Inhibition of miR-22 promotes differentiation of osteoblasts and improves bone formation via the YWHAZ pathway in experimental mice

Abstract

Introduction: In senile osteoporosis countering the age-mediated bone loss, promotion of osteoblastogenesis and identification of responsible micro-RNA (miR) would be a successful strategy. Material and methods: miR microarray screening was carried out to identify the suppressed miRs after real time polymerase chain reaction (RT-PCR) analysis in mesenchymal stem cells (MSCs) derived from adult bone marrow during the proliferation to the mineralization stage. The primary calvarial pre-osteoblasts (human) were harvested and received transfection of miR-22's antagomir or agomir in vitro. Bioinformatics study suggested YWHAZ as the favorable target gene. Next, YWHAZ knockdown was studied for its effect on differentiation of osteoblasts. For in vivo studies, ovariectomized or sham mice were injected with miR-22's antagomir for a period of 6 weeks. The stromal cells were isolated in the 6 th week for ex vivo experiments. Results: miR-22 was found to be down-regulated in bone marrow derived mesenchymal stem cells. miR-22's antagomir converted the pre-osteoblasts to a more differentiated and mineralized phenotype showing upregulated protein expression of COL1A1, ALP and CBFA1. The miR-22's antagomir suppressed YWHAZ, enhanced stability of CBFA1 and promoted the differentiation of osteoblasts. In vivo, miR-22's antagomir promoted mineralization and osteoblastogenesis, elevated bone strength and reversed the ovariectomy mediated bone loss in sham mice. Conclusions: Inhibition of miR-22 may be a potential target for treating osteoporosis clinically. The findings hence suggest that inhibition of miR-22 may be an effective anabolic therapeutic approach in treating osteoporosis clinically.

Therapeutic avenues in bone repair: Harnessing an anabolic osteopeptide, PEPITEM, to boost bone growth and prevent bone loss

“… 13 By contrast, small interfering RNA (siRNA) knockdown of 14-3-3ξ (and thus PEPITEM) significantly reduced osteoblastogenesis in vitro —with osteoblast precursors showing diminished expression of maturation markers ( runx2, alp, col1a1, bmp ) resulting in reduced maturation/alkaline phosphatase activity and mineral production. 14 However, and in contrast to our findings with PEPITEM, overexpression of 14-3-3ξ had no impact on BV/TV or trabecular number in resting Balb/c mice over the 6-week time frame of the experiment. 14 Possible explanations for these differences include the use of different strains of mice; the time point chosen for the analysis (2 vs. 6 weeks); the type and bioactivity of treatment (bioactive peptide vs. microRNA antagomir induced up-regulation of 14-3-3ξ and required subsequent proteolytic cleavage).…”

“… 14 However, and in contrast to our findings with PEPITEM, overexpression of 14-3-3ξ had no impact on BV/TV or trabecular number in resting Balb/c mice over the 6-week time frame of the experiment. 14 Possible explanations for these differences include the use of different strains of mice; the time point chosen for the analysis (2 vs. 6 weeks); the type and bioactivity of treatment (bioactive peptide vs. microRNA antagomir induced up-regulation of 14-3-3ξ and required subsequent proteolytic cleavage). Overexpression of 14-3-3ξ was able to partially reverse ovariectomy-induced loss of cancellous bone density and trabecular number over 6 weeks of treatment in Balb/c mice.…”

“…Overexpression of 14-3-3ξ was able to partially reverse ovariectomy-induced loss of cancellous bone density and trabecular number over 6 weeks of treatment in Balb/c mice. 14 Importantly, these changes were comparable to those we observed with only 2 weeks of PEPITEM therapy, suggesting the pre-processed peptide represents a better therapeutic option. This is further supported by the ubiquitous expression pattern and multitude of cellular responses elicited by 14-3-3ξ and its up-regulation in various cancers (reviewed by Obsilova and Obsil 11 ), which collectively make it a poor candidate for drug development.…”

“…12 Of particular relevance to bone homeostasis, two 14-3-3 family members have been reported to differentially regulate osteoblast function, with 14-3-3β 13 significantly reducing and 14-3-3ξ enhancing osteoblastogenesis. 14 We have previously identified a bioactive 14-amino acid peptide (PEPITEM) cleaved from 14-3-3ξ 15 , which regulates the migration of monocytes (osteoclast precursors) into non-bone tissues during inflammation. 16 , 17 Here we investigated the ability of PEPITEM to directly influence bone remodeling under homeostatic conditions and subsequently the therapeutic efficacy of PEPITEM in models of excessive bone loss.…”

pH-activated, mitochondria-targeted, and redox-responsive delivery of paclitaxel nanomicelles to overcome drug resistance and suppress metastasis in lung cancer

“…The expression of P-gp-related genes was analyzed by real-time PCR [ 30 ]. In brief, RNA was extracted using a TRIzol® Plus RNA purification kit (Invitrogen).…”