Abstract
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 ∼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.
Highlights
AGO2 probably plays a key role in RNA-induced silencing in mammary gland epithelial cells, and mRNAs coimmunoprecipitated with AGO2 may well represent the majority of endogenous miRNA targets
Compared with all expressed genes detected in the input RNA samples, the predicted miRNA targets were significantly overrepresented in AGO2 immunoprecipitation (AGO2-IP) mRNAs (p Ͻ 0.05, 2 test with Yates correction). These results suggest that the majority of the AGO2-IP mRNAs are targets of miRNAs abundantly expressed in MCF7 and MDA-MB-231 cells
We examined endogenous miRNA targets in breast cancer cells by an integrated analysis of AGO2-mRNA interaction, miRNA expression, gene expression, and cell behavior changes in response to inhibition of miRNA processing
Summary
Cell Culture—MCF7 and MDA-MB-231 were purchased from ATCC (Manassas, VA) and cultured in minimal essential medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin-streptomycin. To examine the efficiency of the miR-21 sponge to inhibit miR-21 function, MDA-MB-231 cells were transfected with pCMVluc-miR21, along with CMV--galactosidase and various doses of CMV-d2eGFP-21 or vector control, using Lipofectamine 2000 (Life Technologies). RNA samples prepared from whole cells and AGO2-IP were subjected to qPCR analysis. To examine the effect of miRNA inhibition on mRNA expression, cells (4 ϫ 105) were transfected with 50 nM miRCURY LNA miRNA inhibitor (Exiqon) or a control oligonucleotide using Lipofectamine RNAiMAX (Invitrogen). Genomic DNA from lung tissues (ϳ20 mg from the right lung lobes) was prepared using the Wizard Genomic DNA Purification Kit (Promega) and subjected to qPCR analysis using primers specific for the human Alu sequences (forward, 5Ј-ACG CCTGTAATCCCAGCACTT-3Ј; reverse, 5Ј-TCGCCCAGGCTGGAGTGCA-3Ј) [38]. Analysis of variance and post hoc least significant difference analysis or t tests were performed using GraphPad Prism 5 software. p values of Ͻ0.05 (*) were considered statistically significant
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