miR-34a and miR-200c Have an Additive Tumor-Suppressive Effect on Breast Cancer Cells and Patient Prognosis

Abstract

Breast cancer is the most common women's malignancy in the world and, for subgroups of patients, treatment outcomes remain poor. Thus, more effective therapeutic strategies are urgently needed. MicroRNAs (miRNAs) have emerged as promising therapeutic tools and targets, as they play significant roles in regulating key cellular processes by suppressing gene expression. However, additive opportunities involving miRNAs have been underexplored. For example, both miR-34a and miR-200c individually suppress the development of different types of cancer, but the cellular effects of their combined actions remain unknown. Here, we show that miR-34a and miR-200c levels are reduced in breast tumors compared to adjacent normal tissues and that this additively predicts poor patient survival. In addition, in cell lines, miR-34a and miR-200c additively induce apoptosis and cell cycle arrest, while also inhibiting proliferation, invasion, migration, stemness and epithelial-to-mesenchymal transition (EMT). Mechanistically, both miRNA-34a and miR-200c directly target HIF1-α and subsequently downregulate VEGFR, MMP9 and CXCR4, although combined miRNA-34a and miR-200c delivery suppresses mouse xenograft tumor development as effectively as individual delivery. We establish a model, supported by in vitro and clinical data, which collectively suggest that the co-delivery of miR-34a and miR-200c represents a promising novel therapeutic strategy for breast cancer patients.

Keywords: HIF1-α; apoptosis; breast cancer; cancer stemness; cell cycle arrest; metastasis; miR-200c; miR-34a.

Figure 1

Downregulation of miR-34a and miR-200c is associated with breast cancer development. (A,B) Expression levels of miR-34a (A) and miR-200c (B) in breast cancer (B,C) and adjacent normal tissues, as determined by qRT-PCR. ****, p-value < 0.0001. (C) Pearson correlation analysis between miR-34a and miR-200c expression levels. (D,E) Overall survival curves for ER- breast cancer patients comparing patients whose tumors express high and low levels of miR-34a and miR-200c, respectively, using the median expression as the cut-off. HR: Hazard ratio; CI: confidence interval; GBW: Gehan-Breslow–Wilcoxon. (F) Overall survival curves as in (D,E), but here comparing patients whose tumors express both low miR-34a and low miR-200c to those expressing either low miR-34a or low miR-200c (using median cut-offs). (G) Expression levels of miR-34a and miR-200c in different invasive (MDA-MB-231 and MDA-MB-468), non-invasive BC cell lines (BT, MCF-7 and SKBR3) and normal breast cell line (MCF-10A), as determined by qRT-PCR. Abbreviations: *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001.

Figure 2

Exogenous miR-34a and miR-200c expression additionally induce apoptosis and G₂/M cell cycle arrest. (A–C) Annexin V/propidium iodide assays following transfection of miR-34a, miR-200c and the combination in MCF-7 and MDA-MB-231 breast cancer cell lines. (D,E) Cell cycle profiles and respective quantifications of MCF-7, and (F,G) and MDA-MB-231 cells stained with DAPI and subjected to flow cytometry. Abbreviations: *, p-value < 0.05; **, p-value < 0.01.

Figure 3

MiR-34a and miR200c additionally suppress breast cancer cell migration, invasion, proliferation and expression of markers for the epithelial-to-mesenchymal transition. (A) Light microscope images showing the migration of indicated cells in scratch “wound” assays. Scratch areas are marked by hatched lines. Images show timepoints 0 and 48 h (h) after application of the scratches. Cells were transfected with indicated miRNAs. (B) Quantification of the numbers of cells that migrated into the scratch areas after 48 h, as shown in (A). **, p-value < 0.01. (C) Images of invasion assay plates of transfected breast cancer cell lines, as indicated. (D) Quantification of cell invasion in transfected cells, as shown in (C). **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001. (E) Quantification of BC cell proliferation relative to control transfected cells. (F) Western blots showing changes in expression levels of EMT-promoting proteins after miRNA transfection. (G,H) Correlations between an EMT score and miR-34a or miR200c expression, respectively, in breast tumors (each n = 729). Data are derived from TCGA and show the Spearman’s rank correlation coefficients and p values. Abbreviations: CTRL: control; *, p-value < 0.05.

Figure 4

MiR-34a and miR-200c additionally inhibit stemness features of breast cancer cells. (A) Representative images of colony formation plates pertaining to indicated cell lines transfected with indicated miRNAs. (B) Quantification of colony numbers from experiments shown in (A). *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001. (C–F) Flow cytometry of indicated cells stained for cancer stem cell markers CD133 (C,D) and CD44 (E,F). Histograms are shown in (C,E). Quantification of CD133⁺ and CD44⁺ cells are shown in (D,F). **, p-value < 0.01; ***, p-value < 0.001.

Figure 5

Individual and co-delivery of miR-34a and mir-200c suppress cancer development in a xenograft mouse model. (A) Bioluminescence imaging of serially diluted MDA-MB-231 cells, which stably express luciferase. (B) Bioluminescence imaging of mice with tumor formation prior to miRNA treatment. (C) Bioluminescence imaging of mice after intra-tumor injections with indicated miRNAs. (D) Quantification of bioluminescence intensities of mouse tumors after intra-tumor injections with indicated miRNAs. Measurements of tumor volumes (E), Images of mice (F), excised tumors (G) for indicated groups. ***, p < 0.001. (H,I) H&E staining on each tumor (H) and TUNEL assay to show apoptosis induction (I) after miRNA transfection. (J–N) Immunohistochemistry staining for Ki67, VEGFR, MMP9, HIF1-α and CXCR4, respectively.

Figure 6

A model explaining how miR-34a and miR-200c may cooperate by targeting unique and shared components of key signaling pathways to jointly suppress angiogenesis, metastasis and cancer stemness features in breast cancer.