Hmir0210 mr03

miR-345 mimic (HmiR0210-MR03; GeneCopoeia, Inc., Guangzhou, China) and control vector (CmiR0001-MR03; GeneCopoeia) were transfected into MHCC-97H cells while miR-345 inhibitor (HmiR-AN0437-AM01; GeneCopoeia) and negative control vector (CmiR-AN0001-SN; GeneCopoeia) were transfected into Hep3B cells. YAP1 overexpression plasmid and the control plasmid were transfected into Hep3B cells overexpressing miR-345 while YAP1 siRNA and scramble siRNA were transfected into MHCC-97H cells with miR-345 knockdown. Cell transfection of HCC cells were carried out in 6-well plates with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). Cells after transfection were collected for western blot analysis, qRT-PCR, would healing assay, Transwell assay and in vivo experiments.

The putative binding sequences of MEG3-wild type (WT; 5′-CCAGAGCCTGGTTCAGGG-3′) and MEG3-mutant (MUT; 5′-AACCCGAAGGCGGACAAA-3′) were respectively cloned into a pmirGLO vector (Promega Corporation). HTR-8/SVneo cells were co-transfected with 50 nM either miR-345-3p mimics (HmiR0210-MR03; GeneCopoeia, Inc.) or mimic control (cat. no. CmiR0001-MR03; GeneCopoeia, Inc.) and 100 ng either MEG3-WT or MEG3-MUT using Lipofectamine^® 2000. Following 48 h transfection, Dual-Luciferase Reporter Assay System (Promega Corporation) was used to detect luciferase activity, according to the manufacturer's protocols. All firefly luciferase activities were normalized to Renilla luciferase activity.