Experimental study of expression profile and specific role of human microRNAs in regulating atrophic bone nonunion

Abstract

The expression profile and specific roles of microRNAs (miRNAs) in regulation of atrophic bone nonunion are not fully understood. Here, we present evidence that miRNAs are involved in regulation of several osteogenic genes and may contribute to the development of atrophic bone nonunion. The miRNA expression profile of repairing tissues in atrophic bone nonunion patients (group A) and in callus tissues from patients with healed fractures (group B) were quantitatively measured. microRNA microarrays were used to identify differentially expressed miRNAs, and the bioinformatics methods were used to predict the potential target genes. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase reporter assay were performed in human bone marrow stromal cells (hBMSCs) to validate the microarray results. Nine miRNAs in group A were up-regulated 1.5 times compared to group B, while the other 9 miRNAs in group A were down-regulated 1.5 times. Several target regions of these miRNAs were identified in the osteogenic genes, as well as in the other genes in their families or related regulatory factors. Four miRNAs (hsa-miR-149 ∗ , hsa-miR-221, hsa-miR-628-3p, and hsa-miR-654-5p) could play important roles in regulating bone nonunion development. hBMSCs transfected with these miRNAs significantly decreased mRNA levels of alkaline phosphatase, liver/bone/kidney ( ALPL ), platelet derived growth factor subunit A ( PDGFA ), and bone morphogenetic protein 2 ( BMP2 ). Lower protein expression levels were observed using western blotting, confirming that ALPL , PDGFA , and BMP2 were directly targeted by hsa-miR-149 ∗ , hsa-miR-221, and hsa-miR-654-5p, respectively. In summary, hsa-miR-149 ∗ , hsa-miR-221, and hsa-miR-654-5p may play important biological roles by repressing osteogenic target genes ALPL , PDGFA , and BMP2 , and, therefore, contributing to progression of atrophic bone nonunion.

Biological and molecular profile of fracture non‐union tissue: A systematic review and an update on current insights

“…The electronic literature search retrieved 342 citations, of which 24 met the inclusion criteria for the final analysis (Figure 1 ). 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 Overall, 10 studies 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 assessed non‐union tissue (Table 2 ), whereas 14 studies 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 investigated relevant tissue (Table 3 ).…”

“…The study characteristics of the non‐union tissue and relevant tissue are outlined in Table 4 . 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 Non‐union was defined based upon radiographic and clinical examination, with minor variations between studies. Samples of non‐union tissue and relevant tissue were mostly obtained during the surgical treatment of non‐unions.…”

The Regulation of Collagen Processing by miRNAs in Disease and Possible Implications for Bone Turnover

“…Second, it increases the amount of mineralisation promoting Pi ions by liberating them from PPi [ 136 ]. Lower protein expression levels were observed using Western blotting, confirming that ALPL is directly targeted by hsa-miR-149 [ 128 ]. Inhibition of miR-99a-5p in MC3T3 pre-osteoblastic cells promoted osteogenic differentiation, whereas its overexpression suppressed the levels of osteogenic-specific ALPL expression [ 129 ].…”

The Emerging Role of MicroRNAs in Bone Diseases and Their Therapeutic Potential

“…Consistently, it was detected that nine miRNAs were significantly upregulated in the atrophic non-union tissues (i.e., hsa-miR-149*, hsa-miR-221, hsa-miR-628-3p, hsa-miR-654-5p, hsa-miRPlus-E1114, hsa-miRPlus-E1285, hsa-miRPlus-E1238, hsa-miRPlus-E1101, and hsa-miRPlus-C1115). On the other hand, 9 miRNAs, including hsa-let-7b ∗ , hsa-miR-220b, hsa-miR-513a-3p, and hsa-miR-551a, to name a few, were identified to be downregulated in atrophic non-union samples [ 134 ]. Further analysis performed in transfected human BMSCs elucidated that hsa-miR-149*, hsa-miR-221, and hsa-miR-654-5p can target the ALPL , PDGFA , and BMP2 genes, respectively.…”

“…Further analysis performed in transfected human BMSCs elucidated that hsa-miR-149*, hsa-miR-221, and hsa-miR-654-5p can target the ALPL , PDGFA , and BMP2 genes, respectively. Since these genes are involved in osteogenesis, their miRNA-based repression may contribute to the development of atrophic non-union [ 134 ].…”