<i>EZH2</i> and <i>CTNNB1</i> are downstream targets of miR-214 and both are upregulated in human HCC tissue samples.

<p>(A) Effect of miR-214 on <i>EZH2</i> and <i>CTNNB1</i> expression, as shown by a luciferase reporter assay. The data were normalized by the ratio of Firefly and Renilla luciferase activities measured at 48 h post-transfection. The bar graph showed the mean ± SD in three independent transfection experiments. *<i>P</i><0.05. (B) Western blotting analysis of EZH2, β-catenin, and E-cadherin expression in P-miR-control- and P-miR-214-transfected SK-HEP-1 cells. (C-E) Validation of the expression of <i>EZH2</i> (C), <i>CTNNB1</i> (D) and CDH1 (E) in 20 paired human HCC tissue samples and 10 samples of histologically normal liver tissues were validated by qRT-PCR.</p

Roles of <i>EZH2</i>, <i>CTNNB1</i> and <i>CDH1</i> on the growth and invasion of HCC cells.

<p>(A) Silencing of <i>EZH2</i> significantly inhibited the growth and significant decreased the ability of SK-HEP-1 cells to invade. (i) Western blots showing the silencing of <i>EZH2</i> by pLVTHM-shEZH2. (ii) The effect of silencing <i>EZH2</i> on cell growth at different time points. (iii) The inhibitory effect of silencing <i>EZH2</i> on cell invasion. (B) Silencing of <i>CTNNB1</i> significantly inhibited the growth of SK-HEP-1 cells. (i) Western blots showing the reduction of β-catenin after transfection with shRNA-β-catenin. (ii) Effects of silencing β-catenin on cell growth at different time points. (C) Over-expression <i>CDH1</i> significantly inhibited the ability of SK-HEP-1 cells to invade. (i) Western blots showing the overexpression of CDH1 by pcDNA3.1-CDH1 plasmid transfection. (ii) The inhibitory effects of overexpressing <i>CDH1</i> on SK-HEP-1 cell invasion. (D) Western blots showing the silencing of EZH2 significantly decreased the expression of CTNNB1 and induced CDH1 expression.</p

Downregulation of miR-214 is associated with the early recurrence of human HCC.

<p>The level of expression of miR-214 was analyzed by qRT-PCR and normalized to U6. (A) Expression of miR-214 in 20 paired of HCC tumor tissues was significantly lower compared to 20 matched histologically normal tissues as well as 10 histologically normal liver tissues from colorectal cancer patients with liver metastases (<i>P</i><0.01). (B) Low miR-214 expression was associated with early recurrent HCC disease when studied in an independent cohort of 50 HCC samples. The average expression level of miR-214, analyzed by qRT-PCR, was lower in HCC patients with early recurrence (≤2 years) (n = 29) than patients with no recurrence in the same time period (n = 21). (C) The expression of miR-214 was associated with survival in patients with HCC. The median expression value obtained for miR-214 of the 50 samples studied was employed as the cut-off point. Fisher’s exact test and Kaplan-Meier analysis were used to demonstrate that high miR-214 expression was significantly associated with early recurrent disease and a relative poorer disease-free survival rate.</p

Re-expression of miR-214 significantly inhibits cell growth in vitro and tumorigenic properties of HCC cells.

<p>(A and B) Growth of HLE (A) and SK-HEP-1 (B) cells <i>in vitro</i> at different time points following the re-expression of miR-214 mediated by transfection with P-miR-214, *<i>P</i><0.05. (C) Stable expression of miR-214 inhibited the anchorage-independent growth of SK-HEP-1 cells in soft agar. The upper section shows images of colony formation. The bar graph below the figures showed the mean number of colonies formed (± SD) and counted under the microscope in three randomly selected fields (magnification, ×40). *<i>P</i><0.05. (D) Stable expression of miR-214 inhibited tumorigenicity of SK-HEP-1 cells. The upper section showed images of the tumors obtained in mice at the end of the eighth week. The bar graph indicated the average of overall tumor volume measured each week (n = 6 mice per group).</p

Expression of <i>EZH2, CTNNB1,</i> and <i>CDH1</i>, downstream targets of miR-214, is associated with early recurrent disease and survival of patients with HCC.

<p>The level of expression of <i>EZH2</i>, <i>CTNNB1,</i> and <i>CDH1</i> in the 50 HCC samples described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044206#pone-0044206-g001" target="_blank">Figure 1</a> was analyzed by qRT-PCR and normalized by <i>HPRT1</i>. (A–C) The average expression level of <i>EZH2</i> and <i>CTNNB1</i> was significantly higher in samples from HCC patients with early recurrence (≤2 years) (n = 29) than patients with no recurrence over the same period (n = 21). In comparison, the average expression level of <i>CDH1</i> was significantly lower in in samples from HCC patients with early recurrence (C). (D–F) The median expression value obtained for <i>EZH2</i>, <i>CTNNB1</i>, and <i>CDH1</i> of the 50 samples studied was employed as the cut-off point and employed independently in Kaplan-Meier analysis to study disease-free survival rate. (G) Combining the expression of <i>EZH2</i>, <i>CTNNB1</i> and <i>CDH1</i> to predict tumor recurrence and disease-free survival rate using Kaplan-Meier analysis.</p