Silencing miR-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting BMP2  Ke Liu, Ying Jing, Wen Zhang, Xuejie Fu, Huan Zhao, Xichao Zhou, Yunxia Tao, Huilin Yang, Yan Zhang, Ke Zen, Chenyu Zhang, Donghai Li, Qin Shi  Bone  Volume 97, Pages 130-138 (April 2017) DOI: 10.1016/j.bone.2017.01.014 Copyright © 2017 Terms and Conditions

Fig. 1 Osteoblast differentiation of PMSCs. (a) Cell morphology of PMSCs at different time points upon osteo-induction (scale bar 100μm). (b) ALP staining (left) and relative ALP activity (right) of PMSC-derived osteoblasts at day 10. Data of fold change were presented as mean±SD (⁎⁎⁎p=0.000001, n=3 for all experiments). (c) Relative mRNA expression of osteoblast-related genes (ALP, OSX, RUNX2, COL1A1and BMP2) and miR-106b expression of PMSC-derived osteoblasts at day 10 assayed by qRT-PCR. The gene expressions were referenced by glyceraldehyde 3-phosphate dehydrogenase (GADPH) or U6 snRNA. Data of fold change are presented as mean±SD (⁎p=0.0171(ALP), ⁎⁎p=0.00364 (OSX), ⁎p=0.0238 (RUNX2), ⁎⁎p=0.00687 (COL1A1), ⁎p=0.0133 (BMP2) and ⁎p=0.0265 (miR-106b). n=3 for all experiments). Bone 2017 97, 130-138DOI: (10.1016/j.bone.2017.01.014) Copyright © 2017 Terms and Conditions

Fig. 2 MiR-106b inhibits osteoblast differentiation of PMSCs. (a) Relative levels of miR-106b in PMSCs transfected with the miR-106b mimic or inhibitor. Data of fold change are presented as mean±SD (miR-106b mimic versus mimic negative control (mimic NC), ⁎⁎⁎p=0.00042 at day 3, ⁎p=0.0158 at day 6 and ⁎⁎ p=0.00159 at day 10; miR-106b inhibitor versus inhibitor negative control (inhibitor NC), ⁎⁎⁎p=7.65×10−6 at day 3, ⁎⁎⁎p=0.000286 at day 6 and ⁎p=0.047 at day 10, n=3 for all experiments). (b) ALP staining and relative ALP activity assay of PMSC-derived osteoblasts at day 10 after treatment with the miR-106b mimic or inhibitor. For ALP activity, miR-106b mimic versus mimic NC, ⁎p=0.0107; miR-106b inhibitor versus inhibitor NC, ⁎p=0.0205. n=3 for all experiments. (c) qRT-PCR assay of PMSC-derived osteoblasts at day 10 after treatment with the miR-106b mimic or inhibitor. Data of fold change are shown as mean±SD. Treatment with mimic NC versus miR-106b mimic, ⁎⁎p=0.0087 (ALP), ⁎⁎p=0.0025(RUNX2), ⁎⁎p=0.0059 (COL1A1), ⁎⁎p=0.0049 (BMP2); inhibitor NC versus miR-106b inhibitor, ⁎⁎p=0.0093 (ALP), ⁎p=0.0147 (RUNX2), ⁎⁎p=0.0055(COL1A1), ⁎p=0.0104 (BMP2). n=3 for all PCR experiments. Bone 2017 97, 130-138DOI: (10.1016/j.bone.2017.01.014) Copyright © 2017 Terms and Conditions

