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. 2016 Jul 5;7(27):42513-42526.
doi: 10.18632/oncotarget.9893.

Wnt/β-catenin pathway transactivates microRNA-150 that promotes EMT of colorectal cancer cells by suppressing CREB signaling

Yan-Hua Guo  1   2 , Lu-Qin Wang  1 , Bin Li  1 , Hui Xu  1 , Jian-Hua Yang  1 , Li-Si Zheng  1 , Peng Yu  1 , Ai-Dong Zhou  3 , Yin Zhang  1 , Shu-Juan Xie  1 , Zi-Rui Liang  1 , Chen-Min Zhang  1 , Hui Zhou  1 , Liang-Hu Qu  1
Affiliations

Wnt/β-catenin pathway transactivates microRNA-150 that promotes EMT of colorectal cancer cells by suppressing CREB signaling

Yan-Hua Guo et al. Oncotarget. .

Abstract

A hallmark of aberrant activation of the Wnt/β-catenin signaling pathway has been observed in most colorectal cancers (CRC), but little is known about the role of non-coding RNAs regulated by this pathway. Here, we found that miR-150 was the most significantly upregulated microRNA responsive to elevated of Wnt/β-catenin signaling activity in both HCT116 and HEK293T cells. Mechanistically, the β-catenin/LEF1 complex binds to the conserved TCF/LEF1-binding element in the miR-150 promoter and thereby transactivates its expression. Enforced expression of miR-150 in HCT116 cell line transformed cells into a spindle shape with higher migration and invasion activity. miR-150 markedly suppressed the CREB signaling pathway by targeting its core transcription factors CREB1 and EP300. Knockdown of CREB1 or EP300 and knockout of CREB1 by CRISPR/Cas9 phenocopied the epithelial-mesenchymal transition (EMT) observed in HCT116 cells in response to miR-150 overexpression. In summary, our data indicate that miR-150 is a novel Wnt effector that may significantly enhance EMT of CRC cells by targeting the CREB signaling pathway.

