MCPIP1诱导乳腺癌细胞系MDA-MB-231细胞周期停滞

目的研究单核细胞趋化蛋白诱导蛋白1(MCPIP1)在乳腺癌细胞MDA-MB-231中的作用及机制。方法用脂质体转染法转染细胞,在MDA-MB-231中分别过表达,或敲低MCPIP1并筛选稳定表达shRNA的单克隆;MTT法检测细胞增殖;流式细胞计量技术检测细胞周期;实时荧光定量PCR(q-PCR)检测靶基因半衰期;RNA免疫沉淀法(RNA-IP)检测MCPIP1结合的靶基因;荧光素酶报告基因实验检测调节基因3'非编码区(3'UTR)的能力。结果过表达MCPIP1可显著抑制MDA-MB-231细胞增殖(P0.05),敲低MCPIP1显著促进细胞增殖(P0.05);并分别显著增加或降低G1期细胞百分比(P0.01);MCPIP1显著降低细胞周期素依赖性激酶2(CDK2)、细胞周期素依赖性激酶6(CDK6)、cyclin D1和cyclin E1 mRNAs的半衰期(P0.01);MCPIP1结合细胞周期相关基因(P0.05);MCPIP1显著降低含不同3'UTR的报告基因荧光素酶活性(P0.05)。结论MCPIP1在乳腺癌细胞MDA-MB-231中发挥抑癌作用,诱导细胞周期G_1期停滞可能是其发挥抑癌功能的机制之一。

MCPIP1 induces cell cycle arrest in breast cancer cell line MDA-MB-231

Objective To investigate the functions of Monocyte chemotactic protein-induced protein 1 (MCPIP1) in human breast cancer cell line MDA-MB-231.Methods MDA-MB-231 cells were transfected with GFP-tagged MCPIP1 by Tet-on inducing expression system.Endogenous MCPIP1 was knocked down by stable expressing shRNA.MTT assay was performed to measure the growth of MDA-MB-231 cells after overexpression or knockdown of MCPIP1.FACS method was used to analyze cell cycle in MDA-MB-231 cells.Real-time PCR was used to test the expression of cell cycle-related mRNAs expression and their half-lives.RNA-IP experiment was conducted to detect the mRNA directly enriched by MCPIP1.Luciferase assay was performed to determine whether the mRNA decay was mediated through 3′UTR.Results MCPIP1 overexpression significantly inhibited cell proliferation(P<0.05), while knockdown MCPIP1 promoted cell proliferation with statistical significances (P<0.05).MCPIP1 induced cell cycle arrest in MDA-MB-231 with statistical significance (P<0.01).MCPIP1 overexpression reduced the half-lives of cell cycle mRNAs (CDK2,CDK6,cyclin D1,cyclin E1,respectively) with significance (P<0.01).In addition, cell cycle-related mRNAs were able to be pulled down by GFP-MCPIP1 but not isotype IgG(P<0.05).Compared with control vector, MCPIP1 significant suppressed luciferase activities of all four 3′UTR reporters (P<0.05).Conclusions MCPIP1 functions as a tumor suppressor in human breast cancer cell line MDA-MB-231 through inducing G1 cell cycle arrest.