| MiR-4443通过抑制PEBP1的表达促进乳腺癌细胞的迁移和侵袭 |
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| 引用本文： | 王金焱,王晋秋,顾 诠,杨 艳,马亚军,朱 静,张全安.MiR-4443通过抑制PEBP1的表达促进乳腺癌细胞的迁移和侵袭[J].南方医科大学学报,2020,40(12):1712-1719. |
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| 作者姓名： | 王金焱 王晋秋 顾 诠 杨 艳 马亚军 朱 静 张全安 |
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| 摘 要： | 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-4443inhibitors（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。
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MiR- 4443 promotes migration and invasion of breast cancer cells by inhibiting PEBP1 expression |
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| Abstract: | 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. |
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| Keywords: | 微小RNA 磷脂酰乙醇胺结合蛋白1 肿瘤转移 乳腺癌 |
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