Increased microRNA-93-5p inhibits osteogenic differentiation by targeting bone morphogenetic protein-2

Abstract

Background and purpose: Trauma-induced osteonecrosis of the femoral head (TIONFH) is a major complication of femoral neck fractures. Degeneration and necrosis of subchondral bone can cause collapse, which results in hip joint dysfunction in patients. The destruction of bone metabolism homeostasis is an important factor for osteonecrosis. MicroRNAs (miRNAs) have an important role in regulating osteogenic differentiation, but the mechanisms underlying abnormal bone metabolism of TIONFH are poorly understood. In this study, we screened specific miRNAs in TIONFH by microarray and further explored the mechanism of osteogenic differentiation.

Design: Blood samples from patients with TIONFH and patients without necrosis after trauma were compared by microarray, and bone collapse of necrotic bone tissue was evaluated by micro-CT and immunohistochemistry. To confirm the relationship between miRNA and osteogenic differentiation, we conducted cell culture experiments. We found that many miRNAs were significantly different, including miR-93-5p; the increase in this miRNA was verified by Q-PCR. Comparison of the tissue samples showed that miR-93-5p expression increased, and alkaline phosphatase (ALP) and osteopontin (OPN) levels decreased, suggesting miR-93-5p may be involved in osteogenic differentiation. Further bioinformatics analysis indicated that miR-93-5p can target bone morphogenetic protein 2 (BMP-2). A luciferase gene reporter assay was performed to confirm these findings. By simulating and/or inhibiting miR-93-5p expression in human bone marrow mesenchymal stem cells, we confirmed that osteogenic differentiation-related indictors, including BMP-2, Osterix, Runt-related transcription factor, ALP and OPN, were decreased by miR-93-5p.

Conclusion: Our study showed that increased miR-93-5p in TIONFH patients inhibited osteogenic differentiation, which may be associated with BMP-2 reduction. Therefore, miR-93-5p may be a potential target for prevention of TIONFH.

Fig 1. MRI and X-ray examination of TIONFH patients and the patients without necrosis.

(A) MRI of patients without necrosis showed hollow spike-like images without a significant “double line”. (B) Necrosis patients showed a clear “double line” sign and displayed high signals on fat-suppressed images. (C) X-ray of patients without necrosis showed a smooth surface without collapse. (D) Necrosis patients showed femoral head collapse, density changes, cystic changes, or hollow or pierced femoral head surface.

Fig 2. TIONFH patient bone collapse and osteoblast death.

(A) Micro-CT of a normal area. (B) Micro-CT of a necrotic area, which appeared to indicate trabecular bone disorder, shows structural relaxation. (C) Three-dimensional reconstruction of normal areas. (D) Three-dimensional reconstruction of a necrotic area showing trabecular bone collapse deformation. (E) HE staining of normal areas. (F) HE staining of a necrotic area shows significant bone necrosis and bone marrow tissue necrosis between the trabecular and simultaneous granulation tissue.

Fig 3. miRNA array and miR-93-5p expression.

(A) Cluster analysis of partially differentially expressed miRNAs from TIONFH patients and controls. (B) miR-93-5p expression level in plasma was significantly increased as shown by Q-PCR. (C) miR-93-5p expression increased in bone tissue as shown by Q-PCR.

Fig 4. Association of miR-93-5p and osteoblast differentiation.

(A) ALP and OPN expression in TIONFH patients with necrotic bone tissue samples by immunohistochemistry. Brown represents positive signals. (B) Real time Q-PCR detected the miR-93-5p expression level during differentiation at 0, 3, and 7 d. (C) Effects of miR-93-5p on hBMSCs] proliferation after 24, 48 and 72 h by MTT assays. (D) ALP enzyme activity was detected with a reagent kit. (E) AR staining to assess mineralisation. Two cell groups were seeded on 96-well plastic dishes (Costar). Cells were cultured in differentiation medium for 21 d, and images were captured using a scanner. (F) mRNA expression level of ALP, OPN, Runx-2 and Osterix using real time PCR analysis. (G) Western blotting was performed to detect protein levels of ALP, OPN, Runx-2 and Osterix. (* P < 0.05 and ** P < 0.01).

Fig 5. Identification of BMP-2 as a target of miR-93-5p.

(A) miR-93-5p targets the 3 'UTR of BMP-2. BMP-2-3'UTR WT or MUT was cloned into psiCHECK2.0 and co-transfected with the miR-93-5p mimic. miR-93-5p mimics bind specifically to BMP2-3'UTR WT. (B) Effect of miR-93-5p on BMP-2 RNA 3'UTR region by the luciferase gene reporter assay. (* P < 0.05 and ** P < 0.01).

Fig 6. miR-93-5p regulation of BMP-2 expression.

(A) BMP-2 immunohistochemical analysis of normal bone tissue from patients with necrotic areas and the area of necrosis. (B) BMP-2 protein expression in hBMSCs by Western blotting analysis. (C) BMP-2 mRNA expression level in hBMSCs by real time Q-PCR analysis. (* P < 0.05 and ** P < 0.01).