Melatonin promotes bone marrow mesenchymal stem cell osteogenic differentiation and prevents osteoporosis development through modulating circ_0003865 that sponges miR-3653-3p

Abstract

Background Little is known about the implications of circRNAs in the effects of melatonin (MEL) on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoporosis (OP) progression. The aim of our study was to investigate circRNAs in MEL-regulated BMSC differentiation and OP progression. Methods BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red, and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade was validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on OP development were tested in murine OP model. Results MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 had no effect on proliferation while promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSC osteogenic differentiation induced by MEL, but proliferation of BMSCs induced by MEL had no change whether circ_0003865 was overexpression or not. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSC proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing, but had no effect on proliferation. Finally, circ_0003865 silencing repressed OP development in mouse model. Conclusion MEL promotes BMSC osteogenic differentiation and inhibits OP pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

Melatonin Inhibits Osteoclastogenesis and Osteolytic Bone Metastasis: Implications for Osteoporosis

“…Interestingly, melatonin apparently promotes the osteogenic differentiation of BMMSCs by upregulating microRNA-92b-5p expression, which then enhances the differentiation of BMMSCs into mature osteoblasts by targeting intracellular adhesion molecule-1 (ICAM-1) [54]. In another study, melatonin promoted the osteogenic differentiation of BMMSCs and prevented the progression of osteoporosis by significantly decreasing the expression of circ_0003865, which subsequently increased levels of osteogenic marker genes (ALP, RUNX2 and OPN) and marked induction of osteogenic differentiation [59]. Moreover, melatonin-induced silencing of circ_0003865 increased the expression of miR-3653-3p, which substantially increased ALP, RUNX2, and OPN expression and promoted BMMSC osteogenic differentiation [59].…”

“…In another study, melatonin promoted the osteogenic differentiation of BMMSCs and prevented the progression of osteoporosis by significantly decreasing the expression of circ_0003865, which subsequently increased levels of osteogenic marker genes (ALP, RUNX2 and OPN) and marked induction of osteogenic differentiation [59]. Moreover, melatonin-induced silencing of circ_0003865 increased the expression of miR-3653-3p, which substantially increased ALP, RUNX2, and OPN expression and promoted BMMSC osteogenic differentiation [59].…”

Emerging roles of circular RNAs in osteoporosis

“…Wang et al 56 explored circRNAs expression profiling in the BMSCs after treated with melatonin or not by using RNA sequencing. They found that there were 209 circRNAs were differentially expressed in human BMSCs after treated with melatonin.…”

“…Wang et al 69 demonstrated that the circ_0003865 expression was downregulated in the BMSCs after treated with melatonin. Knock‐down of circ_0003865 promoted the expression of OPN, ALP and RUNX2 in BMSCs.…”

Combination of Melatonin and Zoledronic Acid Suppressed the Giant Cell Tumor of Bone in vitro and in vivo

“…Melatonin (Mlt), a neuroendocrine hormone primarily secreted by the pineal gland, exhibits a wide range of physiological functions, such as improving sleep and promoting poly-differentiation of stem cells, and is an antiaging, antioxidative, and antiosteoporotic agent ( Reiter et al, 2016 ; Nopparat et al, 2017 ; Gao et al, 2018 ; Sharma et al, 2018 ; Qiu et al, 2019 ; Wang et al, 2019 ; Wang et al, 2021 ). In recent years, the therapeutic effect of Mlt on tumors, including lung cancer, liver cancer, and breast cancer, has received great attention ( Ordonez et al, 2015 ; Borin et al, 2016 ; Ma et al, 2019 ) however, the effect of Mlt on GCTB remains unknown.…”