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. 2016 Jul 8;7(31):49859–49877. doi: 10.18632/oncotarget.10489

Figure 2. miR-644a inhibits metastasis, and its expression or gene signature is associated with metastasis of breast cancer patients.

Figure 2

(A) Wound healing assay of MDA-MB-231 cells transfected with miR-Ctrl or miR-644a. Cells were scratched after 48 hours of transfection, and images were taken with 4× Magnification at 0, 15 and 30 hours after transfection. (B and C) Real-time migration of MDA-MB-231 (B) and MDA-MB-436 (C) cells transfected with either miR-Ctrl or miR-644a, monitored using an RTCA assay. (D) Number of invaded cells transfected with miR-Ctrl or miR-644a using Matrigel invasion assay. n = 3. (E) Viability of 231.Ctrl and 231.miR-644a cells grown in anchorage-independent conditions for 7 days, quantified by WST-1 assay (left) together with their fluorescence microscopy images with 10X magnification (right). (F) Fluorescence microscopy images of MD-MB-231 cells transfected with miR-Ctrl or miR-644a. Cell nuclei and filamentous actin were stained with 4,6-diamidino-2-phenylindole (DAPI) and Alexa Fluor 488 phalloidin, respectively. Images were taken after 72 hours of transfection with 20X magnification. Boxes at upper right corners of the images show cell morphology with higher resolution. (G and H) qRT-PCR analysis of epithelial and mesenchymal marker gene expression in MDA-MB-436 (G) and MDA-MB-231. (H) Cells transfected with miR-Ctrl or miR-644a. n = 3. (I) Western blot analysis of epithelial and mesenchymal marker expression in MDA-MB-231 cells transfected with miR-Ctrl or miR-644a. (J) Representative images of lungs collected from nude mice injected intravenously with 231.Ctrl or 231.miR-644a cells. Mice were sacrificed at week 7 and lungs were fixed in Bouin's Solution. (K) Hematoxylin and eosin staining of metastatic nodules in lungs from (J). (L) Luciferase signal coming from metastatic nodules in lungs of (J) as quantified by a luciferase assay. (M) miR-644a expression in 23 normal tissue, 31 primary tumor (IDC) and 13 lymph node metastasis tissues in GSE38167 depicted as box-plot. (N) Enrichment plot of patients from GSE58644 (n = 320) with high or low miR-644a signature score. Genes expressed higher in breast cancer patients with poor outcome as compared to those with good outcome were enriched in patients expressing low levels of miR-644a signature score. (O) Kaplan Meier survival curve representing the percentage distant metastasis-free survival in breast cancer patients based on miR-644a signature score median expression levels in GSE58644 (n = 310).