处理后A2780和A2780T细胞存活率均降低（P<0.01）,A2780T细胞RI为2.8。与A2780细胞相比，A2780T细胞P-gp,MRP1和ABCG2蛋白高表达（P<0.05,P<0.01）。(2)与A2780细胞相比，A2780T细胞miR-152-3p明显高表达（P<0.01）。与转染miR-152-3p阴性对照组比较，转染miR-152-3p抑制物后A2780T细胞存活率（P<0.05,P<0.01）和细胞迁移能力（P<0.05）明显降低，细胞凋亡率升高（P<0.01）;Bax蛋白表达增加（P<0.01）,Bcl-2蛋白表达降低（P<0.05）。(3)生物信息学数据库分析结果提示，PTEN是miR-152-3p的一个靶基因；Western印迹法验证显示，转染miR-152-3p抑制物组A2780T细胞PTEN蛋白表达低于转染miR-152-3p阴性对照组（P<0.05）;RT-qPCR结果显示，A2780T细胞PTEN mRNA表达水平高于A2780细胞（P<0.01）。转染PTEN siRNA沉默PTEN表达后，与转染siRNA阴性对照组相比，A2780T细胞存活率（P<0.05,P<0.01）和IC50值（P<0.01）显著降低，P-gp,MRP1和ABCG2蛋白表达降低（P<0.05,P<0.01）。结论 miR-152-3p在A2780T细胞中高表达，其表达下调可抑制A2780T细胞增殖和迁移，促进细胞凋亡，降低A2780T细胞对紫杉醇的耐药性，该作用可能是通过降低其靶基因PTEN表达发挥的。更多还原

【Abstract】 OBJECTIVE To investigate the effect and mechanism of miR-152-3p on the resistance to paclitaxel（PTX） of PTX-resistant ovarian cancer cells（A2780T cells）. METHODS（1） Ovarian cancer parent cells（A2780 cells） and A2780T cells were treated with PTX(1.875, 3.75, 7.5, 17 and 23 μmol·L^-1)for 48 h. Cell viability was evaluated by MTT assay, and the 50% inhibitory concentration（IC50） and drug resistance index of A2780T cells were calculated. Western blotting was used to detect the expressions of resistance protein P-glycoprotein（P-gp）, multidrug resistance related protein 1（MRP1） and adenosine triphosphate binding transporter G superfamily member 2（ABCG2）.（2） Real-time fluorescent quantitative PCR（RT-qPCR） was used to detect the expressions of miR-152-3p in A2780 and A2780T cells.The lipid-mediated transient transfection technique was employed to transfect the miR-152-3p inhibitor to reduce mi R-152-3p expression in A2780T cells（mi R-152-3p inhibitor group）, while the negative control（miR-152-3p NC） group was established. RT-qPCR was used to detect transfection efficiency, and the MTT method, scratch experiment, and flow cytometry were used to investigate the effects of the transfecting miR-152-3p inhibitor on survival, migration and apoptosis of A2780T cells. Western blotting was used to detect the protein expressions of Bax and Bcl-2 in A2780T cells.（3） Bioinformatics analysis of databases including miRDB, Targetscan, miRWalk, and Starbase predicted the target genes of miR-152-3p that were verified by Western blotting to detect the protein expression of PTEN in A2780T cells of the miR-152-3p inhibitor and miR-152-3p NC groups, and RT-qPCR to detect the PTEN mRNA expression in A2780 and A2780T cells. Then, the lipid-mediated transient transfection technique was used to transfect PTEN siRNA to silence PTEN expression in A2780T cells（PTEN siRNA group）. The siRNA negative control（siRNA NC） group was established. RT-qPCR was used to detect transfection efficiency, the MTT method was employed to measure the survival rate and IC50value, and Western blotting was used to assess the protein expressions of P-gp, MRP1, and ABCG2 in A2780T cells after silencing PTEN expression. RESULTS（1） After treatment with PTX, the cell survival rates were decreased in A2780 and A2780T cells（P<0.05）, and the resistance index of A2780T cells was 2.8. Compared with A2780 cells, the protein expressions of P-gp and MRP1 and ABCG2 were highly expressed in A2780T cells（P<0.05, P<0.01）.（2） RT-qPCR showed that the expression of miR-152-3p in A2780T cells was higher than that of A2780 cells（P<0.01）. Compared with the miR-152-3p NC group, A2780T cell viability（P<0.05, P<0.01） and cell migration capability（P<0.05） were significantly inhibited, while the apoptosis rate increased（P<0.01） in mi R-152-3p inhibitor group. Moreover, the protein expression of Bax was increased（P<0.01）, but Bcl-2 decreased（P<0.05）.（3） Bioinformatics analysis suggested that PTEN was a target gene of the miR-152-3p, and the verified results showed that the PTEN protein expression in A2780T cells of the miR-152-3p inhibitor group was lower than that of the miR-152-3p NC group（P<0.05）, and PTEN mRNA expression in A2780T cells was higher than that in A2780 cells（P<0.01）. After silencing the expression of PTEN in A2780T cells, the cell viability was significantly reduced（P<0.05, P<0.01）,while the IC50value was reduced（P<0.01） compared with the siRNA NC group. In addition, the protein expressions of P-gp, MRP1 and ABCG2 were decreased（P<0.05, P<0.01）. CONCLUSION miR-152-3p is highly expressed in A2780T cells, and down-regulation of its expression may inhibit proliferation and migration, prompt apoptosis and reduce the resistance to PTX of A2780T cells, which is made possible by inhibiting expression of its target gene PTEN.更多还原