Hela, HepG2, Hep3B, SK-Hep-1, and PLC/PRF/5 cells were cultured in modified Eagle’s medium with 10% fetal bovine serum supplement. SNU475, SNU398, and Huh7 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum. Primary hepatocytes were isolated and cultured as described.17 Kupffer cells were isolated with OptiPrep Density Gradient Medium (Sigma, St. Louis, MO) according to a published protocol.18 Stellate cells were isolated and purified by pronase and collagenase.19 Purity of stellate cells, determined by intrinsic vitamin A autofluorescence, was more than 90%. The primary cells were seeded on six-well plates with Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. For knockdown of miR-194 in HepG2 cells, 100 nM miR-194 inhibitors (Ambion, Austin, TX) were transfected into cells with 2 μL HiPerfect (Qiagen, Valencia, CA) on a six-well plate. MDH1-PGK-eGFP-2.0 plasmid (Addgene 11375) BEZ235 nmr was used to transduce a precursor sequence of miR-194 to liver mesenchymal-like cancer cell lines.20 A multiplicity of infection of 3 to 6 was used to generate stable cell lines with miR-194 overexpression. RNAs were extracted using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH). miR-194 expression in human organs was analyzed
with FirstChoice Human Total RNA Survey Panel (Applied Biosystems, Foster City, CA). The reverse transcription was performed with Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). Real-time polymerase chain reaction (PCR) Selleckchem MG132 was performed using the Power SYBR Green PCR Master Mix protocol (Applied Biosystems).13 5S RNA was used to normalize expression levels of miRNAs. Sequences of the primers are provided in Supporting Information Table 1. For analysis of mRNAs, reverse transcription was performed with Superscript III reverse transcriptase and Oligo(dT)20 at 50°C for 1 hour. Primers for miR-194 target genes CDH2, HBEGF, RAC1, IGF1R, and DNMT3B were
provided in Supporting Information Table 2. Gene expression levels for mRNAs were standardized with β-actin (Ambion). Proteins were separated by 10% sodium dodecyl see more sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After blocking in 5% nonfat milk, membranes were incubated with the following primary antibodies: anti–E-cadherin and anti–N-cadherin antibodies from Cell Signaling Technology, Inc. (Danvers, MA); anti-vimentin from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-β-actin from Sigma. Membranes were washed and exposed to peroxidase-conjugated secondary antibodies (Amersham Bioscience, UK). For morphology study, 0.5 × 106 SK-Hep-1 cells transduced by miR-194 virus or control were seeded on a 10-cm dish and incubated at 37°C with 5% CO2 for 36 hours. For cell proliferation study, 5 × 103 SK-Hep-1 cells were seeded in each well of a 96-well plate.