miR-200b-3p/NBR1轴对乳腺癌细胞自噬及生长的影响  

Effects of miR-200b-3p/NBR1 axis on autophagy and growth of breast cancer cells

作　　者：蒋雪梅[1] 易瑛[1] 陈宇 杨诚诚 JIANG Xue-mei;YI Ying;CHEN Yu;YANG Cheng-cheng(Department of Breast Surgery,Deyang Peopled Hospital,Deyang 618000,Sichuan,China)

机构地区：[1]德阳市人民医院乳腺外科,德阳618000

出　　处：《医学研究生学报》2021年第12期1259-1266,共8页Journal of Medical Postgraduates

基　　金：德阳市科技计划项目(2019SZ122)。

摘　　要：目的miR-200b-3P靶向NBR1调控乳腺癌自噬的作用仍未完全探索,文章旨在探讨miR-200b-3p是否靶向NBR1影响乳腺癌细胞自噬及生长。方法介绍些分组选择人乳腺癌细胞(MDA-MB-453)为研究对象,分为阴性对照组(转染mimic NC或inhibitor NC)、miR-20Ob-3p mimic组(转染miR-200b-3p模拟物)、miR-200b-3p inhibitor组(转染miR-200b-3p抑制剂),采用CCK-8法和克隆形成实验检测各组细胞增殖;流式细胞术检测细胞凋亡及细胞周期;Tmnswell实验检测细胞侵袭能力;透射电镜观察细胞超微结构;实时荧光定量PCR(qRT-PCR)检测miR-200b-3p mRNA表达;蛋白质印迹法(Western blot)检测Beclin-1、P62、LC3Ⅱ/Ⅰ、NBR1蛋白表达;双荧光素酶报告实验验证miR-200b-3p与NBR1之间的靶向关系。结果qRT-PCR检测结果显示,与mimic NC组(1.000±0.069)比较,miR-200b-3p mimic组miR-200b-3p mRNA表达(1.314±0.093)明显升高;与inhibitor NC组(1.009±0.055)比较,miR-200b-3p inhibitor组miR-200b-3p mRNA表达(0.581±0.031)明显降低(P<0.05);与miR-200b-3p mimic组比较,miR-200b-3p inhibitor组miR-200b-3p mRNA表达明显降低(P<0.05)。细胞侵袭结果显示,与mimic NC组比较,miR-200b-3p mimic组穿过基膜的细胞数明显降低(P<0.01),与inhibitor NC组比较,miR-200b-3p in-hibitor组穿过基膜的细胞数明显升高(P<0.0l),与miR-200b-3p mimic组比较,miR-200b-3p inhibitor组穿过基膜的细胞数明显升高(P<0.05)。细胞周期阻滞结果显示,与mimic NC组比较,miR-200h-3p mimic组细胞在G2/M期、S期均明显减少,G1/G0期细胞均明显增多,与inhibitor NC组比较,miR-200b-3p inhihitor组细胞在G2/M期、S期均明显增多(P<0.05),G1/G0期细胞均明显减少(P<0.05)。Western blot法检测结果显示,与mimic NC组比较,miR-200b-3p mimic组细胞中Beclin-1、LC3II/I蛋白水平明显升高,P62蛋白表达明显降低(P<0.01),与inhibitor NC组比较,miR-20Ob-3p inhibitor组细胞中Beclin-1、LC3II/I蛋白水平明显降低,P62蛋白表达明显升高(P<0.01)。Weatem blotObjective The role of miR-200b-3p targeting NBR1 to regulate breast cancer autophagy is yet to be further explored.In this study,we aimed to investigate whether miR-200b-3p targets NBR1 to affect breast cancer cell autophagy and growth.Methods Human breast cancer cells(MDA-MB-453)were selected for this study and divided into negative control group(transfected with mimic NC or inhibitor NC),miR-200b-3p mimic group(transfected with miR-200b-3p mimic),and miR-200b-3p inhibitor group(transfected with miR-200b-3p inhibitor).CCK-8 method and clone formation assay were applied to detect cell proliferation;flow cy-tometry to evaluate cell apoptosis and cell cycle;Transwell assay to assess cell invasion ability;transmission electron microscopy to observe cell ultrastructure;Real-time quantitative PCR to analyze miR-200h-3p mRNA expression;Western blot to examine the expression of Beclin-1,P62,LC3II/I,NBR1 protein;dual-luciferase reporter assay to verify the targeting relationship between miR-200b-3p and NBR1.Results qRT-PCR results showed that expression of miR-200b-3p mRNA(1.314±0.093)is notably elevated in the miR-200b-3p mimic group when compared with the mimic NC group(1.000±0.069).In the miR-200b-3p inhibitor group,miR-200b-3p mRNA(0.581±0.031)expressed lower than that in the inhibitor NC group(1.009±0.055).Meanwhile,miR-200b-3p inhibitor showed reduced miR-200b-3p mRNA expression in contrast to the miR-200b-3p mimic group(P<0.05).Cell invasion results suggested that,compared with the mimic NC group,miR-200b-3p mimic group exhibited significantly diminished cell numbers across the basilar membrane(P<0.01).While miR-200b-3p inhibitor group showed increased cell numbers(P<0.01)in contrast to the inhibitor NC group,and the same as miR-200b-3p inhibitor group when compared with the miR-200h-3p mimic group(P<0.05).Cell cycle arrest confirmed that,in contrast to the mimic NC group,cell numbers in G2/M and S phase is remarkably lessened in the miR-200h-3p mimic group,but conspicuously increased in G1/G0 phase.However,ce

关 键 词：乳腺癌 自噬 miR-200b-3p 细胞侵袭 

分 类 号：R737.9[医药卫生—肿瘤]