After incubation with a biotinylaed secondary antibody and DAB (Dako, Carpenteria, CA), the slides were rinsed and counterstained with Mayer‘ hematoxylin. Statistical analysis Two-sided Student’s t test was used to analyze the differences in miR-20a expression [17], proliferation, colony formation number, percent of cells in respective cell cycle and apoptotic rate. Data were presented as mean ± SD from at least three separate experiments. The Fisher

exact test was used for analysis of categorical data. Association of miR-20a expression with overall STA-9090 cell line survival (OS) and recurrence-free survival selleck kinase inhibitor (RFS) was estimated by Kaplan-Meier method, and the resulting curves were compared using the log-rank test. The multivariate Cox proportional hazard regression analysis

were used to evaluate the contribution of independent prognostic factors to patient’s Epigenetics Compound Library high throughput survival by only taking the factors as covariates, that were found to be significant in univariate analysis. Overall survival was calculated as the interval between the date of the LT and either the date of death or the last follow-up date of the patient. Recurrence-free survival was calculated as the time from the date of LT until the date of tumor recurrence and was censored at the time of last following-up or death if at that time there was no evidence of tumor recurrence. All statistical analyses were conducted using the SPSS version 17.0 (SPSS Inc. Chicago, IL). p <0.05 was considered statistically significant. Results MiR-20a was down-regulated in primary HCC tissues especially in those with tumor recurrence following LT With the purpose of revealing the expression and significance of miR-20a in HCC, we first detected the expression of miR-20a in 100 cases of HCC and 10 normal liver tissue by Taqman qPCR. The expression of miR-20a was significantly down-regulated in HCC tissue compared with normal liver tissue (P = 0.001; Figure 1A) and the expression levels of miR-20a were further

down-regulated in HCCs samples of patients with tumor recurrence after LT (P = 0.020; Figure 1B). In accordance with the data between recurrence and non-recurrence patients, the expression of miR-20a Resminostat was much lower in the patients who had died after LT than the patients who still survived (P < 0.001; Figure 1C). At the same time, we also detected the expression level of miR-20a in normal liver cell line, LO2, and three HCC cell lines, HepG2, SMMC-7721 and BEL-7402. We found that the expression level of miR-20a in HCC cell lines was lower than in LO2 cells, which was similar with the results of clinical HCC samples (Figure 1A). Figure 1 Decrease expression of miR-20a in HCC is associated with tumor recurrence and poor prognosis following LT. (A) Expression of miR-20a was measured in 100 FFPE HCC samples, 10 normal liver tissue, normal liver cell line LO2 and 3 HCC cell lines by qRT-PCR, and the expression levels of miR-20a were normalized to U6 RNA expression for subsequent analyses.