MicroRNA-9 inhibition of cell proliferation and identification of novel miR-9 targets by transcriptome profiling in breast cancer cells

Abstract

Although underexpression of miR-9 in cancer cells is reported in many cancer types, it is currently difficult to classify miR-9 as a tumor suppressor or an oncomir. We demonstrate that miR-9 expression is down-regulated in MCF-7 and MDA-MB-231 breast cancer cells compared with MCF-10-2A normal breast cell line. Increasing miR-9 expression levels in breast cancer cells induced anti-proliferative, anti-invasive, and pro-apoptotic activity. In addition, microarray profiling of the transcriptome of MCF-7 cells overexpressing miR-9 identified six novel direct miR-9 targets (AP3B1, CCNG1, LARP1, MTHFD1L, MTHFD2, and SRPK1). Among these, MTHFD2 was identified as a miR-9 target gene that affects cell proliferation. Knockdown of MTHFD2 mimicked the effect observed when miR-9 was overexpressed by decreasing cell viability and increasing apoptotic activity. Despite variable effects on different cell lines, proliferative and anti-apoptotic activity of MTHFD2 was demonstrated whereby it could escape from miR-9-directed suppression (by overexpression of MTHFD2 with mutated miR-9 binding sites). Furthermore, endogenous expression levels of miR-9 and MTHFD2 displayed inverse expression profiles in primary breast tumor samples compared with normal breast samples; miR-9 was down-regulated, and MTHFD2 was up-regulated. These results indicate anti-proliferative and pro-apoptotic activity of miR-9 and that direct targeting of MTHFD2 can contribute to tumor suppressor-like activity of miR-9 in breast cancer cells.

FIGURE 1.

Effects of miR-9 overexpression on cell viability, apoptosis, migration, and invasion. Taqman qRT-PCR analysis of miR-9 in MCF-7 and MDA-MB-231 breast cancer cell lines and MCF-10-2A normal breast cell lines, represented as normalized relative expression ±S.D. (A), and in MCF-7 cells transfected with pre-ctr (control precursor) or pre-9 (miR-9 precursor) synthetic oligonucleotides (B) is shown. All data were analyzed by relative quantification method (2^−ΔΔCt) using RNU6B small RNA as the housekeeping control. Statistical significance was calculated using Student's t test (*, p < 0.05; **, p < 0.001). C, viability and caspase-7 activity in MCF-7 cells 48 h post-transfection with pre-9 or pre-ctr (analyzed by ApoTox-Glo Triplex Assay). Cytotoxicity (dead cell) fluorescence measurements at wavelengths 485_EXT and 520_EM were used for normalizations. Viability (live cell) fluorescence measurements at wavelengths 400_EXT and 505_EM were represented in the graph as normalized percentage (%) change (live/dead cell ratio). Caspase-7 activity luminescence measurements were represented in the graph as normalized percentage (%) change using viability (live cell) measurements in well-to-well normalization (apoptotic/live cell ratio). Three hours of staurosporine (STS) treatment (1 μm) was used as a control for the assay sensitivity to detect caspase-3/7 activation as an indication of apoptosis. D, wound healing assay (migration) of MCF-7 cells at 0, 24, and 48 h of post-transfection of pre-9 or pre-ctr. The images were taken from an inverted microscope under 10× magnification. Solid black lines represent the initial wound boundaries (at 0 h), and dashed white lines represent the boundaries of migrated cells. E, invasion (Matrigel invasion assay) of highly invasive MDA-MB-231 cells at 48 h post-transfection of pre-9 or pre-ctr is shown. Images were taken from an inverted microscope under 10× and 20× magnifications showing invaded cells (black) on the Matrigel surface. The graph represents average number of invading cells counted from the replicate Matrigel images.

FIGURE 2.

Microarray profiling of miR-9 overexpression in MCF-7 cells and qRT-PCR validation of differentially expressed genes. A, shown is a landscape plot of Sylamer analysis for miR-9 seed (7-mer; 1A and 2) enrichment in down-regulated genes is shown. Colored lines represent miRNA seeds with the highest and lowest peaks in gene list ranked by t-statistic (x axis). B, shown is a heat map representation of differentially expressed genes in two dosages of miR-9 expression (1× and 2× pre-9) compared with controls (1× and 2× pre-ctr); each treatment was performed in triplicate, and results represent cutoffs p value 0.01 and -fold change >1.2. C, relative expression analysis (qRT-PCR) of 25 selected genes from microarray results, validating differential expression of 19 down-regulated and 6 up-regulated genes, shown as normalized relative expression ±S.E. GAPDH and β-Actin were used as housekeeping controls in normalizations.

FIGURE 3.

