| Objective To investigate the regulatory role and molecular mechanism of extracellular vesicle (Exo) derived circular RNA (circRNAs) ITCH in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods Exosomes derived from BMSCs from ovariectomized rats (OVX-Exo) and sham-operated rats (Sham-Exo) were co-cultured with macrophages derived from bone marrow to study their effects on osteoclast differentiation. In order to clarify the effect of ITCH on osteogenic differentiation of BMSCs, BMSCs were divided into Sham-Exo group, OVX-Exo group, OVX-Exo+mimic-NC group and OVX-Exo+ITCH mimic group. In order to confirm the potential of ITC to promote osteoblast proliferation and differentiation by inhibiting the expression of miR-23a-5p, BMSCs were divided into OVX-Exo+ITCH+mimic-NC group and OVX-Exo+ITCH+miR-23a-5p mimic group. The expression of ITCH and miR-23a-5p in BMSCs was determined by quantitative real-time PCR (qRT-PCR). Cell proliferation, calcified nodules and cell activity were detected by CCK-8, alizarin red staining and ALP staining respectively. Results Compared with the Sham-Exo group, the number of TRAP positive cells and the mRNA expression of osteoclast markers in OVX-Exo group increased significantly (P<0.05). The expression level of ITCH mRNA in OVX-Exo was lower than that in Sham-Exo(P< 0.01). During the osteogenic differentiation of BMSCs, the expression of ITCH increased significantly on the 5th, 7th and 9th day after osteogenic induction (P<0.01). Compared with Sham-Exo group, the proliferation of BMSCs in OVX-Exo group and OVX-Exo+mimic-NC group decreased significantly (P<0.05), while the proliferation of BMSCs in OVX-Exo+ITCH mimic group was higher than that in OVX-Exo+mimic-NC group (P<0.05). The ALP activity and mineral deposition of BMSCs in OVX-Exo group and OVX-Exo+mimic-NC group were lower than those in Sham-Exo group (P< 0.05), while the ALP activity and mineral deposition of BMSCs in OVX-Exo+ITCH mimic group were significantly higher than those in OVX-Exo+mimic-NC group (P<0.05). MiR-23a-5p mimic significantly reduced the luciferase activity of ITC-WT reporter vector, and miR-23a-5p inhibitor significantly increased the luciferase activity of ITC-WT reporter vector. Compared with OVX-Exo+ITCH mimic+mimic-NC, the proliferation, ALP activity and mineral deposition of BMSCs in OVX-EXO+Itchmimic+Mir-23a-5p Mimic group decreased significantly (P<0.05). Conclusion ITCH plays an important role in mediating Exo to regulate bone formation and bone resorption, and its overexpression promotes osteogenic differentiation of BMSCs through sponge miR-23a-5p. |