MicroRNA-125a represses cell growth by targeting HuR in breast cancer

Abstract

MicroRNAs (miRNAs) are a class of naturally occurring, small, non-coding RNAs that control gene expression during development,normal cell function and disease. Although there is emerging evidence that some miRNAs can function as oncogenes or tumor suppressors, there is limited understanding of the role of miRNAs in cancer. In this study, we observed that the expression of miR-125a was inversely correlated with HuR expression in several different breast carcinoma cell lines. HuR is a stress-induced RNA binding protein whose expression is elevated or localization perturbed in several different cancers. Increased cytoplasmic localization of HuR is a prognostic marker in breast cancer. Real time PCR and gene reporter assays indicated that HuR was translationally repressed by miR-125a. Re-establishing miR-125a expression in breast cancer cells decreased HuR protein level and inhibited cell growth. Using MCF-7 breast cancer cells, we further clarified that miR-125a inhibited cell growth via a dramatic suppression of cell proliferation and promotion of apoptosis.In addition, cell migration was also inhibited by miR-125a overexpression. Importantly, the repression of cell proliferation and migration engendered by miR-125a was partly rescued by HuR re-expression. Our results suggest that miR-125a may function as a tumor suppressor for breast cancer, with HuR as a direct and functional target.

Figure 1

HuR protein level inversely correlates with miR-125 level in breast cancer cells. A, The 1208-nt HuR mRNA 3'UTR is shown (top) along with a predicted Hsa-miR-125 target site at nt 671-692, and the predicted duplex formed with either Hsa-miR-125a (middle) or Hsa-miR125b (bottom) variants. Lines show identity and dots show conservation between the 3'UTR target site and the miR sequence. Mutant indicates the miR-125 seed sequence that was deleted from the HuR 3’UTR for experiments in Figure 6B. B, HuR protein was detected by immunoblotting in the breast epithelial cell lines noted by the numbers and indicated at the bottom of the figure. The bottom graph shows the HuR level relative to MCF10A cells after normalizing to β-actin. C, The levels of miR-125a and miR-125b were analyzed by Northern blotting in the breast epithelial cell lines noted by the numbers and indicated at the bottom of the panel. The bottom graph shows the miR-125a or miR-125b level relative to MCF10A cells after normalization to U6 snRNA. 1. MCF10A is an immortalized breast epithelial cell line. 2–6. MCF-7, T47D, SKBR3, MDA-MB-231 and HMT3522-T4-2 are breast carcinoma cell lines. Representative blots are shown, and similar results were obtained from two additional independent experiments.

Figure 2

Overexpression of miR-125a represses HuR protein level and cell number. The indicated cells were mock transfected or transfected with miR-125a or miR-125b precursors. A, 48 h after transfection, miR-125a or miR-125b was detected by Northern blotting. Blots were reprobed for U6 snRNA as an internal control. B, 72 h after transfection, HuR protein level was detected by immunoblotting. HuR level relative to mock transfected cells after normalization to β-actin is graphed under the blots. C, 72 h after transfection, cell number in each condition was assessed by cell counting and expressed as percent of mock-transfected cells. Data in A is representative of 3 independent experiments. The data shown in B and C are means of three independent experiments.

Figure 3

miR-125a suppression of cell proliferation is partially rescued by HuR. MCF-7 cells were mock transfected or transfected with miR-125a precursor for 24 h followed by transfection of pcDNA3.1 (mock+vec, 125a+vec) or pcDNA3.1mycHuR (mock+HuR, 125a+HuR) for 48 h. A, Cell proliferation was assessed by immunofluorescence analysis of Ki-67, a marker of dividing cells. Representative fluorescence micrographs are shown. Top panels, Ki-67 was visualized with Alexa Fluor 488 conjugated secondary antibody (green). Middle panels, nuclei were stained with DAPI (blue). Bottom panels, merge of Ki-67 and DAPI channels. Magnification, 200×. Percentage of Ki-67⁺ cells was calculated by counting 500 cells for each condition. B, Cell number in each condition was assessed by cell counting and expressed as percent of mock+vec transfected cells. The data shown are representative (micrographs) or means of three independent experiments. ** p< 0.01 vs. mock+vec, # p< 0.05 vs. 125a+vec.

Figure 4

miR-125a promotion of apoptosis is independent of HuR. MCF-7 cells were mock transfected or transfected with miR-125a precursor for 24 h followed by transfection of pcDNA3.1 (mock+vec, 125a+vec) or pcDNA3.1mycHuR (mock+HuR, 125a+HuR) for 48 h. A, HuR and PARP-1 protein levels were detected by immunoblotting. The blot was stripped and reprobed for β-actin as a loading control. B, Cell apoptosis was detected and viewed by fluorescence microscopy using Annexin V Apoptosis Detection Kit. Early apoptotic cells label with Annexin V (green) while late apoptotic cells label with propidium iodide (PI, red staining). All experiments were repeated three times.

Figure 5

miR-125a inhibition of cell migration is partially rescued by HuR. MCF-7 cells were mock transfected or transfected with miR-125a precursor for 48 h or for 24 h followed by transfection with pcDNA3.1 (mock+vec, 125a+vec) or pcDNA3.1mycHuR (mock+HuR, 125a+HuR). 48 h following transfection, 2 × 10⁵ cells in serum-free media were transferred to the top surface of 8-mm transwell chamber inserts. Medium supplemented with 20% serum was used as a chemoattractant in the lower chamber. After 24 h incubation, cells remaining on the top were removed and the lower surface of the membrane stained with Crystal Violet, photographed and counted. A, Representative microscopic fields with stained cells are shown. B and C, Five random fields were counted for each condition and expressed as a percentage of the control cell number (bottom). Experiments were repeated 4 (B) or 3 (C) times. ** p< 0.01 vs. mock or mock+vec, # p< 0.05 vs. 125a+vec transfection.

Figure 6

miR-125a targets the HuR 3’UTR for translational repression. MCF-7 cells were mock transfected or transfected with miR-125a precursor. A, 48 h after transfection, HuR mRNA level was detected by real time RT-PCR. Data were normalized to GAPDH and expressed as relative mRNA levels. B, Cells were co-transfected with miR-125a and pMIR-REPORT β-gal control vector or pMIR-REPORT luciferase vector containing either the HuR 3'UTR (HuR3'UTR-wt) or the HuR 3'UTR with the miR-125a seed sequence deleted (HuR3'UTR-mut). Cell extracts were prepared 48 h after transfection, and luciferase activity measured using the Dual-Light System. The relative luciferase activity after normalization to pMIR-REPORT β-gal control plasmid is shown. The experiments were repeated four times, ** p< 0.01 vs. mock, ## p< 0.01 vs. miR-125a plus HuR3'UTR-wt.