Pre mir 19b

A luciferase reporter assay was performed as previously described39 (link) to test miR-19b binding to its target gene, human SOCS3. The entire human SOCS3 3’-UTR, containing a presumptive miR-19b complementary site (seed sequence, UUUGCAC), was PCR-amplified with the following primers:
sense: 5’-TGGGAGCTCAATGTCAGCCCAGTAAGTATTGGCCAGT-3’; antisense: 5’-GATAAGCTT-GTGCTCTTTATTATAAATTACTGAAATGTTTC-3’. Human genomic DNA was used as a template. PCR products were cloned into the pMIR-REPORT plasmid (Ambion), and insertion was confirmed by sequencing. To test binding specificity, the miR-19b seed sequence was mutated from UUUGCAC to AAACGTG. For luciferase reporter assays, 2 μg firefly luciferase reporter plasmid, 2 μg β-galactosidase (β-gal) expression vector (Ambion), and 100 pmol pre-miR-19b (GenePharma, Shanghai, China), anti-miR-19b (GenePharma), and scrambled ncRNA (GenePharma) were transfected into Caco2 cells or HT29 cells in 6-well plates using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The β-gal vector was used as a control. Twenty-four hours after transfection, luciferase assays were performed (Promega, Madison, WI, USA). The reported data represent three independent experiments.

miR-19b was overexpressed using pre-miR-19b, whereas knockdown was achieved with anti-miR-19b. Synthetic pre-miR-19b and anti-miR-19b and scrambled negative control RNA were purchased from GenePharma (Shanghai, China). Caco2 cells or HT29 cells were seeded in 6-well plates and transfected with Lipofectamine 2000 (Invitrogen) when the cells reached 70% confluence. In miR-19b overexpression experiments, 100 pmol of pre-miR-19b or ncRNA was added. For miR-19b knockdown, 100 pmol of anti-miR-19b was used. After 6 h, the medium was changed to DMEM or McCoy’s 5 A supplemented with 1% FBS. Cells were harvested 24 h or 48 h after transfection. The miR-19b expression level was analysed by quantitative RT-PCR, and the SOCS3 protein level was assessed by Western blot. Protein samples were normalized against α-tubulin.

MiRNA overexpression was achieved by transfecting the cells with a miRNA mimic (a synthetic RNA oligonucleotide duplex mimicking the miRNA precursor sequence), whereas knockdown was achieved by transfecting the cells with a miRNA inhibitor (a chemically modified, single-stranded antisense oligonucleotide designed to specifically target a mature miRNA). Synthetic RNA molecules, including pre- miR- 19b and a scrambled negative control RNA (pre- miR-control and anti-miR- control), were purchased from GenePharma (Shanghai, China). The MCF-7 and MDA-231 cells were seeded on 6-well plates and transfected with Lipofectamine 2000 (Invitrogen) the next day when the cells were approximately 70% confluent. For the overexpression experiments of miRNA, 100 pmol of pre-miR-19b was used, and for the knockdown experiments of miRNA, 100 pmol of anti-miR-19b was used. After 6 h, the medium of the MCF-7 and MDA-231 cells was changed to DMEM or L-15, respectively, supplemented with 2% fetal bovine serum. The cells were harvested at 24 h and 48 h after transfection for the isolation of total RNA and protein, respectively.