Fig. 3 MiR-106b regulates bone mass in Dex-induced osteoporotic mice. (a) Micro-CT 3-dimensional (upper) and 2-dimensional (lower) reconstruction of the distal femur. (b) Structural parameters of the distal femur (BMD (mg/mm3), BV/TV (%), Tb.th (μm) and Tb.N (1/mm)). Data are shown as mean±SD. PBS-inhibitor NC versus Dex-inhibitor NC, ⁎⁎⁎ p=0.0000128(BMD), ⁎⁎⁎p=0.0000176(BV/TV), ⁎⁎p=0.00447 (Tb.th) and ⁎⁎p=0.00249 (Tb·N); Dex-inhibitor NC versus Dex-inhibitor, ⁎⁎p=0.0029 (BMD), ⁎⁎p=0.0023 (BV/TV), ⁎p=0.02125 (Tb.th) and ⁎p=0.05(Tb.N). n=5 for all analysis. Bone 2017 97, 130-138DOI: (10.1016/j.bone.2017.01.014) Copyright © 2017 Terms and Conditions

Fig. 4 MiR-106b influences bone formation in Dex-induced osteoporotic mice. (a) Left: H&E staining for the femurs (yellow arrow, osteoblast; scale bar: 100μm). Middle: Masson staining for the femurs (black arrow, collagen; red arrow, the position of the growth plate, scale bar: 100μm). Right: Minocycline staining to detect bone formation rate of each group (scale bar: 100μm). (b) Serum levels of PINP (pg/ml) and CTx-1 (ng/ml) after miR-106b inhibitor treatment. Data are presented as mean±SD. PBS-inhibitor NC versus Dex-inhibitor NC, ⁎⁎⁎p=0.0000496 (PINP) and ⁎⁎p=0.0057 (CTx-1), Dex-inhibitor NC versus Dex-inhibitor, ⁎p=0.0307 (PINP) and ⁎⁎p=0.00235 (CTx-1). n=5 for all ELISA experiments. (c) The relative expressions of miR-106b and osteoblastic genes of the femurs the after miR-106b inhibitor treatment. Data of fold change are presented as mean±SD. PBS-inhibitor NC versus PBS-inhibitor, ⁎⁎p=0.0019 (miR-106b) and ⁎⁎p=0.0044 (BMP2);PBS- inhibitor NC versus Dex-inhibitor NC, ⁎p=0.04865 (miR-106b), ⁎⁎p=0.00255 (BMP2), ⁎⁎p=0.00444(ALP) and ⁎⁎p=0.0015 (OCN); Dex-inhibitor NC versus Dex-inhibitor, ⁎⁎p=0.00886 (miR-106b), ⁎⁎⁎p=0.0000675 (BMP2), ⁎⁎p=0.00661 (ALP) and p=0.0452(OCN). n=5 for all PCR experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Bone 2017 97, 130-138DOI: (10.1016/j.bone.2017.01.014) Copyright © 2017 Terms and Conditions

Fig. 5 BMP2 is a direct target of miR-106b. (a) Schematics of the miR-106b binding sites in the BMP2 3′UTR. (b) Diagram of the BMP2 3′UTR luciferase reporter system and luciferase assay. (In WT group, ⁎p<0.05 by unpaired two-tailed t-test. There is no significant difference in Mutant group. n=3 for luciferase assay experiments). (c) Western Blot analysis of BMP2 protein levels in PMSC-derived osteoblasts at day 10 after treatment with the miR-106b mimic or inhibitor (shown in cropped gels). Mimic NC versus miR-106b mimic, ⁎⁎⁎p=0.00001 and inhibitor NC versus miR-106b inhibitor, ⁎⁎⁎p=0.000645. n=3 for all experiments. Bone 2017 97, 130-138DOI: (10.1016/j.bone.2017.01.014) Copyright © 2017 Terms and Conditions

Fig. 6 MiR-106b affects SMAD1 and SMAD5 gene expression. The relative expressions of SMAD1 and SMAD5 genes after miR-106b inhibitor treatment. Data of fold change are presented as mean±SD. ⁎p<0.05; ⁎⁎p<0.01; ⁎⁎⁎p<0.001. n=5 for all PCR experiments. Bone 2017 97, 130-138DOI: (10.1016/j.bone.2017.01.014) Copyright © 2017 Terms and Conditions