Comprehensive Analysis of MicroRNA (miRNA) Targets in Breast Cancer Cells
Comprehensive Analysis of MicroRNA (miRNA) Targets in Breast Cancer Cells
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DOI：
10.1074/jbc.m113.491803
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发表时间：
2013-09-20
影响因子：
4.8
通讯作者：
Pfeffer, Lawrence M.
中科院分区：
文献类型：
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作者：
Fan, Meiyun;Krutilina, Raisa;Pfeffer, Lawrence M.
MicroRNA (miRNA) 通过 RNAi 诱导的沉默复合物的作用来调节 mRNA 的稳定性和翻译。在本研究中，我们通过使用 AGO2 免疫沉淀 (AGO2-IP) 和微阵列分析，在两种乳腺癌细胞系 MCF7 和 MDA-MB-231（分别代表管腔型乳腺癌和基底样乳腺癌）中系统地鉴定了内源 miRNA 靶基因。通过 DROSHA 或 DICER1 敲低，大约 70% 的 AGO2-IP mRNA 的表达水平增加。此外，对 miRNA 表达谱、mRNA-AGO2 相互作用和 mRNA 3'-UTR 的综合分析表明，> 60% 的 AGO2-IP mRNA 是 50 个表达最丰富的 miRNA 的推定靶标。总之，这些结果表明大多数 AGO2 相关 mRNA 是真正的 miRNA 靶标。功能富集分析发现，AGO2-IP mRNA 参与细胞周期、细胞凋亡、粘附/迁移/侵袭、应激反应（例如 DNA 损伤和内质网应激和缺氧）和细胞间通讯（例如 Notch 和 Ephrin 信号通路）的调节。通过检查 DROSHA 敲低对细胞行为的影响，进一步明确了 miRNA 在调节细胞迁移/侵袭和应激反应中的作用。我们证明，DROSHA 敲除增强了细胞迁移和侵袭，而它使细胞对悬浮培养、葡萄糖消耗和展开蛋白应激诱导的细胞死亡敏感。来自原位异种移植模型的数据表明，DROSHA 敲低导致原发肿瘤的生长减少，但增强了肺转移。总而言之，这些结果表明 miRNA 共同发挥作用，促进肿瘤细胞在压力下的存活，但抑制乳腺癌细胞中的细胞迁移/侵袭。
MicroRNAs (miRNAs) regulate mRNA stability and translation through the action of the RNAi-induced silencing complex. In this study, we systematically identified endogenous miRNA target genes by using AGO2 immunoprecipitation (AGO2-IP) and microarray analyses in two breast cancer cell lines, MCF7 and MDA-MB-231, representing luminal and basal-like breast cancer, respectively. The expression levels of similar to 70% of the AGO2-IP mRNAs were increased by DROSHA or DICER1 knockdown. In addition, integrated analysis of miRNA expression profiles, mRNA-AGO2 interaction, and the 3 '-UTR of mRNAs revealed that >60% of the AGO2-IP mRNAs were putative targets of the 50 most abundantly expressed miRNAs. Together, these results suggested that the majority of the AGO2-associated mRNAs were bona fide miRNA targets. Functional enrichment analysis uncovered that the AGO2-IP mRNAs were involved in regulation of cell cycle, apoptosis, adhesion/migration/invasion, stress responses (e. g. DNA damage and endoplasmic reticulum stress and hypoxia), and cell-cell communication (e.g. Notch and Ephrin signaling pathways). A role of miRNAs in regulating cell migration/invasion and stress response was further defined by examining the impact of DROSHA knockdown on cell behaviors. We demonstrated that DROSHA knockdown enhanced cell migration and invasion, whereas it sensitized cells to cell death induced by suspension culture, glucose depletion, and unfolding protein stress. Data from an orthotopic xenograft model showed that DROSHA knockdown resulted in reduced growth of primary tumors but enhanced lung metastasis. Taken together, these results suggest that miRNAs collectively function to promote survival of tumor cells under stress but suppress cell migration/invasion in breast cancer cells.