Journal: iScience
Article Title: Regulatory BC200 RNA promotes breast carcinogenesis by repressing BRCA1 gene expression
doi: 10.1016/j.isci.2026.115267
Figure Lengend Snippet: BRCA1 cDNA rescues the BC200 RNA-induced phenotype (A) MCF-10A cells were transfected and seeded at low density in an agarose gel. After transfection, the ability of the cells to grow in soft agarose medium was assessed by measuring absorbance at 450 nm using a microtiter plate reader. Consistent with previous observations, MCF-10A cells transfected with 30 nM BC200 RNA exhibited a pronounced growth phenotype. Co-transfection with 30 nM of a DsiRNA specific for BC200 RNA significantly reduced this oncogenic phenotype. Finally, rescue experiments were performed in which BRCA1 expression was restored in BC200 RNA-expressing cells. BC200 RNA exerts its translational repression activity by inhibiting the activities of eIF4A and eIF4B, two translation initiation factors essential for the translation of mRNAs containing higher-order structural elements in their 5′ UTRs. To overcome BC200 RNA-mediated inhibition, cells in these rescue experiments were co-transfected with a plasmid expressing the coding sequence of BRCA1 mRNA. This cDNA lacks the structured 5′ UTR and is therefore not targeted by BC200 RNA-mediated translational repression. The results showed a significant decrease in malignant transformation. Quantitative analysis: one-way ANOVA (∗∗∗ p < 0.001) followed by Dunnett’s multiple comparison analysis (comparison with cells transfected with 30 nM BC200 RNA): ∗∗∗ p < 0.001 for untransfected cells, ∗∗ p = 0.002 for cells transfected with 30 nM BC200 RNA and 30 nM dsiRNA, ∗ p = 0.016 for cells transfected with 30 nM BC200 RNA and eGFP-BRCA1 ( n = 5). Error bars: SD. (B) Levels of BC200 RNA following transfection were measured by RT-qPCR 48 h after transfection with 30 nM wild-type BC200 RNA, co-transfection with 30 nM BC200 RNA and 20 nM BC200 RNA-specific DsiRNA, or co-transfection with 30 nM BC200 RNA and 1.5 μg eGFP-BRCA1 plasmid per culture. BC200 RNA levels were measured and compared with those of untransfected MCF-10A cells. BC200 RNA levels in all samples were normalized to those of untransfected cells ( n = 4). Statistical analysis: one-way ANOVA (∗∗∗∗ p < 0.0001) followed by Dunnett’s multiple comparison analysis (comparison with untransfected cells): ∗∗∗ p < 0.001 for cells transfected with 30 nM BC200 RNA, ∗∗∗ p < 0.001 for cells transfected with 30 nM BC200 RNA and eGFP-BRCA1. ∗∗∗ p < 0.0004 for cells transfected with 30 nM BC200 RNA and DsiRNA vs. cells transfected with 30 nM BC200 RNA. Error bars: SD. (C) Representative photomicrographs show differential expression of eGFP fluorescence in the samples. MCF-10A cells were transfected with 30 nM BC200 RNA, 30 nM BC200 RNA and 20 nM BC200 RNA-specific DsiRNA, or co-transfected with 30 nM BC200 RNA and 1.5 μg eGFP-BRCA1 plasmid per culture. 48 h after transfection, the differential expression of eGFP was examined. Cells were then plated in soft agarose for the transformation assay. The top row of images presents brightfield photomicrographs, while the bottom row of images presents fluorescence photomicrographs documenting differential eGFP expression levels. Only the sample transfected with the eGFP-BRCA1 plasmid shows a fluorescence signal. Scale bar, 100 μm.
Article Snippet: BRCA1 , Cell Signaling Technology , 9010; RRID: AB_2228244.
Techniques: Transfection, Agarose Gel Electrophoresis, Cotransfection, Expressing, Activity Assay, Inhibition, Plasmid Preparation, Sequencing, Transformation Assay, Comparison, Quantitative RT-PCR, Quantitative Proteomics, Fluorescence
Santa Cruz Biotechnology is a verified supplier 
