1Department of Orthopaedics, The Third Xiangya Hospital of Central South University, Changsha, China. 2Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. 3Department of Anatomy, Histology and Embryology, Changsha Medical University, Changsha, China. 4Four Gynecological Wards, Ningbo Women & Children’s Hospital, Ningbo, Zhejiang, China. 5The Second Xiangya Hospital of Central South University, Changsha, China. 6School of Basic Medical Science, Central South University, Changsha, China.
The switch between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) plays a key role in aging-induced osteoporosis. In this study, miR-19a-3p was obviously downregulated in BMSCs from aged humans and mice. Overexpressed miR-19a-3p evidently reduced aging-induced bone loss in mice and promoted osteogenic differentiation of BMSCs, while silenced miR-19a-3p manifestly increased aging-induced bone loss in mice and repressed osteogenic differentiation of BMSCs. Hoxa5 was significantly downregulated in the BMSCs from aged mice and contribute to miR-19a-3p-induced osteoblast differentiation as a direct target gene of miR-19a-3p. Furthermore, lncRNA Xist was found as a sponge of miR-19a-3p to repress BMSCs osteogenic differentiation. In conclusion, our study reveals the critical role of the lncRNA Xist/miR-19a-3p/Hoxa5 pathway in aging-induced osteogenic differentiation of BMSCs, indicating the potential therapeutic target for osteoporosis.
Figure 1. Aging induces miR-19a-3p expression in BMSCs. A The miR-19a-3p expression in BMSCs from mice with different ages. B, The relationship between miR-19a-3p expression with age in BMSCs from 61 human females and C, 62 males. Data shown as mean ± SD. *p < 0.05.
Figure 2. The age-associated bone loss in miR-19a-3p Tg mice. (A) The μCT images of WT mice and miR-19a-3p Tg mice at 1, 3 and 12 months old. B-E, quantitative μCT analysis of trabecular bone microarchitecture. (B) Tb. BV/TV, trabecular bone volume per tissue volume. (C) Tb.N, trabecular number. (D) Tb.Th, trabecular thickness. E, Tb.Sp, trabecular separation. (F, G) Representative images of calcein double labelling of trabecular bone, (G) with quantiﬁcation of BFR per bone surface BFR/BS, H, and mineral apposition rate MAR. Data shown as mean ± SD, *p <0.05.
Figure 3. Age-associated bone loss in miR-19a-3p knockout mice. (A) μCT images of WT mice and miR-19a-3p -/- mice at 1, 3 and 12 months old. B-E, quantitative the trabecular bone microarchitecture. (B) Tb. BV/TV, trabecular bone volume per tissue volume. (C) Tb.N, trabecular number. (D) Tb.Th, trabecular thickness. (E) Tb.Sp, trabecular separation. (F, G) quantification of BFR/BS and MAR. (F) with quantiﬁcation of BFR per bone surface (BFR/BS); (G) mineral apposition rate (MAR). Data shown as mean ± SD. *p <0.05.
Figure 4. miR-19a-3p regulates the osteogenic differentiation in BMSCs. (A) miR-19a-3p expression in the BMSCs during osteogenic differentiation. (B) The antagomiR-19a-3p or agomiR-19a-3p were used to silence or overexpress miR-19a-3p in BMSCs. (C, D) Alizarin Red staining revealed the role of miR-19a-3p on the osteogenic differentiation of BMSCs. Scale bars: 100 μm. (E) ALP activity, (F) osteocalcin secretion, (G) RUNX2 and H, osterix expression in BMSCs. Data shown as mean ± SD. *p <0.05.
Figure 5. miR-19a-3p regulated Hoxa5 expression by targeting the 3’UTR of Hoxa5 directly. (A) the predict binding role of miR-19a-3p on 3′-UTR of Hoxa5. (B) luciferase reporter assay revealed the target role of miR-19a-3p on Hoxa5. (C) miR-19a-3p regulated Hoxa5 expression. (D) Hoxa5 expression was increased in BMSCs of the aging mice. (E, F) The role of Hoxa5 and miR-19a-3p on ALP activity of BMSCs. (G, F) The role of Hoxa5 and miR-19a-3p on osteocalcin secretion of BMSCs. Data shown as mean ± SD. *p <0.05.
Figure 6. LncRNA Xist was involved osteoblast differentiation via miR-19a-3p/Hoxa5 pathway. (A) Xist expression in BMSCs from mice with different age. (B) RNA FISH assay for miR-19a-3p and Xist. Scale bar=10 µm. (C) BMSCs were incubated with biotin-labeled miR-19a-3p and Xist, and qPCR analyses revealed the production of pulled down. (D) RIP assay to reveal the relationship between miR-19a-3p and Xist. (E) Luciferase reporter assay revealed the target role of miR-19a-3p and Xist. (F) The role of Xist and miR-19a-3p on Hoxa5 expression in BMSCs. (G) The effects of Xist and miR-19a-3p on ALP activity. H, The role of Xist and miR-19a-3p on osteocalcin secretion in BMSCs. Data shown as mean ± SD. *p <0.05.
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