Pre mir control

miRNA mimic (a synthetic RNA oligonucleotide duplex mimicking miRNA precursor) was used for the overexpression of miRNA. The synthetic RNA molecules were purchased from GenePharma (Shanghai, China) including pre-miR-200c and pre-miR-control (scrambled negative control RNA). MDA-MB-231 and 4T1 cells seeded in 6-well plates were transfected with Lipofectamine 2000 (Invitrogen) when the cells were nearly 70% confluent. Then 100 pmol of pre-miR-200c was added. 6 h later, changed the culture medium to DMEM supplemented with 2% fetal bovine serum (FBS). 24 h or 48 h after the transfection, harvested the cells for the isolation of total RNA or protein.

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.

miRNA overexpression was achieved by transfecting cells with a miRNA mimic, which is a synthetic RNA oligonucleotide duplex mimicking the miRNA precursor sequence. Synthetic RNA molecules, including pre-miR-135b and a scrambled negative control RNA (pre-miR-control), were purchased from GenePharma (Shanghai, China). HT-29 and SW-480 cells were seeded on 6-well plates and were 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-135b were used. After 6 h, the medium of the HT-29 and SW-480 cells was changed to RPMI-1640 medium 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.

Overexpression of miR-23a/b was achieved by transfecting gastric cancer cells with miRNA mimics (synthetic RNA oligonucleotides mimicking precursors of miR-23a/b). Knockdown of miR-23a/b was achieved by transfecting with miRNA inhibitors (chemically modified single-stranded antisense oligonucleotides designed to specifically sequester mature miR-23a/b). Synthetic miR-23a/b mimics (pre-miR-23a/b), inhibitors (anti-miR-23a/b) and scrambled negative control RNAs (pre-miR-control and anti-miR-control) were purchased from GenePharma (Shanghai, China). MKN-45 and AGS cells were seeded in 6-well plates using RPMI 1640 medium supplemented with 10% FBS. The cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) using Opti-MEM Reduced Serum Medium (Gibco, Carlsbad, CA, USA) on the following day when the cells were ~60–70% confluent. For each well, equal amounts of pre-miR-23a/b, pre-miR-control, anti-miR-23a/b or anti-miR-control were used. After 6 h, the medium was changed to RPMI 1640 supplemented with 2% FBS. The cells were collected at 24 or 48 h after transfection and subjected to analysis by quantitative RT-PCR or western blotting.

miRNA overexpression was achieved by transfecting cells with miRNA mimics, whereas knockdown was achieved by transfecting cells with a miRNA inhibitor according to previous publications42 (link). miRNA overexpression was achieved by transfecting cells with miRNA mimics (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 absorb target miRNA). Synthetic RNA molecules, including pre-miR-221, anti-miR-221 and scrambled negative control RNA (pre-miR-control and anti-miR-control), were purchased from GenePharma (Shanghai, China). MCF-7 or MDA-MB-231 cells were seeded in 6-well plates and transfected with Lipofectamine 2000 (Invitrogen) on the following day when the cells were approximately 70% confluent. Cells were transfected with 50 pmol pre-miR-221 or anti-miR-221 to overexpress or knockdown cellular miR-221. After 6 h, the medium was changed to DMEM or L-15 medium that was supplemented with 2% fetal bovine serum. The cells were harvested 24 or 48 h posttransfection for RNA and protein analysis.

Synthetic RNA molecules, including pre-miR-30a, anti-miR-30a and scrambled negative control RNAs (pre-miR-control and anti-miR-control) were purchased from GenePharma (Shanghai, China). The cells were seeded onto 6-well plates and transfected using Lipofectamine 2000 (Invitrogen) on the following day when the cells were approximately 70% confluent. In each well, equal 100 pmol of pre-miR-30a, anti-miR-30a or scrambled negative control RNA were used. The cells were harvested at 24 h after transfection for quantitative RT-PCR analysis and Western blotting.

miRNA overexpression was achieved by transfecting cells with a pre-miR-10a (a synthetic RNA oligonucleotide duplex mimicking the miRNA precursor), whereas knockdown was achieved by transfecting cells with an anti-miR-10a (a chemically modified single-stranded antisense oligonucleotide designed to specifically target mature miRNA). Synthetic pre-miR-10a, anti-miR-10a, pre-miR-control and anti-miR-control RNAs were purchased from Genepharma (Shanghai, China). Next, 3 × 10⁶ OCI-LY7 or OCI-LY3 cells were seeded per well in 6-well plates and were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocols. The cells were harvested 24 h or 48 h after transfection for quantitative RT-PCR or Western blotting. The transfection efficiency was determined by quantitative RT-PCR for hsa-miR-10a. Transfection experiments were repeated three times independently and in each case were done in triplicate.