N MS023 biological activity breast cancer metastasis.Figure 4 Vascular endothelial growth factor C (VEGF-C
N breast cancer metastasis.Figure 4 Vascular endothelial growth factor C (VEGF-C) is involved in nuclear factors of activated T cells 5 (NFAT5)-driven progression of metastatic breast cancers. (A) In vitro tube formation of human umbilical vein endothelial cells (HUVECs) after co-culture for indicated time with MDA-MB-231 cells either mocked transfected or transfected with indicated siRNAs or miRNA mimic (?00). (B-D) ELISA using the serum samples for comparison of VEGF-C levels in healthy volunteers, patients with benign lesions, and patients with breast cancers (B), in patients with breast cancers in various stages (C), and in patients with non-metastatic and metastatic breast cancers (D).Li et al. Breast Cancer Research 2014, 16:454 http://breast-cancer-research.com/content/16/5/Page 9 ofNFAT5 is a direct target of miR-568 in metastatic breast cancersWe next addressed the regulation of NFAT5 in metastatic breast cancers with particular interests in the responsible miRNAs, which abrogate gene expression at the posttranscriptional or translational level [17]. We searched for potential miRNAs which may target NFAT5, and identified miR-568 as a candidate regulator of NFAT5 (Figure 5A). Consistent with our previous findings in T lymphocytes [23], the expression of NFAT5 inversely correlated with miR-568 levels in breast cancer cell lines, with significantly lower miR-568 expression in highly invasive cell lines (Figure 5B). Suppression of miR-568 in MCF-7 cells caused the upregulation of NFAT5, whereas introduction of a miR-568 mimic dose-dependently decreased NFAT5 levels in MDA-MB-231 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27597769 cells (Figure 5C). Consistently, miR-568 significantly inhibited the expression of a luciferase reporter gene fused to a specific wild-type but not mutated 3 UTR of NFAT5, suggesting the direct targeting of NFAT5 by miR-568 (Figure 5D). The role of miR-568 in breast cancer metastasis was next evaluated. Similar to siRNA-mediated knockdown of NFAT5 or S100A4, miR-568 significantly decreased the levels of metastasis executioners (Figure 5E). Transfection with miR-568 significantly suppressed the invasiveness of MDA-MB-231 cells, while inhibition of miR-568 enhanced the invasion of MCF-7 cells in a transwell assay (Figure 5F). These results are consistent with the observation that introduction of miR-568 into MDA-MB-231 cells suppressed a mesenchymal phenotype and concurrently improved epithelial marker expression in these cells (Figure 5G). MDA-MB-231 cells were next infected with a control or pre-miR-568-expressing recombinant lentivirus, and were implanted subcutaneously in nude mice to allow for tumor development. Compared with the tumors derived from control cells, primary tumors that ectopically expressed miR-568 showed decreased invasion and infiltration of peripheral tissues (Figure 5H). Significantly lower frequency of lung metastasis was detected in xenograft tumor models with miR-568-overexpressing cells than in tumors from control breast cancer cells (Figure 5H). Clinically, miR-568 expression was much lower in metastatic breast cancers than in non-metastatic breast cancers cells or normal breast tissues (Figure 5I). These data suggest an essential role for miR-568 in suppressing distal metastasis probably by targeting NFAT5 in breast cancers.Hotair suppresses miR-568 to maintain NFAT5 expression in metastatic breast cancerslines, while only modest expression of Hotair was detected in breast cancer cell lines with lower invasiveness (Figure 6A). In.
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