Emerging evidence indicates that microRNAs (miRNAs) have important roles in regulating osteogenic differentiation and bone formation. Thus far, no study has established the pathophysiological role for miRNAs identified in human osteoporotic bone specimens. Here we found that elevated miR-214 levels correlated with a lower degree of bone formation in bone specimens from aged patients with fractures. We also found that osteoblast-specific manipulation of miR-214 levels by miR-214 antagomir treatment in miR-214 transgenic, ovariectomized, or hindlimb-unloaded mice revealed an inhibitory role of miR-214 in regulating bone formation. Further, in vitro osteoblast activity and matrix mineralization were promoted by antagomir-214 and decreased by agomir-214, and miR-214 directly targeted ATF4 to inhibit osteoblast activity. These data suggest that miR-214 has a crucial role in suppressing bone formation and that miR-214 inhibition in osteoblasts may be a potential anabolic strategy for ameliorating osteoporosis.

Metabolic skeletal disorders associated with impaired bone formation are a major clinical challenge. One approach to treat these defects is to silence bone-formation-inhibitory genes by small interference RNAs (siRNAs) in osteogenic-lineage cells that occupy the niche surrounding the bone-formation surfaces. We developed a targeting system involving dioleoyl trimethylammonium propane (DOTAP)-based cationic liposomes attached to six repetitive sequences of aspartate, serine, serine ((AspSerSer)(6)) for delivering siRNAs specifically to bone-formation surfaces. Using this system, we encapsulated an osteogenic siRNA that targets casein kinase-2 interacting protein-1 (encoded by Plekho1, also known as Plekho1). In vivo systemic delivery of Plekho1 siRNA in rats using our system resulted in the selective enrichment of the siRNAs in osteogenic cells and the subsequent depletion of Plekho1. A bioimaging analysis further showed that this approach markedly promoted bone formation, enhanced the bone micro-architecture and increased the bone mass in both healthy and osteoporotic rats. These results indicate (AspSerSer)(6)-liposome as a promising targeted delivery system for RNA interference-based bone anabolic therapy.

Currently, major concerns about the safety and efficacy of RNA interference (RNAi)-based bone anabolic strategies still exist because of the lack of direct osteoblast-specific delivery systems for osteogenic siRNAs. Here we screened the aptamer CH6 by cell-SELEX, specifically targeting both rat and human osteoblasts, and then we developed CH6 aptamer–functionalized lipid nanoparticles (LNPs) encapsulating osteogenic pleckstrin homology domain-containing family O member 1 (Plekho1) siRNA (CH6-LNPs-siRNA). Our results showed that CH6 facilitated in vitro osteoblast-selective uptake of Plekho1 siRNA, mainly via macropinocytosis, and boosted in vivo osteoblast-specific Plekho1 gene silencing, which promoted bone formation, improved bone microarchitecture, increased bone mass and enhanced mechanical properties in both osteopenic and healthy rodents. These results indicate that osteoblast-specific aptamer-functionalized LNPs could act as a new RNAi-based bone anabolic strategy, advancing the targeted delivery selectivity of osteogenic siRNAs from the tissue level to the cellular level.

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