Together, these data contribute to the characterization of the molecular mechanisms of HCC metastasis, Selleck Proteasome inhibitor and might provide new potential biomarkers for HCC. The human HCC cell lines HepG2, PLC-PRF-5, and QGY-7703 were cultured in MEM-α or RPMI 1640 media (Gibco, Gaithersburg, MD), respectively. Hep3B was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA). Transient transfection was performed using the Lipofectamine 2000 reagent (Invitrogen). Total RNA was extracted using the Trizol reagent (Invitrogen), and miRNAs were obtained using the mirVana miRNA isolation kit (Ambion, Austin, TX). Forty

paired human HCC and adjacent nontumor liver tissues were collected from the cancer center of Sun Yat-sen University. Among them, 30 paired specimens were from male and 10 paired specimens were from female patients. At collection, six patients were defined as stage I, 20 patients were defined as stage II, and 14 patients were classified as stage III (TNM classification). Venous invasion or tumor microsatellite formation was observed in 22 patients. Informed consent was obtained from each patient and ethics approval was granted by the Ethics

Committee of Sun Yat-sen University. EGFP, enhanced green fluorescent protein; EMT, epithelial-mesenchymal transition; EphA4, Eph tyrosine kinase Vadimezan receptor A4; HCC, hepatocellular check details carcinoma; MET, mesenchymal-to-epithelial transition; miRNA, microRNA; miR-10a, microRNA-10a; qRT-PCR, quantitative real-time polymerase chain reaction. The vector constructions used in this study are

shown in the Supporting Materials and Methods. The analysis of miR-10a predicted targets was performed using the TargetScan, PicTar, miRanda algorithms and the complementary DNA (cDNA) microarray results of colon carcinoma in our previous study. The related functions of the targets were also considered. QGY-7703 cells were transiently cotransfected with EGFP reporter plasmid and pcDNA3-pri-10a, pcDNA3, ASO-miR-10a, and ASO-NC. The RFP expression vector, pDsRed2-N1, was used as the internal control. The intensities of EGFP and RFP fluorescence were detected with an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Target genes and controls were analyzed by qRT-PCR using SYBR Premix Ex TaqTM (TaKaRa, Dalian, China). Details and antibodies used in western blot are in the Supporting Materials and Methods. Cell viability and proliferation were assessed by MTT and colony formation assays as described in the Supporting Information. The 24-well Boyden chamber with 8-μm pore size polycarbonate membrane (Corning, Cambridge, MA) was used to analyze the migration and invasion of tumor cells. For invasion assay, the membrane was coated with Matrigel to form a matrix barrier. Details are in the Supporting Information. Transfected QGY-7703 (2.