miR-152-3p表达下调降低紫杉醇耐药人卵巢癌细胞A2780T对紫杉醇的耐药性  

Down-regulation of miR-152-3p expression reduces resistance to paclitaxel of paclitaxel-resistant ovarian cancer cells A2780T

作　　者：张洋[1,2] 赵辰戈 程荔春 吕慧怡[1,3] 吴迪[1] ZHANG Yang;ZHAO Chen′ge;CHENG Lichun;LYU Huiyi;WU Di(Department of Pharmacy,the Second Affiliated Hospital of Dalian Medical University,Dalian 116023,China;College of Pharmacy,Dalian Medical University,Dalian 116044,China;Dalian Kexiang Technology Development Co.Ltd.,Dalian 116085,China)

机构地区：[1]大连医科大学附属第二医院药学部,辽宁大连116023 [2]大连医科大学药学院,辽宁大连116044 [3]大连科翔生物科技有限公司,辽宁大连116085

出　　处：《中国药理学与毒理学杂志》2024年第1期22-30,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：大连市医学科学研究计划(2112017);大连市重点领域创新团队支持计划(2021RT14)。

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�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).METHODS①Ovarian cancer parent cells(A2780 cells)and A2780T cells were treated with PTX(1.875,3.75,7.5,17 and 23μmol·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 expres⁃sions of resistance protein P-glycoprotein(P-gp),multidrug resistance related protein 1(MRP1)and adenosine triphosphate binding transporter G superfamily member 2(ABCG2).②Real-time fluorescent quantita⁃tive 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 miR-152-3p expression in A2780T cells(miR-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 trans⁃fecting 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.③Bioinformatics 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 IC50 value,and Western b

关 键 词：卵巢癌 miR-152-3p 紫杉醇 耐药 细胞增殖 细胞凋亡 

分 类 号：R966[医药卫生—药理学]