Keywords: CREB; EMT; Wnt/β-catenin; colorectal cancer; miR-150.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. A group of miRNAs were responsive to the activation of Wnt/β-catenin signaling
A-C. β-catenin Western blot (A), Wnt pathway reporter assay (B) and Axin2 qRT-PCR (C) in HCT116/HEK293T cells treated with LiCl as indicated. The same molarity of NaCl was used as a negative control (NC) for LiCl stimulation. D. The relative expression of miRNAs conserved between humans and mice were analyzed by qRT-PCR in LiCl-treated HCT116 and HEK293T cells; miR-150-5p is indicated by an arrow. All qRT-PCR experiments were performed at least three times. Error bars represent SEM. *p< 0.05 by Student's t-test.
Figure 2
Figure 2. miR-150 is directly transactivated by β-catenin/LEF1
A. qRT-PCR for pri-mir-150 and miR-150 expression after overexpression of β-catenin (HEK293T cells) or LEF1 (HCT116 cells). B. qRT-PCR for pri-mir-150 and miR-150 expression after siRNA-mediated β-catenin knockdown in SW480 and SW620 cells. C. Schematic of the genomic miR-150 region. The transcriptional start site (TSS) is marked as +1, and the three transcription termination sites (TTSs) are indicated. The sequence and location of the TCF/LEF1 binding site (TBE) is be noted by a black dot. The locations of the primers used in this study to generate plasmid constructs are indicated by arrows below the schematic. D. Deletion analysis of the miR-150 gene promoter in SW480 cells. The luciferase activity of each reporter construct is presented relative to that of the pGL4.11-basic vector. E, F. Luciferase reporter assays showing the dose-dependent effect of β-catenin (left panel) or dnTCF4 (right panel) overexpression on miR-150 promoter activity. G. Luciferase reporter assays showing the effect of β-catenin on the activity of the deleted or mutated TBE promoter. H. Gel shift and supershift assays showing binding of LEF1 to the miR-150 promoter in SW480 cells. 10 or 50-fold molar excess of unlabeled wild-type (WT) cold probe or mutate-type (MT) cold probe was used in the competition EMSA assay. I. Chromatin immunoprecipitation (ChIP) assays demonstrating an in vivo interaction between the β-catenin/LEF1 complex and the miR-150 promoter. The TBE site in the SP5 gene promoter was used as a positive control, and the coding region of Myo was used as a negative control (NC). All experiments were repeated at least three times with similar results. Error bars represent SEM. *p < 0.05 by Student's t-test.
Figure 3
Figure 3. Ectopic expression of miR-150 promoted EMT, migration and invasion of HCT116 cells
A. Morphological changes in HCT116 cells transfected with miR-150-5p mimics or NC after 48 h. B. Western blotting for EMT markers (adherens junction protein E-cadherin, tight-junction protein ZO-1 and mesenchymal intermediary filament vimentin) in HCT116 cells treated with miR-150-5p mimics. C. Immunofluorescent microscopy analysis of the localization and expression of EMT markers in HCT116 cells. Cells were counterstained with Hoechst 33342 dye. Scale bars, 50 μm. D. Migration and invasion assays in HCT116 cells treated with miR-150-5p mimics. Representative images are shown in the left panel. The mean number of cells per visual field was determined while viewing the entire chamber, and the experiments were performed in triplicate (right panel). Scale bars, 100 μm. E. A schematic of the structure of the plasmid for lentiviral overexpression of miR-150 (upper panel). qRT-PCR analysis of miR-150 expression in HCT116 stable cell lines and xenograft tumors (lower panel). Experiments were performed in triplication. Error bars represent SEM. *p< 0.05 by Student's t-test. F. Photographs of mice injected with HCT116-pLSNCG-miR-150 cells illustrate the metastatic sites found. The white arrow indicates clusters of metastatic cells. G, H. Microscopic images of HE staining (G) and GFP expression (H) in livers isolated from mice 35 days after the subcutaneous injection of HCT116 cells stably overexpressing miR-150. Scale bars, 100 μm.
Figure 4
Figure 4. miR-150 suppressed CREB signaling by directly targeting EP300 and CREB1
A. The 45-pathway assay revealed the effects of miR-150 on the activity of various pathways in HCT116 stable cell lines. The data were log2-transformed. B. Relative luciferase activity of WT and Mut EP300 and CREB1 reporters in HEK293T and HCT116 cells after co-transfection with the miR-150 expression vector or an empty vector. C. Western blotting for EP300 and CREB1 expression in HEK293T and HCT116 cells after transfection with miR-150-5p mimics or NC, or in SW480 cells after transfection with miR-150-5p inhibitors or NC. D. The mRNA levels of EP300 and CREB1 were determined by qRT-PCR after transfecting HCT116 cells with miR-150-5p mimics. E, F. The activity of CREB signaling and mRNA levels of c-Fos were measured by luciferase assay and qRT-PCR in HCT116 cells transfected with the miR-150-5p mimics, EP300 siRNA or CREB1 siRNA. G, H. Western blot analysis showing the effects of activation of Wnt signaling by LiCl treatment or LEF1 overexpression on EP300 and CREB1 expression in HCT116 cells. I. Western blot analysis showing the effects of knockdown of Wnt signaling by special siRNA on EP300 and CREB1 expression in SW480 cells. For D and F, the RT-PCR experiments were performed three times with similar results. Error bars represent SEM. *p< 0.05 by Student's t-test.
Figure 5
Figure 5. CREB1 and EP300 were the key mediators of miR-150-regulated EMT and CRC cell migration
A. Morphological changes in HCT116 cells 48 h after transfection with siRNA against EP300 or CREB1 or with NC. Scale bars, 50 μm. B. Western blotting for EMT markers (E-cadherin, ZO-1 and vimentin) in HCT116 cells treated with siRNA against EP300 or CREB1 or with NC. C. Immunofluorescent microscopy analysis of the localization and expression of EMT markers in HCT116 cells. Cell nuclei were labeled with Hoechst 33342 dye. Scale bars, 50 μm. D. Migration and invasion assays were performed in HCT116 cells after transfection with NC, si-EP300 and si-CREB. E. Western blotting for CREB1 expression in HCT116 cells after knockout by CRISPR/Cas9. F. Migration and invasion assays were performed in HCT116 cells after knockout CREB1 by CRISPR/Cas9. G. Transwell migration and invasion assays using HCT116 cells after co-transfection with miR-150-5p mimics and pcDNA3-CREB1, and NC. For D, F and G, representative images are shown in the left panel. The mean number of cells per visual field was determined in three randomly selected visual fields per chamber (right panel). The data were representative of three independent experiments. Error bars represent SEM. *p < 0.05 by Student's t-test.
Figure 6
Figure 6. A model of the Wnt/β-catenin-miR-150-CREB signaling regulation axis in colorectal cancer
The Wnt/β-catenin signaling pathway transcriptionally activates the expression of miR-150, and miR-150-5p subsequently suppresses the CREB pathway by directly targeting EP300 and CREB1, thereby inducing EMT in CRC cells.
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