In recent years, BMSCs have been used for osteoporosis treatment because of their potential for differentiating into osteoblasts . Previous studies have revealed that MEL promotes osteogenic differentiation of BMSCs, which indicates that they may be effectively used for BMSC-based osteoporosis treatment [14, [15, . However, the underlying mechanisms remain largely undefined. In the present study, we focused on the potential role of circRNAs in the MEL-activated osteogenic differentiation of human BMSCs and their association with osteoporosis pathogenesis and treatment. We first characterized the significant alterations of circRNA and mRNA expression profiles in BMSCs following MEL treatment by deep RNA sequencing, which was associated with multiple biological processes.. Subsequently, circ_0003865 was further shown to be repressed by MEL in BMSCs and sponges miR-3653-3p to modulate GAS1 gene expression and osteogenic differentiation in human BMSCs. Overexpression of miR-3653-3p promoted BMSC osteogenic differentiation, whereas miR-3653-3p inhibitors abrogated this effect induced by circ_0003865 silencing. Finally, we verified the function of circ_0003865 in repressing BMSC osteogenic differentiation and promoting osteoporosis pathogenesis by infecting a murine osteoporosis model with AAVs designed to express sh_circ_0003865. These investigations revealed the role of a new circ_0003865/miR-3653-3p/GAS1 signaling axis in MEL-regulated BMSC differentiation and osteoporosis treatment.
BMSCs refer to the mesenchymal stem cells derived from the bone marrow. They serve as the progenitors for osteocyte, chondrocytes, and other cell types involved in the formation of skeletal tissues, hematopoiesis-supporting stroma, and adipose tissues . Additionally, BMSCs exhibit a high potential to differentiate into a large spectrum of other specialized human cell types, including cardiac myocytes, neural cells, renal cells, liver hepatocytes, corneal cells, blood cells, and even myogenic cells [28, [29, . Benefiting from their multi-faceted differentiation capabilities, high portability, and relatively low immunogenicity, BMSCs have been regarded as ideal candidate stem cells for the treatment of various human disorders over the past decades [31, [32, . In adult skeletal tissues, BMSCs primarily differentiate into two cell types: osteoblasts and adipocytes. The biased differentiation of BMSCs toward adipocytes may lead to a decrease of osteoblast cells and osteoporosis pathogenesis . Therefore, the various regulatory factors that can promote BMSC osteogenic differentiation may represent potential therapeutic drugs for treating osteoporosis [34, . As described above, the indole hormone, MEL (MEL), was previously reported to modulate BMSC stemness and osteogenic differentiation [14, [15, , but little is known about its underlying mechanism(s). In the present study, we first confirmed the role of MEL in enhancing the osteogenic differentiation of human BMSCs. This hormone was then further used to develop a cell model for investigating the molecular mechanisms underlying its role in BMSC differentiation.
Epigenetic events mediated by non-coding RNAs such as circRNAs and microRNAs perform critical roles in the regulation of stem cell fates and differentiation [36, . Although multiple circRNAs are known to mediate the osteogenic differentiation of BMSCs and osteoporosis pathogenesis [19, [20, [21, , the implications of circRNAs in MEL-regulated BMSC differentiation and osteoporosis development remain largely unknown. To study this phenomenon, we conducted a large-scale characterization of differentially expressed circRNAs in human BMSCs following MEL treatment by deep RNA sequencing. The results showed significant alterations of the circRNA profiles in MEL-treated human BMSCs, suggesting a potential role for circRNAs in BMSC differentiation regulation by MEL. It is well known that the major biological functions of circRNAs are mediated by their substantial effects on gene expression . We also identified changes in the gene expressional profiles by RNA sequencing and observed significant expression differences for many functional genes in MEL-treated BMSCs. These were closely associated with various biological processes and signaling pathways, including metabolism, PI3K-AKT, and FOXO signaling cascades. Among them, the FOXO signaling pathway was previously shown to mediate osteogenesis and glucocorticoid-induced osteoporosis . Furthermore, the PI3K-AKT signaling pathway has been implicated in the osteogenic differentiation of BMSCs resulting from glucagon-like peptide 1 receptor activation . The significant changes in circRNA and mRNA expression identified in the present study indicate that the regulation of BMSC differentiation by MEL is driven by complex signaling mechanisms. This warrants further investigations and may lead to new discoveries for osteoporosis prevention and treatment.
In the present study, circ_0003865 was identified for the first time as a differentially expressed circRNA in MEL-treated human BMSCs. A bioinformatics analysis indicated that circ_0003865 is formed by back-splicing of pre-mRNA encoded by the ANKRD12 gene. Previous reports have shown that ANKRD12 gene expression is correlated with metastasis and poor survival of colorectal cancer patients . Furthermore, the ANKRD12 circRNA modulates the invasiveness of cancer cells , but little is known about the role of circRNAs derived from the ANKRD12 gene in BMSC differentiation and osteoporosis. We demonstrated that the expression of circ_0003865, encoded by the ANKRD12 gene, was significantly decreased in human BMSCs by MEL treatment. The silencing of circ_0003865 expression increased the expression of osteogenic marker genes, including ALP, RUNX2, and OPN and resulted in the marked induction of osteogenic differentiation. Furthermore, overexpression of circ_0003865 effectively abrogated the MEL-induced expression of osteogenic marker genes and BMSC osteogenic differentiation. This validates the mediating role of circ_0003865 in MEL-induced BMSC differentiation. Finally, we verified that circ_0003865 silencing significantly elevated the expression of osteogenic marker gene expression and repressed the progression of osteoporosis in a murine model. This convincingly establishes circ_0003865 as a potent inhibitor of BMSC osteogenic differentiation and a potential biomarker for osteoporosis diagnosis and treatment.
Previous reports demonstrated that the gene expression-regulating functions of circRNAs are mainly mediated by targeting miRNAs as sponges to modulate gene expression [42, . We also predicted by a bioinformatics analysis that circ_0003865 sponges miR-3653-3p, miR-4775, and miR-6509-3p to regulate the expression of the GAS1 gene. The expression of all three microRNAs was significantly increased by MEL treatment in human BMSCs, but only the expression of miR-3653-3p was increased by circ_0003865 silencing. This suggests that miR-3653-3p is a specific target of circ_0003865 in human BMSCs. Furthermore, circ_0003865 overexpression promoted the expression of miR-3653-3p in human BMSCs treated with MEL. Additionally, we showed that miR-3653-3p overexpression greatly elevated ALP expression, RUNX2, and OPN and promoted the osteogenic differentiation of BMSCs. More importantly, miR-3653-3p inhibitors significantly mitigated the circ_0003865 silencing-induced promotion of BMSC osteogenic differentiation, which validates the mediating role of miR-3653-3p in circ_0003865-regulated BMSC differentiation. GAS1 is an inhibitor of the G0 to S phase transition during cell cycle progression and regulates the Hedgehog concentration gradient and related signaling pathways [44,  that also suppress muscle stem cell renewal . In the present study, we showed that the GAS1 expression in BMSCs could be suppressed by MEL treatment, enhanced by circ_0003865, and repressed by miR-3653-3p. We also confirmed the direct association between circ_0003865 and miR-3653-3p and the binding of miR-3653-3p with GAS1 gene sequences using a luciferase reporter assay. This demonstrates the existence of a novel circ_0003865/miR-3653-3p/GAS1 axis in MEL-regulated BMSC osteogenic differentiation.