Pre mir mirna precursor

Transfection was conducted using Lipofectamine 2000 (Invitrogen), negative control Pre-miR, Pre-miR29, Pre-miR-7f (50 nM) and negative control Anti-miR, Anti-miR29 and Anti-miR-7f (100 nM) (Ambion Pre-miR miRNA Precursors and Ambion Anti-miR miRNA Inhibitors, respectively).

HCT116/GFP or DLD-1/GFP (2 × 10⁵ cells)(11 (link)) were transfected with 6 pmol siRNA (Qiagen; Invitrogen, Maryland, MD, USA) or 4 pmol miRNA mimics (pre-miR miRNA precursors; Ambion, Carlsbad, CA, USA) in the presence of 20 μL HiPerFect (Qiagen, Hilden, Germany) for 2 days. Subsequently, the GFP-expressing cells were mixed with HCT116/red fluorescent protein (RFP) or DLD-1/RFP at a 1:1 ratio, suspended in normal growth medium containing 50% Matrigel (BD Biosciences, Bedford, MA, USA), and liver metastasis assays were carried out as previously described.(11 (link)) A fraction of the cell mixture before the injection was evaluated by flow cytometry to measure original ratios of GFP-expressing cells/RFP-expressing cells. Two to 10 days after splenic injection, the inhibitory effects of siRNA or miRNA on liver metastasis were evaluated by counting ratios of GFP-positive foci versus RFP-positive foci by in vivo imaging (OV110; Olympus, Tokyo, Japan), or by measuring the ratios of the number of GFP- or RFP-expressing cells by flow cytometry (FACSCalibur; BD Biosciences). All mouse procedures were carried out according to the Guidelines for Animal Experiments, approved by the committee for ethics of animal experimentation of the National Cancer Center (Tokyo, Japan), and carried out in accordance with institutional policies.