Differentially expressed miRNAs in bone after methotrexate treatment

Abstract

Previous studies have shown that administration of antimetabolite methotrexate (MTX) caused a reduced trabecular bone volume and increased marrow adiposity (bone/fat switch), for which the underlying molecular mechanisms and recovery potential are unclear. Altered expression of microRNAs (miRNAs) has been shown to be associated with dysregulation of osteogenic and/or adipogenic differentiation by disrupting target gene expression. First, the current study confirmed the bone/fat switch following MTX treatment in precursor cell culture models in vitro. Then, using a rat intensive 5-once daily MTX treatment model, this study aimed to identify miRNAs associated with bone damage and recovery (in a time course over Days 3, 6, 9, and 14 after the first MTX treatment). RNA isolated from bone samples of treated and control rats were subjected to miRNA array and reverse transcriptionpolymerase chain reaction validation, which identified five upregulated miRNA candidates, namely, miR-155-5p, miR-154-5p, miR-344g, miR-6215, and miR-6315.Target genes of these miRNAs were predicted using TargetScan and miRDB. Then, the protein-protein network was established via STRING database, after which the miRNA-key messenger RNA (mRNA) network was constructed by Cytoscape.Functional annotation and pathway enrichment analyses for miR-6315 were performed by DAVID database. We found that TGF-β signaling was the most significantly enriched pathway and subsequent dual-luciferase assays suggested that Smad2 was the direct target of miR-6315. Our current study showed that miR-6315 might be a vital regulator involved in bone and marrow fat formation. Also, this study constructed a comprehensive miRNA-mRNA regulatory network, which may contribute to the pathogenesis/prognosis of MTX-associated bone loss and bone marrow adiposity.

miR-6315 Attenuates Methotrexate Treatment-Induced Decreased Osteogenesis and Increased Adipogenesis Potentially through Modulating TGF-β/Smad2 Signalling

“…Using a rat acute MTX treatment model, previous studies have shown that administration of MTX caused a reduced trabecular bone volume and an increased marrow adiposity [ 30 , 31 , 32 ]. Our recent investigation also confirmed the MTX-induced reduced osteogenesis/increased adipogenesis in vitro [ 24 ]. Consistent with these previous observations, using MC3T3.E1 preosteoblastic cells and 3T3 F442A preadipocytic cells, the current study also observed the reduced osteogenic differentiation and increased adipogenic differentiation following MTX treatment from the precursor cells.…”

“…In both patients and animal models, reduced bone formation and increased marrow adiposity have been observed, and yet the underlying mechanisms are unclear [ 3 , 4 , 13 , 14 ]. Previously, using a rat MTX intense treatment model (five daily injections, mimicking intensive MTX treatment for childhood leukemia), our investigations have identified five upregulated miRNA candidates, namely, miR-155-5p, miR-154-5p, miR-344g, miR-6215, and miR-6315, as promising candidates associated with MTX-induced bone defects [ 24 ]. Based on bioinformatic analyses, among them, miR-6315 was indicated as a potentially important factor involved in bone/bone marrow fat formation defects following MTX treatment.…”

“…Thus, our current study suggests that miR-6315 plays a vital role in regulating osteogenesis and adipogenesis and that supplementary treatment with miR-6315 preserves osteoblast differentiation but attenuates adipocyte differentiation in vitro after MTX treatment. Based on these, it could be proposed that the induction of miR-6315 expression in bone of MTX-treated rats, as shown in our recent study, may be associated with its inhibitory effect, attempting to limit the extent of MTX-induced increased adipogenesis, and/or with its osteogenesis-promoting effect, attempting to enhance the bone recovery mechanism following MTX treatment [ 13 , 24 , 32 , 33 ].…”

“…Previous work found that miR-6315 might be involved in MTX-induced bone defects by targeting Smad2 [ 24 ]. Smad2 is known to respond to TGF-β ligands interacting with the receptor complex, mediating the gene expression of various transcription factors, including osteogenic gene DLX5 (an activator of Runx2) [ 46 ].…”

“…Previously, the regulation of the miRNA-6315 network was found to be potentially involved in hyperplasia of mammary glands and cell proliferation during liver regeneration [ 22 , 23 ]. Our recent study has shown that miR-6315 was upregulated in bones of MTX-treated rats, which might be associated with bone/bone marrow fat imbalance by directly targeting Smad2 [ 24 ]. However, the underlying mechanisms by which miR-6315 regulates osteogenic and adipogenic differentiation remain unknown.…”