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. 2019 Mar;17(3):1903-1911.
doi: 10.3892/etm.2019.7146. Epub 2019 Jan 2.

Regulatory effect of miR-421 on humeral fracture and heterotopic ossification in elderly patients

Chunfang Ju  1 , Zhiyun Lv  2 , Cheng Zhang  3 , Yujuan Jiao  4
Affiliations

Regulatory effect of miR-421 on humeral fracture and heterotopic ossification in elderly patients

Chunfang Ju et al. Exp Ther Med. 2019 Mar.

Abstract

The present study aimed to investigate the role of miR-421 and bone morphogenetic protein-2 (BMP-2) in the bone tissues and blood of elderly patients with humeral fractures and heterotopic ossification. A total of 38 patients with humeral fractures, including 16 patients who received surgery within 1-7 days of fracture and 22 patients who received surgery within 8-14 days of fracture, were enrolled. An additional 18 patients who had heterotopic ossification and 26 patients who had humeral fracture and not heterotopic ossification were also included. Bone tissues and blood were collected. Reverse transcription-quantitative polymerase chain reaction was performed to determine the miR-421 and BMP-2 mRNA expression levels in the samples. Western blotting and ELISA were performed to detect BMP-2 protein levels in bone tissues and blood, respectively. Dual-luciferase reporter assays were performed to verify whether BMP-2 is the direct target gene of miR-421. Compared with the patients who received surgery 1-7 days after fracture, the patients who accepted the surgery 8-14 days after fracture had significantly increased levels of BMP-2 mRNA and protein in their bone tissues and blood (P<0.05). Contrastingly, the expression level of miR-421 decreased in the samples from patients who accepted the surgery 8-14 days after fracture compared with the level in those who received surgery 1-7 days after fracture (P<0.05). Compared with the patients without heterotopic ossification, the patients with heterotopic ossification had increased BMP-2 mRNA and protein expression levels in their bone tissues and blood, whereas the expression of miR-421 was significantly decreased (P<0.05). The dual-luciferase reporter assay demonstrated that BMP-2 was the direct target gene of miR-421. The upregulation of BMP-2 may be associated with the downregulation of miR-421. miR-421 may regulate the recovery of humeral fracture and heterotopic ossification through BMP-2. The results of the present study may provide a theoretical basis for the diagnosis and treatment of humeral fracture and heterotopic ossification.

Keywords: bone morphogenetic protein-2; heterotopic ossification; humeral fracture; miR-421.

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Figures

Figure 1.
Figure 1.
Expression levels of BMP-2 mRNA and protein in bone tissues and blood of patients with humeral fractures. Bone tissues and blood were collected from patients who received surgery 1–7 days after fracture and from patients who received surgery 8–14 days after fracture. (A) The expression changes of BMP-2 mRNA in the fracture bone tissues and blood. The expression changes of BMP-2 protein in the (B) fracture bone tissues and (C) serum. Student's t-tests were used for the comparison between two groups. *P<0.05 and **P<0.01 vs. patients who received surgery 1–7 days after fracture. BMP-2, bone morphogenetic protein-2.
Figure 2.
Figure 2.
Expression levels of miR-421 in bone tissues and blood of patients with humeral fractures. Bone tissues and blood were collected from patients who received surgery 1–7 days after fracture and patients who accepted surgery 8–14 days after fracture. The expression changes of miR-421 in the (A) fracture tissues and (B) blood are shown. Student's t-tests were used for the comparison between two groups. *P<0.05 and **P<0.01 vs. patients who received surgery 1–7 days after fracture.
Figure 3.
Figure 3.
Expression levels of BMP-2 mRNA and protein in bone tissues and blood of patients with and without HO. Bone tissues and blood were collected from patients with HO and from those without HO (non-HO). (A) The expression changes of BMP-2 mRNA in the fracture bone tissues and blood. The expression changes of BMP-2 protein in the (B) fracture bone tissues and (C) serum. Student's t-tests were used for the comparison between two groups. *P<0.05 and **P<0.01 vs. non-HO. BMP-2, bone morphogenetic protein-2; HO, heterotopic ossification.
Figure 4.
Figure 4.
Expression levels of miR-421 in bone tissues and blood of patients with and without HO. Bone tissues and blood were collected from patients with HO and from those without HO (non-HO). The expression changes of miR-421 in the (A) fracture tissues and (B) blood are shown. Student's t-tests were used for the comparison between two groups. *P<0.05 and **P<0.01 vs. non-HO. HO, heterotopic ossification.
Figure 5.
Figure 5.
miR-421 targets BMP-2. The wild and mut type binding sequences between BMP-2 and miR-421 are demonstrated on the upper panel. The relative luciferase activity is indicated on the lower panel. One-way analysis of variance was used to compare the measured data of the three groups, and the Student-Newman-Keuls method was used for the pairwise comparison between two groups. **P<0.01 vs. NC group. BMP-2, bone morphogenetic protein-2; wild, wild-type; mut, mutant; NC, negative control.
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