MicroRNA-672-5p Identified during Weaning Reverses Osteopenia and Sarcopenia in Ovariectomized Mice

Abstract

Post-menopausal condition augments the biological aging process, characterized by multiple metabolic disorders in which bone loss is the most prevalent outcome and usually coupled with sarcopenia. Coexistence of such associated pathogenesis have much worse health outcomes, compared to individuals with osteoporosis only. Pre- and post-natal bone development demands calcium from mother to fetus during pregnancy and lactation leading to a significant maternal skeletal loss. It follows an anabolic phase around weaning during which there is a notable recovery of the maternal skeleton. Here, we have studied the therapeutic effect of microRNA-672-5p identified during weaning when it is predominantly expressed, in ovariectomized mice for both osteopenia and sarcopenia. miR-672-5p induced osteoblast differentiation and mineralization. These actions were mediated through inhibition of Smurf1 with enhanced Runx2 transcriptional activation. In vivo, miR-672-5p significantly increased osteoblastogenesis and mineralization, thus reversing bone loss caused by ovariectomy. It also improved bone-mineral density, load-bearing capacity, and bone quality. Sarcopenia was also alleviated by miR-672-5p, as we observed increased cross-sectional area and Feret's diameter of muscle fibers. We hypothesize that elevated miR-672-5p expression has therapeutic efficacy in estrogen-deficiency-induced osteopenia along with sarcopenia.

Keywords: micro-computed tomography; microRNA-672-5p; osteopenia; sarcopenia; trabecular microarchitecture.

Graphical abstract

Figure 1

miR-672-5p Represses Smurf1 to Promote Osteoblast Differentiation (A) Stem-loop structure of miR-672 predicted by miR-base (the mature miR-672-5p sequence is shown in green). (B and C) Expression (qPCR, in triplicate) of miR-672-5p in different tissues (B) and cells, i.e., in osteoblasts during proliferation, differentiation, or mineralization (C). Data are mean ± SE. *p < 0.05 and ***p < 0.001 compared with proliferation stage of osteoblasts. (D–F) Effect of transfection of osteoblasts with the mimic (miR-672-5p), inhibitor (anti-miR-672-5p), and miR-C (control) on ALP (alkaline phosphatase) activity (OD, n = 8) (D) and mineralization (OD, n = 4) (E). (F) Representative wells showing alizarin-positive colony (Cfu-ob) formation in osteoblast cell cultures on day 15 in osteogenic medium. Data are mean ± SE. *p < 0.05 and **p < 0.01 compared with miR-C. (G) Identification of miR-672-5p target gene in osteoblasts and computational analysis was performed for the complementarities of miR-672-5p to the 3′ UTR of Smurf1 and (H) schematic presentation of the reporter plasmid used to illustrate the effect of Smurf1 3′ UTR on luciferase activity. CMV, cytomegalovirus promoter; Luc, luciferase; RLuc, renilla luciferase. (I) Effect of miR-672-5p overexpression on a dual luciferase reporter plasmid containing Smurf1 3′ UTR was analyzed. Cells were co-transfected with the WT-pEZX-MT06-Smurf1 or an empty vector (NC, negative control) and miR-672-5p or miR-C. Firefly and renilla luciferases were measured in the cell lysate. Data are mean ± SE of three independent measurements. **p < 0.01 compared with miR-C. (J) Expression (qPCR, in triplicate) of Smurf1 in osteoblasts during proliferation, differentiation, or mineralization. Data are mean ± SE. *p < 0.05 compared with proliferation stage of osteoblasts. (K) Transfection of osteoblasts with the mimic (miR-672-5p) enhanced the release of BMP2 in conditioned medium (BMP2 ELISA). Data are mean ± SE of three independent experiments. *p < 0.05 compared with miR-C. (L) Effect of miR-672-5p or scrambled miR-C on Runx2 promoter activity using a luciferase (Luc) reporter. Data are mean ± SE of three independent experiments. *p < 0.05 compared with miR-C. (M) qPCR (in triplicate) was performed for Smurf1 (a direct target gene of miR-672-5p) or the osteoblast differentiation genes, including Runx2, BMP2, Smad1, and ALP after 48 h of transfection. Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with miR-C. (N) Western blot analysis was done for Smurf1 (ab38866-Abcam; 1:1,000), Runx2 (ab76956-Abcam; 1:1000), BMP2 (ab14933-Abcam; 1:1000), and Smad1 (ab63356-Abcam; 1:1,000) after 48 h of transfection. β-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as a loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were horseradish peroxidase (HRP) conjugated (Sigma-Aldrich). (O–S) miR-672-5p represses Smurf1, which promotes the upregulation of Runx2 and osteoblast differentiation. (O) ALP activity (OD, n = 8), (P) Cfu-ob formation, (Q) mineralization (OD, n = 4), and (R) mRNA expression of Runx2, BMP2, Smurf1, ALP, and Smad1 (qPCR, in triplicate), and (S) protein expression of Smurf1 (ab38866-Abcam; 1:1,000), Runx2 (ab76956-Abcam; 1:1000), and BMP2 (ab14933-Abcam; 1:1,000) in either control osteoblasts or transfected with siRNA against Smurf1 or scrambled siRNA (Si-Scr). β-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as a loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were HRP conjugated (Sigma-Aldrich). Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with miR-C or control. Other comparisons, ˆp < 0.05, ˆˆp < 0.01, and ˆˆˆp < 0.001 as shown.

