Lipofectamine 2000 transfection

ERMP1 cDNA and miR-328-3p mimics were synthesized by Molbase biological reagent company. After PCR amplification, ERMP1 cDNA was inserted into the pcDNA3.1 plasmid. Huh-7 cells and HepG2 cells were seeded in 6-well plates. After reaching 70–80% confluence, the cells were transfected with Lipofectamine 2000 transfection reagents (Invitrogen, Carlsbad, CA, USA). Upon transfection for 6 h, fresh medium was replaced. Cells were harvested at 48 h after transfection. Transfection efficiency was confirmed using RT-PCR and Western blot. Cells were performed in five groups: Control group, mimic-NC group, miR-328-3p mimic group, pcDNA-ERMP1 group, and miR-328-3p mimic + pcDNA-ERMP1 (mimic + ERMP1) group. Epithelial-mesenchymal transition (EMT) of each group cells were observed by optical microscope (Leica, Germany).

A dual-luciferase reporter gene assay was used to verify the binding association of HCG11 and SOX4 to miR-214-5p. The 3′ untranslated region (UTR) containing HCG11 or SOX4, and sequence fragments of miR-214-5p predicted binding sites, were ligated into luciferase reporter plasmids and used to co-transfect CRC cells together with miR-214-5p mimics or miR-214-5p inhibitor using Lipofectamine^® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.); the imported mutant sequence fragment was used as the reference group. To determine the gene binding relationship, the dual-luciferase reporter gene system (Promega Corporation) was used to measure luciferase activity. Luciferase activity was measured 24 h after transfection and the results were normalized to Renilla luciferase activity.

Lipofectamine 2000 transfection and TRIzol LS reagents were purchased from Invitrogen (Grand Island, NY, USA). Antibodies against Cullin7 were purchased from Abcam (Cambridge, MA, USA). E-cadherin, N-cadherin, vimentin, and β-actin antibodies were from Cell Signaling technology (Danvers, MA, USA). Anti-α-catenin antibody was from BD (Franklin Lakes, NJ, USA). Unless otherwise noted, all other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).