Pre mir 145

miRNA overexpression was achieved by transfecting cells with a miRNA mimic, a synthetic RNA oligonucleotide duplex mimicking the miRNA precursor. Synthetic RNA molecules, including pre-miR-143, pre-miR-145 and a scrambled negative control RNA (pre-miR-control), were purchased from GenePharma (Shanghai, China). Caco2, HT29 and SW480 cells were seeded in 6-well plates and transfected using Lipofectamine 2000 (Invitrogen) on the following day when the cells were approximately 70% confluent. For the miRNA overexpression experiments, 100 pmol of pre-miR-143, 100 pmol of pre-miR-145 or 50 pmol of both pre-miR-143 and pre-miR-145 were used. At 6 h after transfection, the medium for the Caco2 cells was changed to DMEM supplemented with 2% fetal bovine serum, and the medium for the HT29 and SW480 cells was changed to RPMI-1640 supplemented with 2% fetal bovine serum. The cells were harvested at 24 h or 48 h post-transfection for the isolation of total RNA or protein, respectively.

miRNA overexpression was achieved by transfecting cells with a miRNA mimic (a synthetic RNA oligonucleotide duplex mimicking miRNA precursor), whereas knockdown was achieved by transfecting cells with a miRNA inhibitor (a chemically modified single-stranded antisense oligonucleotide designed to specifically target mature miRNA). Synthetic RNA molecules, including pre-miR-143, pre-miR-145, anti-miR-143, anti-miR-145 and scrambled negative control RNA (pre-miR-control and anti-miR-control), were purchased from GenePharma (Shanghai, China). MCF-7 cells were seeded in 6-well plates and transfected with Lipofectamine 2000 (Invitrogen) on the following day when the cells were approximately 70% confluent. For overexpression of miRNAs, 100 pmol of pre-miR-143, 100 pmol of pre-miR-145 or 50 pmol of both pre-miR-143 and pre-miR-145 were used. For knockdown of miRNAs, 100 pmol of anti-miR-143, 100 pmol of anti-miR-145 or 50 pmol of both anti-miR-143 and anti-miR-145 were used. After 6 h, the medium was changed to DMEM that was supplemented with 2% fetal bovine serum. The cells were harvested 24 h or 48 h after the transfection for the isolation of total RNA or protein, respectively.