Figure 2

miR-672-5p Treatment Restores Ovariectomy-Induced Trabecular Bone Loss in Tibia Bones (A) Schematic diagram illustrating in vivo experimental setup. (B) Enhanced miR-672-5p expression (qPCR, in triplicate) in bone following injection of a mimic of miR-672-5p using Invivofectamine 3.0 reagent into 12-week-old female BALB/c mice at the end of 6 weeks of treatment, and this indicates sufficient tissue uptake. Data are mean ± SE. ***p < 0.001 compared with the PBS-treated group. (C) Representative 3D images of the tibia bones generated by micro-CT. (D) Volumetric bone-mineral density (vBMD) in the tibia at the end of 6 weeks of treatment was improved. Data are mean ± SE. **p < 0.01 and ***p < 0.001 compared with the PBS-treated group, or as shown ˆˆp < 0.01; n ≥ 6 mice per group. (E) Structural micro-CT parameters for trabecular bone, including BV/TV, Tb.Sp, Tb.N, Tb.Pf, SMI, and Conn.Dn (units are shown) in the tibia epiphyses. Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the PBS-treated group, or as shown ˆˆˆp < 0.001; n = 8 mice per group. See also Figure S1 and Table 1 for femur and vertebrae, respectively.

Figure 3

miR-672-5p Treatment Has an Anabolic Effect and Promotes Bone Formation (A) Immunofluorescence assay in decalcified bone sections showed that miR-672-5p overexpression promoted Runx2 expression (red) and suppressed Smurf1 expression (green) as compared with the PBS-treated group (n = 3, magnification 20×). (B and C) Representative photomicrographs of Goldner’s trichrome (GT) staining (B) of distal femurs from different groups and histomorphometric quantification; BS/TV (C) using the Bioquant software. Data are mean ± SE. *p < 0.05 compared with the PBS-treated group, or as shown ˆp < 0.05; (n = 3, magnification 20×). (D) Osteogenic marker; serum P1NP concentration of different groups by ELISA. Data are mean ± SE. **p < 0.01 compared with the PBS-treated group, or as shown ˆˆˆp < 0.001; (n = 6). (E) Representative photomicrographs of the calcein double labeling in the femur (n = 3, magnification 20×). (F) Quantification of data showing mineralizing surface normalized with bone surface (MS/BS), mineral apposition rate (MAR), and rate of bone formation, surface referent (BFR/BS). Data are mean ± SE. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the PBS-treated group, or as shown ˆˆp < 0.01 and ˆˆˆp < 0.001. See also Figures S2A–S2C for histological examination of the femur metaphysis for osteoclast numbers and bone resorption surface estimates for in vivo experiments.

Figure 4

miR-672-5p Treatment Represses Smurf1 In Vivo to Promote Osteoblastogenesis (A) ALP activity (OD, n = 6), (B) mineralization (OD, n = 6), and (C) representative wells of Cfu-ob formation. Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the PBS-treated group, or as shown ˆˆp < 0.01. mRNA expression (qPCR, in triplicate) of Smurf1 (D), Runx2 (E), and BMP2 (F) and protein expression (G) (western blotting) of Smurf1 (ab38866-Abcam; 1:1,000), Runx2 (ab76956-Abcam; 1:1,000), and BMP2 (ab14933-Abcam; 1:1,000) in osteoblasts from sham-operated (Sham) or ovariectomized (OVx) mice that were given PBS (0.2 mL), scrambled miR (miR-C, 7.0 mg kg⁻¹), or miR-672-5p (7.0 mg kg⁻¹). β-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were HRP conjugated (Sigma-Aldrich). Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the PBS-treated group. See also Figures S2D–S2F for in vitro actions of microRNA-672-5p on osteoclastogenesis from bone marrow cell cultures.

Figure 5

miR-672-5p Treatment Mitigates Ovariectomy-Induced Sarcopenia (A) Body weight, (B) lean mass, and (C) serum CK levels. Data are mean ± SE. *p < 0.05 and **p < 0.01 compared with the PBS-treated group, or as shown ˆp < 0.05, ˆˆp < 0.01, and ˆˆˆp < 0.001 (n ≥ 6 mice per group). (D) Representative photomicrographs of H&E-stained sections of gastrocnemius muscle from the indicated groups (n = 3, magnification 40×). (E) Cross-sectional area (CSA) and (F) Feret’s diameter. Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the PBS-treated group, or as shown ˆˆˆp < 0.001. (G and H) Immunostaining for laminin (G) and immunostaining for Atrogin-1 (H) (n = 3, magnification 40×). mRNA expression (qPCR, in triplicate) of MyoD (I), Atrogin-1 (J), and MuRF-1 (K) and protein expression (L) (western blotting) of muscle differentiation marker; MyoD (MA1-41017-Thermo Scientific; 1:1,000), catabolic markers, atrogin-1 (ab74023-Abcam; 1:1,000), and muscle ring-finger protein-1 (MuRF-1, ab183094-Abcam; 1:1,000) from gastrocnemius muscles of indicated groups. β-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as a loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were HRP conjugated (Sigma-Aldrich). Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the PBS-treated group.