Figure 5

Stable miR-342 expression alters gene expression and sensitizes MCF-7/HER2Δ16 cells to tamoxifen. (A) Three independent total RNA samples purified from MCF-7/HER2Δ16 (Parental), stable MCF-7/HER2Δ16 cell lines expressing pCMV-puro-NC (Vector) or pCMV-miR-342 (miR-342) cultured for 48 hr in CS-FBS MEM were analyzed for miR-342 expression by qRT-PCR. (B) Each indicated cell line was cultured for 24 hr in 5% CS-FBS MEM and treated for 96 hr with 100 pM of E2 alone or in combination with 1.0 μM or 5 μM TAM. Each MTT experiment was repeated three times and the data is represented as the mean +/- SE relative to the E2 treated sample. Asterisks indicates significant changes (p < 0.04). (C) Heat map of microarray expression analysis of genes significantly altered (p < 0.001) by at least 1.5 fold in stable miR-342 expressing MCF-7/HER2Δ16 cells. (D) Microarray validation by qRT-PCR of three suppressed miR-342 direct target genes (SEMAD, BMP7, GEMIN4) and an upregulated indirect miR-342 target gene (TXNIP), normalized to β-actin, and expressed relative to parental MCF-7/HER2Δ16 cells. Asterisks indicate significant differences (p < 0.05). (E) MiR-342 inhibition of GEMIN4 and BMP7 3' UTRs. MCF-7 cells were transfected with 20 nM of hsa-miR-342-3p (Ambion) or pre-miR negative control (Ambion) followed by pMir target firefly luciferase reporter plasmid containing 3' UTR sequences from BMP7 or GEMIN4 (Origene) and a renilla luciferase expression vector. At 48 hrs post-transfection cells were analyzed using the Dual Luciferase Assay Kit (Promega) according to the manufacturer's instructions. Each sample was prepared in duplicate and the entire experiment was repeated three times. Data represents mean +/- SE percent inhibition of luciferase activity of miR-342 transfected cells relative to pre-miR negative control.