Comprehensive analysis of microRNA (miRNA) targets in breast cancer cells

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.

Keywords: Breast Cancer; Gene Regulation; Metastasis; MicroRNA; Stress Response.

FIGURE 1.

Identification of mRNAs associated with AGO2 in MCF7 and MDA-MB-231 cells. A, specificity and efficiency of anti-AGO2 antibody. AGO2 protein from whole cell lysate, AGO2-IP, and IP flow-through was detected using immunoblotting. GAPDH was used as loading control. B, enrichment of miRNA targeted mRNAs by AGO2-IP. Total RNA was prepared from cell lysate or AGO2-IP and subjected to qPCR analysis. The mRNA levels were normalized to GAPDH. The -fold enrichment was calculated with the equation, -fold enrichment = mRNA level detected in AGO2-IP/mRNA level in cell lysate, and is presented as mean ± S.E. (error bars) (n = 3). C, Venn diagrams and enriched cell signaling pathways of AGO2-IP mRNAs in MCF7 and MDA-MB-231 cells.

FIGURE 2.

miR-21 sponge inhibits AGO2 association of miR-21 targets in MDA-MB-231 cells. A, a luciferase reporter that harbors miR-21 target sites in the 3′-UTR was used to monitor the efficiency of miR-21 inhibition by sponge mRNA. B, enrichment of mRNAs by AGO2-IP in the absence (black bars) and presence (gray bars) of miR-21 sponge. The results are presented as mean ± S.E. (error bars) (n = 3). *, p < 0.05 (Student's t test). C, increased expression of miR-221 targets in MDA-MB-231 cells transfected with miRCURY LNA miR-221 inhibitor. D, increased expression of miR-200a targets in MCF7 cells transfected with miRCURY LNA miR-200a inhibitor. The results were presented as mean -fold change (miRNA inhibitor versus control oligonucleotide) ± S.E. (n = 3). EV, empty vector.

FIGURE 3.

DROSHA knockdown in MDA-MB-231 cells leads to pri-miRNA accumulation and mature mRNA reduction. A, DROSHA expression levels in cells stably expressing shRNA (MDA-MB-231/DROSHA-KD) or scramble RNA (MDA-MB-231/C). DROSHA mRNA and protein levels were examined by qPCR and immunoblotting, respectively. B, -fold changes of pri-miRNAs in response to DROSHA knockdown. Pri-miRNA levels were examined by using TaqMan pri-miRNA assays. The results are presented as mean -fold change (DROSHA-KD versus control) ± S.E. (error bars) (n = 3). *, p < 0.05 (Student's t test). C, expression levels of mature miRNA in DROSHA knockdown and control (C) cells. The results are presented as mean expression levels of miRNAs (normalized to U6) ± S.E. (n = 3).

FIGURE 4.

DROSHA knockdown in MDA-MB-231 cells increases expression levels of putative miRNA targets identified by AGO2-IP. A, -fold change of individual mRNAs in response to DROSHA knockdown. Gene expression was examined by array analysis using Illumina HT-12 expression BeadChips. B, box plots of expression levels of putative miRNA targets and non-miRNA targets. The box shows the 25th to 75th percentile with a line at the median. C, control. Error bars, S.D.

FIGURE 5.

Identification of the 50 most abundantly expressed miRNAs in MCF7 and MDA-MB-231 cells. A, heat map of miRNAs abundantly expressed in MCF7 and MDA-MB-231 cells. The relative expression levels miRNA were calculated according to Z scores from seven publicly available data sets. B, relative miRNA levels detected in AGO2-IP from MCF7 and MDA-MB-231 cells. The results are presented as mean ± S.E. (error bars) (n = 3).

FIGURE 6.

Alternative polyadenylation contributes to cell type-specific AGO2-interaction of mRNAs. The left panel shows the presence of alternative polyadenylation sites (red arrows) in the 3′-UTRs of mRNAs. The qPCR primers used to detect the expression of the extended 3′-UTR regions (between the proximal and distal polyadenylation site) are showed as purple bars. The middle panel shows the cell type-specific interaction with AGO2 of the indicated mRNA. The right panel exhibits the expression ratio of the extended 3′-UTR region relative to the coding region of mRNAs. Error bars, S.D.

FIGURE 7.

Global miRNA inhibition by DROSHA knockdown in MDA-MB-231 cells enhances cell migration and invasion, but promotes cell death in response to various types of stress. A, DROSHA knockdown increases cell potential for migration and invasion, which were detected by Boyden chamber assays with uncoated or Matrigel-coated membrane, respectively. The results are presented as mean number of cells/field ± S.E. (error bars) (n = 3). C, control. B, DROSHA knockdown impairs autophagy flux, indicated by the lack of response of MAP1LC3A to glucose depletion (GD) or chloroquine (CQ) treatment. In control cells with normal autophagy activity, glucose depletion induces conversion of MAP1LC3A from cytosolic (LC3A-I) to membrane-bound lipidated form (LC3A-II) due to increased autophagosome assembly, whereas chloroquine causes accumulation of LC3A-II by inhibiting autophagosome degradation by lysosome. C, DROSHA knockdown sensitizes cells to apoptosis induced by various types of stress. Apoptotic cells with compromised membrane integrity were detected with YO-PRO-1 dye, followed by flow cytometer analysis.

FIGURE 8.

DROSHA knockdown in MDA-MB-231 cells reduces growth of orthotopic xenografts but increases lung metastasis. A, growth rates of xenograft tumors derived from DROSHA knockdown or control cells inoculated in mammary gland fat pads. The results are presented as average tumor volume ± S.D. (n = 12) (top). For wet weight of tumors at 7 weeks after inoculation, the results are presented as average tumor weight ± S.D. (n = 8) (bottom). B, H&E staining of tumor sections. Necrosis loci were observed in tumors derived from DROSHA knockdown cells but not in tumors from control cells. C, metastatic burden in lungs. H&E staining showed the presence of clusters of human tumor cells in mouse lung sections. Human tumor cells in mouse lung tissues were quantified by qPCR using primers specific for human Alu sequences. The cell number was calculated using standard curve of genomic DNA purified from cultured MDA-MB-231 cells and presented as mean ± S.D. (error bars) (n = 8).