microRNA-101a enhances trabecular bone accrual in male mice

High-throughput microRNA sequencing was used to identify microRNAs (miRs) that regulate osteoblast differentiation. We establish that miR-101a, which targets the epigenetic enzyme Ezh2, is up-regulated in differentiated MC3T3 cells and robustly expressed in mouse calvaria. Our previous studies showed that loss of Ezh2 function enhances osteogenesis and bone formation. Thus, we tested the attractive mechanistic model that increased miR-101s expression may suppress Ezh2 to stimulate the osteogenic process. Transient miR-101a over-expression suppresses Ezh2 levels and reduces tri-methylation on lysine 27 of histone 3 (H3K27me3), a heterochromatic mark catalyzed by Ezh2. Importantly, over-expression of miR-101a stimulates osteogenic differentiation of MC3T3 cells as quantified by alizarin red staining. Therefore, we examined skeletal phenotypes of mice transgenic for miR-101a under the control of Col1a1promoer and doxycycline administration. Experimental controls and mir-101 over-expressing mice were exposed to doxycycline during pregnancy and postnatal stages (phenotyping at 8 weeks) to maximize penetrance of skeletal phenotypes. Our analyses revealed that miR-101a over-expressing male mice are bigger as measured by total body weight and exhibit an increase in long bone length. These mice exhibit significant increases in trabecular bone volume fraction, trabecular number, trabecular thickness, as well as a reduction in trabecular spacing based on microCT analysis. Histomorphometric examination established a significant reduction in osteoid volume to bone volume and osteoid surface to bone surface. These findings suggest that the enhancement in trabecular bone may be due to accelerated bone mineralization in miR-101a over-expressing male mice. Remarkably, while female mice exhibit a significant increase in bone length, no significant changes were noted by microCT (trabecular bone parameters) and histomorphometry (osteoid parameters). We conclude that miR-101a upregulation during osteoblast maturation enhances trabecular bone parameters in male mice. Thus, miR-101 and Ezh2 may form part of an intricate epigenetic feed-forward mechanisms to regulate bone formation. MC3T3 E1 sc4 murine calvarial osteoblasts were purchased from American Type Culture Collection and maintained in α-minimal essential medium without ascorbic acid (Gibco) containing 10% fetal bovine serum (Atlanta Biologicals), 100 units/ml penicillin (Gibco), and 100 μg/ml streptomycin (Gibco) in cell culture incubators (37oC and 5% CO2). Cells were plated at 10,000 cells/cm2 in maintenance medium. Osteogenic differentiation was induced the next day by the addition of 50μg/ml ascorbic acid (Sigma) and 4mM β-glycerol phosphate (Sigma). Osteogenic medium was replaced every two to three days. RNA was harvested in QIAzol and stored at -80oC until further processing. Parietal and frontal bones (calvaria) were harvested from 2-3 days old C57/B6 Prrx1-Cre+ female mice, washed in cold phosphate buffered saline (PBS), incubated in collagenase digestion medium (αMEM, 2mg/ml Collagenase type II, and 0.005% trypsin) for 20 minutes at 37oC, washed in PBS, snap-frozen in liquid nitrogen, and stored at -80oC until further processing. Before isolation, calvaria (2 to 3 day old mice) were homogenized for 30 seconds in ice-cold QIAzol using the Ultra Turrax T25 tissue homogenizer (IKA). Total RNA from MC3T3 cells and calvaria was isolated using the Direct-zol™ RNA kit (Zymo Research). RNA was quantified using the NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific). For MC3T3 samples, three biological samples were pooled (equal amounts of RNA) for each time point assessed (days 0, 6, 10, 17, and 24).