Expression profiling of mitochondria-associated microRNAs during osteogenic differentiation of human MSCs

The goal of this study was to determine expression profiles of mitochondria-associated microRNAs (mitomiR) in human bone marrow-derived mesenchymal stem/stromal cells (MSCs) as well as in MSCs during osteogenic differentiation. MicroRNAs are epigenetic regulators that commonly function by targeting specific mRNAs resulting in suppression of protein expression. In addition to their location in the cytosol, microRNAs have also been found in other sub-cellular compartments including the mitochondria. While some studies suggest that mitomiRs may affect mitochondrial function, research on mitomiRs is still in its infancy. To date, there is no information on mitomiR expression in MSCs or osteoblasts. A standard in vitro osteogenesis assay system was used to differentiate MSCs toward the osteoblast lineage. Purified mitochondrial extracts were obtained from MSCs or from MSCs at specific time points of osteogenic induction. RNA was isolated from mitochondrial extracts and then biotin-labeled in preparation for microRNA array (Affymetrix miRNA array 4.0). Array data was analyzed to generate information on most abundantly-expressed mitomiRs in non-induced MSCs as well as in MSCs at set time points (day 3, 7, or 14) of osteogenic induction. Information on significantly differentially-expressed mitomiRs during osteogenesis (comparing day 0 with either day 3, 7 or 14) was also obtained. In this study, 3 independent MSC cell lines were used. Each MSC cell line was differentiated toward the osteoblast lineage and mitochondrial RNA was isolated from MSCs at day 3, 7, or 14 of differentiation. As a control, mitochondrial RNA from non-induced MSCs (day 0) was also collected, Therefore, 12 samples in total were analyzed.