Abstract
Previously, we developed a novel microRNA (miRNA) delivery system based on bacteriophage MS2 virus-like particles (MS2 VLPs). In this current study, we used this system to transport miR-146a into human peripheral blood mononuclear cells (PBMCs), and demonstrated the inhibition of osteoclastogenesis in precursors. Two cytokines, receptor activator of NF-κB ligand (RANKL), and macrophage-colony stimulating factor (M-CSF) were used to induce osteoclastogenesis. MS2 VLPs were transfected into PBMCs. qRT-PCR was applied to measure expression levels of miR-146a and osteoclast (OC)-specific genes. Western blot (WB) was conducted to evaluate miR-146a downstream target proteins: epidermal growth factor receptor (EGFR) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). The formation and activity of OCs were assessed by cytochemical staining and bone resorption assay, respectively. In PBMCs treated with MS2-miR146a VLPs, qRT-PCR assays showed increased expression of miR-146a (p < 0.01) and decreased expression of all four OC-specific genes (p < 0.05). WB results indicated decreased expression of EGFR (p < 0.01) and TRAF6 (p < 0.05). The number of OCs decreased markedly and bone resorption assay demonstrated inhibited activity. This miR-146a delivery system could be applied to induce overexpression of miR-146a and to inhibit the differentiation and function of OCs.
Highlights
Bone remodeling is a continuous process kept in balance by two types of cells: bone-forming osteoblasts and bone-resorbing osteoclasts (OCs)
It is clear that this delivery system is capable of upregulating the expression of miR-146a in human peripheral blood mononuclear cells (PBMCs)
Compared to the negative control group, the experimental groups treated with MS2-miR-146a VLPs displayed apparently downregulated expression of epidermal growth factor receptor (EGFR) and TRAF6 (p < 0.01 for EGFR and p < 0.05 for TRAF6; Figure 2)
Summary
Bone remodeling is a continuous process kept in balance by two types of cells: bone-forming osteoblasts and bone-resorbing osteoclasts (OCs). OCs, the major cells for bone resorption, play a pivotal role in the development and progression of bone loss [3,4]. Some miRNAs play critical roles in immunoregulation, and are involved in the pathogenesis of osteoporosis or RA [2,8,9,10]. It has been reported that miR-146a, a negative regulator of autoimmunity [15,16], plays a role in osteoclastogenesis. We used the monocyte/macrophage lineage of human PBMCs as OC precursors [23,24] to demonstrate that this system can be applied for the upregulation of miR-146a and inhibition of osteoclastogenesis. As expected, upregulated miR-146a displayed an inhibitory role and there was a marked decrease in the expression of downstream proteins and OC-specified marker genes. Tartrate-resistant acid phosphatase (TRAP) staining and pits formation assay demonstrated the inhibition of OC formation and activity
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