Loss of SMARCA4 expression or activity derepresses miR-27a.

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Loss of SMARCA4 expression or activity derepresses miR-27a. Loss of SMARCA4 expression or activity derepresses miR-27a. (A) Differential expression analysis by RNA-seq for genes associated with chromatin-modifying complexes in Rh30 and U23674 cells after treatment for 72 hours with ENT (1 μM) or PAN (45 nM) relative to cells treated with DMSO. SMARCA4 average transcripts per million for DMSO-treated cells, ENT-treated cells, and PAN-treated cells were 86.86, 56.15, and 72.19, respectively. (B) SMARCA4 protein abundance in Rh30 and U23674 aRMS cultures treated with ENT or PAN. Blots are representative of n = 3 independent experiments. (C) SMARCA4 protein abundance in Rh30 cells transfected with HDAC2- or HDAC3-targeted siRNA (100 nM for 72 hours). Blots are representative of n = 3 independent experiments. (D) PAX3:FOXO1 protein abundance in Rh30 cells transfected with SMARCA4-targeted siRNA (100 nM for 72 hours). Blots are representative of n = 3 independent experiments. (E) Quantitative polymerase chain reaction (qPCR) analysis of miR-27a expression in Rh30 cells treated with the SMARCA4 bromodomain inhibitor PFI-3 (10 μM for 24 hours). (F) qPCR analysis of miR-485 in Rh30 cells treated with PFI-3. **P < 0.01, two-sided Student’s t test. (G and H) qPCR analysis of miR-27a expression in Rh30 and CF-1 cells transfected with SMARCA4-targeted siRNA (100 nM for 72 hours). Data were normalized to the expression of U6snRNA. Gene expression was quantified using the 2−ΔCt method. Data are means ± SD (n = 3 independent experiments each in triplicate). *P < 0.05, two-sided Student’s t test. (I) Diagrammatic representation of how ENT and PFI-3 disrupt SMARCA4-mediated interference of miR-27a transcription. Narendra Bharathy et al., Sci. Signal. 2018;11:eaau7632 ©2018 by American Association for the Advancement of Science