[12] Cell migration was assessed using cell-culture inserts (BD Biosciences, Bedford, MA), according to the manufacterer’s guidelines. Ibrutinib Details are provided in the Supporting Materials. The sphere formation assay was performed as previously described[13] (and in the Supporting Materials). Flow cytomety analysis was performed as previously described[13] and detected using a FACSCanto

II flow cytometer (BD Biosciences). Antibodies (Abs) and the procedure are described in the Supporting Materials. The 3′-UTR (untranslated region) sequence of PTEN and SMAD7 are predicted to interact with miR-216a, and miR-217 or a mutated sequence within the predicted target sites was synthesized and inserted into the XbaI and FseI sites of the pGL3 control luciferase reporter vector (Promega, Madison, WI) (Supporting Fig. 3). The luciferase reporter assay was performed as previously described[11, 13] (and in the Supporting Materials). All experiments on mice were approved by the SingHealth Institutional Animal Care and Use Committee. Details of animal studies are provided in the Supporting Materials. Experimental data are presented as the mean ± standard deviation (SD). All statistical analyses were performed using analysis of variance (ANOVA) or a two-tailed Student t test with either GraphPad

Prism 5 (GraphPad Software, Inc., La Jolla, CA) or Partek Genomics Suite software (Partek Incorporated, St. Louis, MO). Survival curves were

calculated using Kaplan-Meier’s method. Differences selleckchem were considered statistically significant when P values were less than 0.05. Because there have only been a few reports on the expression of miRNAs and their relation to early recurrent disease in patients with HCC, we conducted Molecular motor comprehensive miRNA profiling of liver biopsies from HCC patients with early and nonrecurrent diseases to identify miRNAs that are relevant to early recurrent disease. Early recurrence was defined as a recurrence within 2 years after a curative resection. miRNA expression profiles of 30 cases of liver biopsies, including 10 samples from HCC patients who had early recurrent disease over the 24-month observation period and 10 from patients who did not have early recurrent disease, were compared to 10 histologically normal tissue samples using the GeneChip miRNA 2.0 Array (Affymetrix, Inc., Santa Clara, CA). A panel of miRNAs with significant differential expression between early and nonrecurrent HCC and histologically normal liver tissue was derived through a series of ANOVA contrasts, and these miRNAs were selected for further validation and functional characterization. Among these miRNAs, expression of the miR-216a/217 cluster was significantly up-regulated in biopsies from HCC patients associated with early recurrent disease. Expression of miR-216a and miR-217 was >20- and >16-fold, respectively, when compared between HCC and normal liver tissue.