Luciferase analysis of miR-9 predicted targets. A, binding sites of miR-9 on the 3′-UTR of six predicted targets (TargetScan) (AP3B1, CCNG1, LARP1, MTHFD1L, MTHFD2, and SRPK1) and mutated miR-9 seed regions in mutant 3′-UTR constructs generated are compared with wild type 3′-UTR constructs. B, shown is a luciferase assay for the six predicted targets using wild type 3′-UTR (WT UTR) and mutant 3′-UTR (MUT UTR) constructs co-transfected with targeting miRNA (pre-9) or non-targeting miRNA (pre-ctr) in MCF-7 cells. Data are represented as the log 2 ratio of targeting to non-targeting miRNA (pre-9/pre-ctr) luciferase activity. PLight luciferase activity is shown as the EV control. C, shown is a Western blot of six predicted miR-9 targets proteins in pre-9- or pre-ctr-transfected MCF-7 cells. Numbers represent relative band intensities measured by densitometry analysis normalized using β-Actin housekeeping control. D, shown is a luciferase assay for MTHFD2 wild type 3′-UTR (WT UTR) and mutant 3′-UTR (MUT UTR) constructs co-transfected with targeting miRNA (pre-9) or non-targeting miRNA (pre-ctr) in MDA-MB-231 cells. E, shown is relative expression analysis of MTHFD2 (qRT-PCR) in MDA-MB-231 cells transfected with pre-ctr or pre-9. All data were analyzed by relative quantification method (2^−ΔΔCt) using GAPDH and β-Actin as housekeeping controls in normalizations. Western blot analysis shows relative MTHFD2 protein level (densitometry analysis) after pre-ctr or pre-9 transfections in MDA-MB-231 cells.

FIGURE 4.

Knockdown effects of MTHFD2. A, shown is relative expression analysis (qRT-PCR) of MTHFD2. B, shown is Western blot analysis of MTHFD2 protein in MCF-7 and MDA-MB-231 cells at 48 h post-transfection of siRNAs (siMTHFD2 or siControl). C, shown is viability and caspase-3/7 activity (ApoTox-Glo Triplex Assay) in MCF-7 and MDA-MB-231 cells at 48 h post-transfection of siRNAs (siMTHFD2 or siControl) represented in graphs as percentage (%) change (t test; *, p < 0.05; **, p < 0.001). Invasion (Matrigel invasion assay) of MDA-MB-231 cells at 48 h post-transfection of siRNAs are shown as images (10× magnification) from an inverted microscope, also represented as average number of invading cells in the graph.

FIGURE 5.

Overexpression effects of MTHFD2. A, linear maps of pCMV empty vector control (EV) and MTHFD2 overexpression constructs pCMV-MTHFD2-UTR (wild type 3′-UTR), pCMV-MTHFD2-UTRmut (3′-UTR with mutated miR-9 binding sites) and relative expression analysis (qRT-PCR) of MTHFD2 in MCF-7 and MDA-MB-231 cells are shown. The graphs represent MTHFD2 expression detected after co-transfections of overexpression constructs and pre-9 or pre-ctr oligonucleotides; pre-9 results are normalized using pre-ctr (pre-9/pre-ctr ratio). B, Western blot of MTHFD2 protein at 48 h post-transfection of MCF-7 and MDA-MB-231 cells with overexpression constructs (EV, UTR, and UTRmut). Numbers represent band intensities calculated using densitometry analysis. No measurable band intensity was detected for MDA-MB-231 cells. C, viability and caspase-3/7 activity in MCF-7 and MDA-MB-231 cells at 48 h post-transfection of overexpression constructs, presented as percentage (%) change (t test; *, p < 0.05; **, p < 0.001). Invasion of MDA-MB-231 cells at 48 h post-transfection of overexpression constructs were shown as images (10× magnification) from an inverted microscope, also represented as average number of invading cells in the graph.

FIGURE 6.

MTHFD2 and miR-9 expression in primary breast tumor samples. A, relative expression analysis (qRT-PCR) of MTHFD2 and miR-9 (Taqman qRT-PCR) in primary breast tumor samples (n = 16; non-metastatic tumor samples #1–8 and metastatic tumor samples #9–16) compared with normal breast tissues (n = 14). The graph represents relative expressions (log₂ ± S.D.) in individual primary tumor samples compared with the mean of normal breast samples (n = 14, the mean set to log 2 = 1). GAPDH and RNU6B were used as housekeeping controls in MTHFD2 and miR-9 relative expression analysis, respectively. B, MTHFD2 expression in normal breast and breast tumors using Richardson_breast_2 and Zhao_breast datasets obtained from the Oncomine™ data base is shown. Box plots represent normalized expression of MTHFD2, higher in breast tumor samples compared with normal breast tissues (p = 6.95e-5 in Zhao_breast dataset). n indicates the number of patients used in the study.