MiR-542-3p Attenuates Bone Loss and Marrow Adiposity Following Methotrexate Treatment by Targeting sFRP-1 and Smurf2.

Ya-Li Zhang,Cory J Xian,Yu-Wen Su,Liang Liu

doi:10.3390/ijms222010988

Ya-Li Zhang, Cory J Xian + Show 2 more

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https://doi.org/10.3390/ijms222010988

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Abstract

Intensive methotrexate (MTX) treatment for childhood malignancies decreases osteogenesis but increases adipogenesis from the bone marrow stromal cells (BMSCs), resulting in bone loss and bone marrow adiposity. However, the underlying mechanisms are unclear. While microRNAs (miRNAs) have emerged as bone homeostasis regulators and miR-542-3p was recently shown to regulate osteogenesis in a bone loss context, the role of miR-542-3p in regulating osteogenesis and adipogenesis balance is not clear. Herein, in a rat MTX treatment-induced bone loss model, miR-542-3p was found significantly downregulated during the period of bone loss and marrow adiposity. Following target prediction, network construction, and functional annotation/ enrichment analyses, luciferase assays confirmed sFRP-1 and Smurf2 as the direct targets of miR-542-3p. miRNA-542-3p overexpression suppressed sFRP-1 and Smurf2 expression post-transcriptionally. Using in vitro models, miR-542-3p treatment stimulated osteogenesis but attenuated adipogenesis following MTX treatment. Subsequent signalling analyses revealed that miR-542-3p influences Wnt/β-catenin and TGF-β signalling pathways in osteoblastic cells. Our findings suggest that MTX treatment-induced bone loss and marrow adiposity could be molecularly linked to miR-542-3p pathways. Our results also indicate that miR-542-3p might be a therapeutic target for preserving bone and attenuating marrow fat formation during/after MTX chemotherapy.

Highlights

Methotrexate (MTX) is known as a useful chemotherapy reagent that is utilised to treat autoimmune diseases and cancers, such as rheumatoid arthritis, head cancer, osteosarcoma, and leukemia, the major childhood cancer acute lymphoblastic leukemia (ALL) [1,2,3,4]
To verify if miR-542-3p influences on Wnt/β-catenin signalling in osteoblastic cells, we examined the mRNA expression of downstream genes of Wnt/β-catenin signalling, namely lymphoid enhancer factor 1 (LEF1) and T-cell factor 7 (TCF7) in MC3T3.E1 cells after miR-542-3p treatment (Figure 7)
We demonstrated that miR-542-3p transfection increased mRNA expression of Smad3 and RUNX2, which are direct targets of Smurf2, suggesting that miR-542-3p might function in regulating osteogenesis through modulating TGF-β/Smad signalling, which is consistent with a previous study [33]

Summary

Introduction

Methotrexate (MTX) is known as a useful chemotherapy reagent that is utilised to treat autoimmune diseases and cancers, such as rheumatoid arthritis, head cancer, osteosarcoma, and leukemia, the major childhood cancer acute lymphoblastic leukemia (ALL) [1,2,3,4]. Clinical studies have shown that intensive use of MTX induces skeletal defects, including reduced bone mineral density (BMD) or bone loss, bone growth arrests and marrow adiposity [5,6]. In an acute intensive MTX treatment model in rats (5 daily administrations at 0.75 mg/kg), altered expression of Wnt/β-catenin signalling pathway components was observed, including increased mRNA expression of Wnt antagonists secreted frizzled related protein 1 (sFRP-1) and dickkopf-1 (DKK1) in rat bones, decreased activation of β-catenin in BMSCs, and reduced mRNA expression of β-catenin target genes lymphoid enhancer factor 1 (LEF1), cyclin D1 and survivin in bones, indicating that MTX treatment attenuated Wnt/β-catenin signalling in bones [7]. Subsequent studies showed that the preservation of Wnt/β-catenin signalling partially abrogated

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