MICRORNA-301A TARGETS DICER TO ATTENTUATE PRIMARY C-KIT+ HUMAN ATRIAL CELL DIFFERENTIATION

Abstract

Cardiac fibroblasts (CFs) and bone marrow-derived mesenchymal progenitor cells (BM-MPCs) both contribute to cardiac fibrosis by differentiating into pro-fibrotic cell types. These pro-fibrotic cells can exacerbate fibrosis by producing excess extracellular matrix proteins that cause the scar to infiltrate viable cardiac muscle that can lead to congestive heart failure. We have previously identified microRNAs (miR) as key regulators of differentiation. These 22-25 nucleotide long non-coding segments of RNA target multiple mRNA transcripts simultaneously to prevent translation of their respective proteins. We found that miR-301a in particular is influential in attenuating the differentiation of both primary CFs isolated from atrial tissue, and primary BM-MPCs, both isolated from patients undergoing open heart surgery. One of the potential targets of miR-301a is Dicer, a protein responsible for processing miRNA and facilitating its inhibitory action. We sought to determine if miR-301a had a similar effect on the recently discovered c-kit+ adult cardiac stem cells (CSCs). C-kit+ adult cardiac stem cells (CSCs) were isolated from atrial appendages isolated from patients undergoing open heart surgery. Atrial tissue was digested using collagenase in SMEM media, and then cells were isolated and co-incubated for 2 hrs at 37°C with Dynabeads coated with c-kit antibody. These magnetic beads were washed to remove c-kit- cells, and then remaining c-kit+ cells were plated. At 70% confluency, cells were transfected with miR-301a or scrambled negative control, and then protein and RNA was collected 48hrs later. We performed qRT-PCR and Western blot analysis to investigate Dicer expression and expression of pro-fibrotic markers. miR-301a transfection of c-kit+ CSCs significantly reduced expression of both Dicer1a and Dicer 1b. In addition, miR-301a over-expression decreased the mRNA level of myosin heavy chain 9, and in the protein levels of non-muscle myosin IIA and alpha-smooth muscle actin. These results provide insight into a potential cellular mechanism that influences the differentiation of AFs, BM-MPCs, and c-kit+ CSCs which could be caused by changes in Dicer, the key protein in activating micro-RNAs.