Turbofect reagent kit

The miR-214-expressing plasmid was cloned into the expression vector pcDNA6.2 followed by transfection into Huh7 cells using the TurboFect reagent (Thermo Fisher Scientific, Waltham, MA, USA). After 48 hrs of incubation, the cells were transferred to medium containing blasticidin for selection. After 2 weeks, the specific overexpression of miR-214 was confirmed via qRT-PCR. The lentivirus, miRZip-214, was purchased from System Biosciences (Mountain View, CA, USA). The plasmid and lentiviral package plasmids were cotransfected into Her-293T cells using the TurboFect reagent kit (Thermo Fisher Scientific) and produced viruses in the cells. After 24 hrs, the viral soup was collected for the infection of the HepG2-TR and SK-Hep1 cells. After 48 hrs of incubation, the cells were transferred into medium containing puromycin for selection. Specific anti-sense miR-214 was confirmed using qRT-PCR.

The pcDNA3‐CRNDE expression plasmid was generated from the pcDNA3 vector using EcoRI and XhoI restriction sites. pLAS5w.PeGFP-I2-Bsd and pcDNA3.1/Myc-His A vectors were used to generate YY1-expressing pLAS5w.PeGFP-I2-Bsd-YY1 and pcDNA3.1/Myc-His A-YY1, respectively, followed by 48 h of incubation with DNA and TurboFect Reagent kit (Thermo Fisher Scientific, Waltham, MA, USA). All plasmids were prepared using an EasyPrep EndoFree Maxi Plasmid Extraction kit (DPT-BA17; Biotools Co., Ltd., Taipei, Taiwan).

The FAM215A plasmid was cloned into expression vector pLAS5w.PeGFP-I2-Bsd and prepared using an EasyPrep EndoFree Maxi Plasmid Extraction Kit (DPT-BA17; Biotools Co., Ltd., Taipei, Taiwan). The generated plasmid was transfected to Mahlavu and J7 cells using a lentivirus-based method and a FAM215A-encoding lentivirus (System Biosciences). The plasmid and lentiviral packaging plasmids were co-transfected to Hek-293T cells using a Turbofect Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA), and produced a virus in Hek-293T cell. The Mahlavu and J7 cells were infected with viruses collected from 24 h cultures of virus-producing Hek-293T cells. After 48 h of incubation, cells were transferred to medium containing blasticidin (3 μg/mL) for selection of infected cells, and FAM215A expression was confirmed by qRT-PCR.