Viral vectors containing the miR-539 (GenBank ID: 664612) construct and the green fluorescent protein (GFP) construct were purchased from Takara (Shiga, Japan). The miR-539 sequence was sub-cloned into the pLVX-IRES-Hyg vector (Takara) to generate pLV-miR-539. The viral particles were harvested 48 h after transfection of pLV-miR-539 into HEK293T cells using the Lenti-HT Packaging Mix (Takara). HCCLM3 and SK-HEP1 cells were infected with the harvested recombinant lentivirus in the presence of 6 µg/ml Polybrene (Sigma, St. Louis, MO, USA). The empty pLV-GFP vector encoding GFP was used as the control (Fig. 1 ). The viral particles were harvested as previously described (11 (link)) and used in all subsequent studies.
The full-length 3′-UTR of the FSCN1 gene containing the putative miR-539 binding sites was amplified by PCR and was inserted into the pGL3 vector under the control of the CMV promoter (Promega, Madison, WI, USA). The coding sequences of FSCN1 were amplified by PCR and cloned into the pCDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA) to generate pCDNA-FSCN1. The primer sequences used to amplify the target genes are listed inTable I . The correct insertion of the PCR-amplified sequences was confirmed by sequencing. The plasmid was transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
The full-length 3′-UTR of the FSCN1 gene containing the putative miR-539 binding sites was amplified by PCR and was inserted into the pGL3 vector under the control of the CMV promoter (Promega, Madison, WI, USA). The coding sequences of FSCN1 were amplified by PCR and cloned into the pCDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA) to generate pCDNA-FSCN1. The primer sequences used to amplify the target genes are listed in