Anti mir 21 5p

Pre-miR-21-5p and a control miR, and anti-miR-21-5p and a scramble miR control were purchased from Ambion; Thermo Fisher Scientific, Inc. The sequences were as follows: Pre-miR-27a-3p, 5′-AACAUCAGUCUGAUAAGCUAUU-3′; control miR, 5′-AACCAUUUGAGAGUCAUCAAGA-3′; anti-miR-21-5p, 5′-UCAACAUCAGUCUGAUAAGCUA-3′; scramble miR control, 5′-CAGUACUUUUGUGUAGUACAA-3′.

Expression plasmids encoding wild-type EZH2 was constructed as described previously [30] (link). pcDNA3-intracellular domain of NOTCH1 (NICD1) plasmid was kindly provided by Dr. Jon C. Aster (Brigham and Women's Hospital, Boston, MA, USA). Luciferase reporter plasmid containing the 3′-flanking region (3’-UTR) of JAG1 were amplified from cDNA library of Huh7 cells and constructed in pGL3-Basic vector as described previously [30] (link). All plasmid constructs were confirmed by DNA sequencing. Plasmid transfections were performed using Lipofectamine 2000 (Invitrogen, USA). The shRNA against EZH2 gene shEZH2s and corresponding scrambled shRNA (Sigma, USA) were used for RNA interference as described previously [30] (link). Control, NOTCH1, JAG1 siRNAs (Santa Cruz Biotechnology, USA), miR-21-5p mimic, miR-26a-1-5p mimic, negative control miRNA (miR-NC), anti-miR-21-5p, anti-miR-26a-1-5p, and anti-miR-NC (Thermo fisher, USA) transfections were conducted with X-tremeGENE (Roche Applied Science, Germany). Gene silencing effect was confirmed by immunoblotting and real-time PCR at 48 hours posttransfection. The sequences are listed in Supplementary Table S1.