Regional lymph node metastasis and distant metastasis are critical in the prognosis of laryngeal squamous cell carcinoma (LSCC). suppression of epithelial-mesenchymal transition as determined by increased E-cadherin and α-catenin and reduced fibronectin and vimentin expression. Additionally ETS-1 is a molecular target of miR-144-3p and silencing ETS-1 expression inhibited FaDu and Hep2 cell invasion and migration as well as reduced Hep2 xenograft tumor volume. In LSCC the expression of ETS-1 is upregulated with disease progression and higher Cilomilast ETS-1 expression which was negatively associated with Cilomilast miR-144-3p levels adversely corresponded with prognoses. Thus upregulated ETS-1 levels may promote LSCC metastasis resulting in poor patient prognosis. ≤ 0.002; Figure ?Figure1B).1B). Analysis of 3D cultures revealed that upregulation of miR-144-3p resulted in fewer and shorter processes than observed in the NC cells (Figure ?(Figure1C) 1 indicating a reduced propensity for invasion and migration. Figure 1 miR-144-3p inhibits FaDu and Hep2 cell invasion and migration To confirm the effects of miR-144-3p on cell migration and invasion FaDu and Hep2 cells were next transfected with a miR-144-3p inhibitor which Cilomilast significantly reduced miR-144-3p levels (Supplementary Figure 1). As shown in Figure ?Figure2A 2 cells expressing the miR-144-3p inhibitor migrated faster than the NC cells and had greater invasive capacity (≤ 0.002; Figure ?Figure2B).2B). In addition 3 cultures transfected with a miR-144-3p inhibitor had a greater number of longer cellular processes as compared to the NC group (Figure ?(Figure2C).2C). Taken together these results suggest that miR-144-3p inhibits cell Rabbit Polyclonal to SIN3B. migration invasion and possibly metastasis = 124.055 < 0.001). In contrast after transfection with a miR-144-inhibitor the proliferation of Hep2 cells increased significantly (= 702.700 < 0.001). Similarly transfection with miR-144-3p-mimics significantly reduced the number of colonies formed by Hep2 cells relative to the control group (= 26.361 = 0.000); miR-144-3p-inhibitors increased the number of colonies formed by Hep2 cells (= ?24.200 = 0.000; Figure ?Figure4B).4B). As shown in Figure Cilomilast ?Figure4C 4 transfection with miR-144-3p mimic significantly increased the proportion of cells in the G0/G1 phase (73.62% vs. 57.16%) and reduced the proportion of cells in the G2/M phase (12.67% vs. 19.22%) as well as the S phase (13.72% vs. 23.62%). After transfection with a miR-144-3p inhibitor the proportion of cells in the G0/G1 phase increased (48.41% vs. 58.38%); the proportion in the G2/M phase decreased (13.45% vs. 15.45%) and those in the S phase increased (26.16% vs. 38.14%; Figure ?Figure4C).4C). Taken together these data suggest that miR-144-3p inhibits the proliferation of Hep2 cells. Figure 4 miR-144-3p inhibits Hep2 cell growth 2.4 miR-144-3p binds to ETS-1 3′UTR to downregulate ETS-1 We previously identified ETS-1 as a putative target of miR-144-3p . Given that ETS-1 can induce the invasion and migration of rat C6 glioma cells  we next analyzed whether it was a miR-144-3p target. Analysis of the ETS-1 3′-UTR revealed a putative miR-144-3p target site (Figure ?(Figure5A).5A). As shown in Figure ?Figure5B 5 Western blot analyses of ETS-1 from FaDu and Hep2 cell lysates showed that was less abundant in cells overexpressing miR-144-3p. In contrast ETS-1 was more abundant in cells expressing a miR-144-3p inhibitor as compared to NC cells. Similarly Western blot analyses of GFP-ETS-1-3′UTR showed that it was less abundant in the miR-144-3p-transfected Cilomilast cells and more abundant with miR-144-3p inhibition as compared to NC cells (Figure ?(Figure5C).5C). These studies suggest that miR-144-3p downregulates ETS-1 protein expression. Figure 5 miR-144-3p binds to the 3′UTR of ETS-1 to downregulate its expression together with downregulation of MMP2 and MMP9 Dual luciferase reporter gene analyses of < 0.001; Figure ?Figure5D).5D). However no such downregulation in reporter activity was seen with the mir-144-3p + pGL3-ETS1-3′UTR-mu group (Figure ?(Figure5D).5D). Similarly as compared to NC + pGL3-ETS-1-3′UTR group relative luciferase activities were significantly reduced in the mir-144-3p-in + pGL3-ETS1-3′UTR-mu groups at either the 20 or 50 nmol assay concentrations (all < 0.001; Figure ?Figure5E).5E). Luciferase activity was significantly higher in the mir-144-3p-in + pGL3-ETS-1-3′UTR-mu groups compared to the NC + pGL3-ETS-1-3′UTR groups (all < 0.001). These findings confirm that miR-144-3p downregulates ETS-1 protein expression by targeting its 3′-UTR. The impact of.