LncRNA GAS5调控miR-223-5p逆转乳腺癌他莫昔芬耐药性分析

Analysis of LncRNA GAS5 Reversing Tamoxifen Resistance in Breast Cancer by Regulating miR-223-5p

目的:探究LncRNA GAS5在乳腺癌他莫昔芬耐药性菌株中的表达情况,初步探究其可能的作用机制。方法:体外培养MCF-7、MCF-7/TAM细胞,采用实时荧光定量PCR(qRT-PCR)检测细胞LncRNA GAS5、miR-223-5p表达情况;LncRNA GAS5 mimics、对照序列转染至MCF-7/TAM细胞分为LncRNA GAS5过表达组、阴性对照组(NC组)、空白对照组(Control);MTT法检测细胞活性及IC50变化;双荧光素酶报告系统检测LncRNA GAS5、miR-223-5p的靶向关系;免疫印迹法对耐药蛋白P糖蛋白(P-gp)(SBPVR1)、多药耐药相关蛋白1(MRP1)及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)通路蛋白进行检测。结果:MCF-7/TAM细胞中LncRNA GAS5表达低于MCF-7细胞,miR-223-5p高于MCF-7细胞(P<0.05)。与空白对照组、NC组相比,LncRNA GAS5过表达组LncRNA GAS5表达、细胞增殖抑制率显著升高,IC50值、miR-223-5p表达、蛋白P-gp、MRP1、P-PI3K、P-Akt表达显著降低(P<0.05)。双荧光素酶实验结果表明LncRNA GAS5与miR-223-5p具有靶向关系。结论:过表达LncRNA GAS5可能通过靶向抑制miR-223-5p,抑制PI3K/Akt信号通路,逆转MCF-7细胞的TAM耐药性。

Objective:To investigate the expression of LncRNA GAS5 in tamoxifen-resistant breast cancer strains and its possible mechanism.Methods:MCF-7 and MCF-7/TAM cells were cultured in vitro,and real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expressions of LncRNA GAS5 and miR-223-5 p;LncRNA GAS5 mimics and control sequence were transfected into MCF-7/TAM cells and divided into LncRNA GAS5 overexpression group,NC group and blank control group;cell viability and IC50 were measured by MTT assay;dual luciferase reporting system was used to detect the targeting relationship between LncRNA GAS5 and miR 223-5 p;immunoblotting was used to detect resistance protein P glycoprotein(P-gp)(SBPVR1),multidrug resistance related protein 1(MRP1)and phosphatidylinositol 3 kinase(PI3 K)/protein kinase B(Akt)pathway proteins.Results:The expression of LncRNA GAS5 in MCF-7/TAM cells was lower than that in MCF-7 cells,and the expression of miR 223-5 p was higher than that in MCF-7 cells(P<0.05).Compared with those in the control group and NC group,the expression of LncRNA GAS5 and the inhibition rate of cell proliferation in the over-expression group of LncRNA GAS5 were higher significantly,while the expression of IC50 value,the expression of miR-223-5 p and the expressions of P-gp,MRP1,P-PI3 K and P-Akt proteins were lower significantly(P<0.05).The results of dual luciferase assay showed that LncRNA GAS5 had a targeted relationship with miR-223-5 p.Conclusion:Overexpression of LncRNA GAS5 can reverse the drug resistance to TAM of MCF-7 cells by inhibiting miR-223-5 p and phosphoinositide 3-kinase PI3 K/Akt signaling pathway.