Toward a novel bone anabolic strategy for aged postmenopausal osteoporosis: Inhibiting miR-214 in osteogenic cells for promoting bone formation

Intermittent injection of recombinant human parathyroid hormone (iPTH) is the only bone anabolic agent clinically approved for stimulating bone formation in aged postmenopausal osteoporosis. However, iPTH is limited to a 2-year treatment period due to increasing bone resorption over bone formation besides other concerns. This limitation has been an incentive to search for alternative bone anabolic drugs.

Emerging evidence indicates that microRNAs play an important role in regulating osteogenic differentiation and osteoblastic bone formation. However, no study has established a pathophysiological role for miRNAs by studying bone samples isolated from patients with reduced bone formation. The applicants found that elevated miR-214 correlated with reduced bone formation in bone specimen from postmenopausal fractured patients and ovariectomized mice (Xiaogang Wang et al. Journal of Orthopeadic Research-Suppl, 2012 Annual Meeting). Further, we found that osteogenic differentiation and matrix mineralization in C2C12 mesenchymal cells or pre-osteoblast cells in vitro were promoted by miR-214 inhibitor (Antagomir-214) and reduced by miR-214 mimics, respectively. Importantly, it was confirmed that miR-214 directly targeted ATF4 (one of the main transcription factor required for osteogenesis) for inhibiting osteoblast function. Impressively, reduced osteoblastic bone formation and decreased ATF4 protein level were found without altered osteoclastogenesis in our established osteoblast-specific miR-214 transgenic mice (Baosheng Guo et al. Journal of Orthopeadic Research-Suppl, 2012 ORS Annual Meeting). Thereafter, we used Antagomir-214 to rescue the osteoblast-specific miR-214 transgenic mice. Not unexpectedly, the bone phenotype after the rescue treatment was toward that of the corresponding wild type (Xiaogang Wang et al. Under review by Nature Medicine, NMED-A57387). Accordingly, we have the following hypothesis that inhibiting miR-214 in osteogenic cells might promote bone formation in aged postmenopausal osteoporosis.

Recently, the applicants have developed a targeted delivery system for ribonucleic acid specifically approaching osteogenic cells (Zhang G et al. Accept by Nature Medicine 2011). It means we could specifically deliver Antagomir-214 to osteogenic cells at various stages to realize cell-specific inhibition of miR-214 by employing the targeted delivery system encapsulating Antagomir-214. On the other hand, an aged ovariectomized rat model has been regarded as a golden one for evaluating bone anabolic agent. So, it strongly facilitated the applicants to test the above hypothesis using the targeted delivery system with Antagomir-214 by achieving the following five specific aims in the aged ovariectomized rats: (1) To investigate the effect of miR-214 inhibition on intraosseous miR-214 expression/bone formation related gene expression by Q-PCR and ATF4 protein expression by western blot; (2) To examine the effect of miR-214 inhibition on bone formation by bone histomorphometry analysis; (3) To quantify the effect of miR-214 inhibition on bone mass and bone structure by microCT analysis; (4) To evaluate the effect of miR-214 inhibition on bone mechanical properties by mechanical testing; (5) To test the safety of miR-214 inhibition on non-skeletal organs (liver and kidney).

This proposal would help establish miR-214’s role as a pivotal negative regulator of bone formation in the aged skeleton, and also would provide new insight that therapeutically inhibiting miR-214 in osteogenic cells may be a novel bone anabolic strategy to reverse aged postmenopausal osteoporosis, thereby reducing fracture risk.