lncRNA ENST 933在乳腺癌中的作用及其机制研究

Role of long non-coding RNA ENST 933 in breast cancer and its underlying mechanism

目的探讨长链非编码RNA ENST00000579933(简称lncRNA ENST 933)对乳腺癌(breast cancer,BC)发展的调控机制。方法通过实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RT-qPCR)检测lncRNA ENST 933在BC组织和BC细胞系(MCF-7、MDA-MB-231、MDA-MB-453)中的表达;运用细胞计数试剂盒-8(cell counting kit-8,CCK-8)、克隆形成、EdU、Transwell、流式细胞仪、蛋白质印迹法(Western blot)、体内移植瘤实验检测lncRNA ENST 933和miR-588在BC细胞增殖、迁移、侵袭与周期中的作用;采用双荧光素酶报告基因、RNA免疫共沉淀实验、RT-qPCR、Western blot和挽救实验来评估lncRNA ENST 933、miR-588、真核起始因子6(eukaryotic initiation factor 6,EIF6)之间的互相作用。结果lncRNA ENST 933在BC组织和BC细胞系中表达均显著上调(P<0.05),过表达lncRNA ENST 933能够提升BC细胞增殖、迁移与侵袭能力,促进移植瘤生长,而敲低lncRNA ENST 933表达后BC的发展受到抑制,并导致细胞周期阻滞(P<0.05)。miR-588在BC组织中表达明显下调,过表达miR-588可抑制BC细胞的增殖、迁移与侵袭(P<0.05)。挽救实验结果表明lncRNA ENST 933可通过与miR-588进行竞争性结合,从而促进靶基因EIF6的表达。Western blot结果显示miR-588降低了AKT和mTOR的磷酸化水平(P<0.05)。结论lncRNA ENST 933可通过调控miR-588/EIF6轴促进BC的进展。

Objective To investigate the regulatory mechanism of long non-coding RNA(lncRNA)ENST00000579933(hereinafter referred to as lncRNA ENST 933)on the development of breast cancer(BC).Methods The expression level of lncRNA ENST 933 in BC tissues as well as in BC cell lines(MCF-7,MDA-MB-231,MDA-MB-453)was detected by fluorescence-based real-time quantitative polymerase chain reaction RT-qPCR.The effects of lncRNA ENST 933 and miR-588 on the proliferation,migration,invasion and cell cycle in BC were determined using CCK-8 assay,clone formation assay,EdU,Transwell assay,flow cytometry,Western blotting and in vivo transplantation tumor assay.Finally,dual luciferase reporter gene,RNA immunoprecipitation assay,RT-qPCR,Western blotting and rescue assay were used to evaluate the interactions among lncRNA ENST 933,miR-588,and eukaryotic initiation factor 6(EIF6).Results The expression of lncRNA ENST 933 was up-regulated in both BC tissues and BC cell lines(P<0.05).Overexpression of lncRNA ENST 933 could enhance the proliferation,migration and invasion abilities of BC cells and promote the growth of transplanted tumors,while knockdown of lncRNA ENST 933 resulted in inhibited development of BC and arrested cell cycle(P<0.05).The expression of miR-588 was obviously down-regulated in BC tissues,and its overexpression inhibited cell proliferation,migration and invasion abilities in the BC cells(P<0.05).Moreover,the results of rescue assay indicated that lncRNA ENST 933 promoted the expression of target gene EIF6 by competitively binding with miR-588.Western blotting revealed that miR-588 decreased the phosphorylation of AKT and mTOR(P<0.05).Conclusion lncRNA ENST 933 promotes the progression of BC by modulating the miR-588/EIF6 axis.