A MicroRNA-Transcription Factor Blueprint for Early Atrial Arrhythmogenic Remodeling

Figure 5

MicroRNAs which can be potentially responsible for the downregulation of Pitx2, Tbx5, and Myocd expression in atrial myocardium after pacing. (a) miRNA:mRNA alignments. Shown are the schematics of porcine Pitx2 3′-UTR sequence targeted by miR-21, porcine Tbx5 3′-UTR sequence targeted by miR-10a and miR-10b, and human Myocd 3′-UTR sequence targeted by miR-1. The porcine Tbx5 3′-UTR was identified within the pig genomic chromosome-14 sequence (NC_010456.4), starting from nt 40259322. (b) Overall relative levels of Pitx2c (grey boxes) and Mef2c (white boxes) transcripts in HL-1 cells transfected with 50 nM miR-21 mimic, anti-miR-21 inhibitor, and FAM-labeled pre-miR negative control (Con). The results of qRT-PCR analysis are shown. Data from 3 replicates of each transfection were pooled and averaged. . (c) Control and transfected cells were pooled (from triplicate wells in each setup), lysed, electrophoresed, and immunoblotted with antibodies against PITX2A,B,C. MW values (kDa) of the bands detected are shown. (d) Membrane stained with Amido Black 10B. (e) The histogram demonstrating inverse expression correlations of selected miRNA/target gene pairs in paced pigs as revealed by qRT-PCR (miR-21, miR-10b, and miR-1) and Western blot (PITX2C, TBX5, and MYOCD). See text for details.