Thirty-six hours after the transfections with RNA duplex, 2 × 104 QGY-7703 cells were added to coverslips that had been precoated with 240 μg Matrigel (R&D Systems) in 24-well plates. The
cells were allowed to spread for 1 hour at 37°C and were fixed, permeabilized and stained with fluorescent phalloidin (Invitrogen, catalog no. A34055), a probe for filamentous actin. Recurrence-free survival (RFS) was calculated from the date of the HCC resection to the time of first recurrence. Patients who were lost to follow-up or who died from causes unrelated to HCC were treated as censored events. Kaplan-Meier plots and Cox proportional hazard regression analysis, which were applied to identify the prognostic factors, were performed with SPSS version 13.0 (SPSS Inc., Chicago, IL). Associations between the RFS and the molecular changes or clinical characteristics were analyzed initially by a univariate Cox proportional AG-014699 price hazards regression analysis. Staurosporine molecular weight Significant prognostic factors found in the univariate analysis were evaluated further by a multivariate Cox regression analysis. The data are expressed as the mean ± SEM from at least three independent experiments. The values for the capillary tube formation and luciferase activity
assays are from three independent experiments that were performed in duplicate. The differences between the groups were analyzed by Student t test when two groups were compared or by one-way analysis of variance when more than two groups were compared. Analyses were performed with GraphPad Prism, version 5 (GraphPad Software, Inc., San Diego, CA). Correlations between two variables were explored with the Spearman’s correlation coefficient. All statistical tests were two-sided; P < 0.05 was considered statistically significant. Previously, we observed frequent down-regulation of miR-195 in HCC tissues. To investigate the biological significance of this finding, we analyzed the correlation between miR-195 levels and the clinical features of HCC patients in this study. The Kaplan-Meier
plots revealed an association of lower miR-195 levels with shorter RFS (P = 0.004; Fig. 1A). Multivariate Cox regression analysis further confirmed miR-195 not down-regulation as an independent risk factor for RFS (HR, 1.773; P = 0.017; Supporting Table 1). Importantly, lower miR-195 levels were associated significantly with higher microvessel densities (MVDs; Fig. 1B) and the presence of metastasis (Fig. 1C), suggesting that miR-195 down-regulation may contribute to HCC progression by promoting tumor angiogenesis and metastasis. Angiogenesis is a prerequisite for cancer growth and metastasis, and migration and invasion are key steps in the metastatic cascade. To clarify the effect of miR-195 on HCC angiogenesis, we first performed in vitro endothelial recruitment and capillary tube formation assays with two HCC cell lines, QGY-7703 and MHCC-97H.