MiR-4443通过抑制PEBP1的表达促进乳腺癌细胞的迁移和侵袭

MiR-4443 promotes migration and invasion of breast cancer cells by inhibiting PEBP1 expression

目的探讨miR-4443对乳腺癌转移的影响。方法使用高通量测序技术测定检测3例乳腺癌患者原位癌组织及其配对的癌旁组织中miR-4443的表达。使用TCGA数据库验证miR-4443在乳腺癌组织以及正常乳腺组织中的表达水平。以MCF-7细胞(低侵袭性乳腺癌细胞)和MDA-MB-231细胞(高侵袭性乳腺癌细胞)为研究对象,采用RT-qPCR验证miR-4443在这两种细胞株中的表达水平。通过电转染法将miR-4443 mimics(miR-4443模拟物)、mimics-NC(miR-4443模拟物的对照物)或miR-4443 inhibitors(miR-4443抑制物)、inhibitors-NC(miR-4443抑制物的对照物)转染至不同的细胞株,利用RT-qPCR检测miR-4443在转染前后的表达水平,使用流式细胞分析仪分析miR-4443表达水平的变化对乳腺癌细胞凋亡的影响,使用划痕实验和Transwell侵袭实验检测miR-4443表达水平的变化对乳腺癌细胞迁移和侵袭能力的影响。使用生物信息学软件预测miR-4443潜在的靶基因,并进一步使用双荧光素酶报告基因系统验证。采用RT-qPCR和Western blot分别检测不同细胞株中PEBP1(重组人磷脂酰乙醇胺结合蛋白1)在mRNA和蛋白水平的表达。结果高通量测序技术检测发现在乳腺癌组织中miR-4443的表达远高于癌旁组织(P<0.01)。TCGA数据分析显示,与正常乳腺组织想比,miR-4443在乳腺癌组织中的表达升高(P<0.01)。RT-qPCR显示,与MCF-7细胞相比,MDA-MB-231细胞中miR-4443的表达升高(P<0.01)。与转染mimics-NC相比,MCF-7细胞转染miR-4443 mimics后,miR-4443的表达上调(P<0.01);而转染miR-4443 inhibitors的MDA-MB-231细胞,其表达下调(P<0.01)。转染miR-4443 mimics的MCF-7细胞的凋亡率与阴性对照和空白对照差异无统计学意义(P>0.05),并且转染miR-4443 inhibitors的MDA-MB-231细胞的凋亡率与阴性对照和空白对照亦差异无统计学意义(P>0.05)。划痕实验及Transwell侵袭实验显示转染miR-4443 inhibitors的MDA-MB-231细胞的迁移及侵袭能力减弱(P<0.01)。相反的,转染miR-4443 mimics的MCF-7细胞迁移及侵袭能力加强(P<0.01)。生物信息学软件预测发现PEBP1可能是miR-4443的潜在靶基因且与转移的相关程度最高。进一步行双荧光素酶报告基因实验,验证PEBP1是miR-4443的靶基因(P<0.01)。RT-qPCR和Western blot显示,MDA-MB-231细胞中PEBP1在mRNA及蛋白中的水平均低于MCF-7细胞(P<0.01)。转染miR-4443 mimics的MCF-7细胞中,PEBP1在mRNA及蛋白水平均低于对照(P<0.01);转染miR-4443 inhibitors的MDA-MB-231细胞中,PEBP1在mRNA及蛋白水平均高于对照(P<0.01)。结论miR-4443通过抑制PEBP1的表达水平促进乳腺癌细胞的迁移和侵袭能力,而抑制乳腺癌细胞中miR-4443的表达可能是未来潜在的治疗方法。

Objective To investigate the effect of miR-4443 expression on migration and invasion of breast cancer.Methods We examined the expression of miR-4443 in breast carcinoma in situ and paired adjacent tissues from 3 breast cancer patients with high-throughput sequencing and verified the results using TCGA database.We also detected miR-4443 expressions using real-time quantitative PCR(RT-qPCR)in low invasive and highly invasive breast cancer cells(MCF-7 and MDA-MB-231 cells,respectively).The changes in apoptosis,migration and invasion of MCF-7 and MDA-MB-231 cells after transfection with miR-4443 mimics,mimics-NC,miR-4443 inhibitor or inhibitor-NC were analyzed using flow cytometry,wound healing assay and Transwell invasion assay.The target gene of miR-4443 was predicted by bioinformatics software and validated by a dual luciferase reporter gene system.RT-qPCR and Western blotting were performed to detect the expression of recombinant human phosphatidyl ethanolamine binding protein 1(PEBP1)in the transfected cells.Results The expression of miR-4443 was significantly higher in the breast cancer tissues than in the adjacent tissues(P<0.01),and was significantly up-regulated in MDA-MB-231 cells as compared with MCF-7 cells(P<0.01).Transfection with miR-4443 mimics or inhibitors did not obviously affect apoptosis rate of the breast cancer cells(P>0.05),but significantly enhanced or weakened the migration and invasion abilities of the cells,respectively(P<0.01).Bioinformatic analysis identified PEBP1 as the target gene of miR-4443 with a close correlation with metastasis of breast cancer(P<0.01),and the result was confirmed by double luciferase reporter gene assay.The mRNA and protein expression of PEBP1 were significantly lower in MDA-MB-231 cells than in MCF-7 cells(P<0.01),and miR-4443 over-expression or knockdown significantly down-regulated or up-regulated PEBP1 expressions in the cells,respectively(P<0.01).Conclusion MiR-4443 promotes the migration and invasion of breast cancer cells by inhibiting the expression of PEBP1,suggesting the possibility of suppressing miR-4443 expression as a potential therapeutic strategy for breast cancer.