1. HDAC1 positively regulates the abundance of miRNAs.
HDAC1 positively regulates the abundance of miRNAs. (A) We took advantage of the lentiviral expression system to achieve HDAC1 overexpression and knockdown in HEK293 and K562 cells, respectively. After 48 h, we sorted transduced cells using flow cytometry, isolated total cellular RNAs, including small RNAs, and subjected them to miRNA expression profiling using microarrays. Left panel: the y axis in the upper panel indicates the percentage of miRNAs affected by HDAC1 overexpression and knockdown. We defined >1.5‐fold increase and >50% reduction as upregulation and downregulation, respectively. Representative results of the miRNA set with an expression cutoff value >100 are shown. We obtained the same tendency by analysing miRNA sets with different cutoff values (supplementary Fig S1A online). The efficiency of overexpression and knockdown was monitored by immunoblotting HDAC1 and GAPDH (lower panel). Right panel: we performed linear regression analysis of 78 miRNAs regulated by HDAC1 commonly in HEK293 (x axis) and K562 (y axis) cells. (B) RNA samples isolated as described above were subjected to real‐time quantitative RT–PCR. The data were quantified with the 2−ΔΔCt method using simultaneously amplified U6 snRNA, GAPDH and β‐actin as references. The y axes indicate relative gene expression in HDAC1‐ and siHDAC1‐transduced cells against corresponding Mock‐ and siControl‐transduced cells with the expression levels of the latter being set at 1.0. The means±s.d. (bars) of five independent experiments are shown (*P<0.05 by Student's t‐test). (C) We transfected HEK293 and K562 cells with KAT2B expression vector and lentiviral shRNA expression vectors for p300, respectively, and carried out RT–PCR analysis as described above. The means±s.d. (bars) of three independent experiments are shown (*P<0.05 by Student's t‐test). (D) We cultured normal human CD34+ bone marrow mononuclear cells in serum‐free liquid medium containing appropriate cytokines (Wada et al, 2009). After 24 h, total cellular RNA was isolated and subjected to real‐time quantitative RT–PCR for miRNAs (upper panel) and semiquantitative RT–PCR for HDAC1 (lower panel). The means±s.d. (bars) of three independent experiments are shown (*P<0.05 by Student's t‐test). BMMNC, bone marrow mononuclear cell; Ex, exon; HAT, histone acetyltransferase; HDAC, histone deacetylase; miRNA, micro RNA; pri‐miR, primary transcript; pre‐miR, precursor transcript; shRNA, short hairpin RNA.
Taeko Wada et al. EMBO Rep. 2012;13:
© as stated in the article, figure or figure legend