A NOTCH1/LSD1/BMP2 Co-Regulatory Network Mediated by miR-137 Negatively Regulates Osteogenesis of Human Adipose-Derived Stem Cells

Abstract

Background: MicroRNAs have been recognized as critical regulators for the osteoblastic lineage differentiation of human adipose-derived stem cells (hASCs). Previously, we have displayed that silencing of miR-137 enhances the osteoblastic differentiation potential of hASCs partly through the coordination of lysine-specific histone demethylase 1 (LSD1), bone morphogenetic protein 2 (BMP2), and mothers against decapentaplegic homolog 4 (SMAD4). However, still numerous molecules involved in the osteogenic regulation of miR-137 remain unknown. This study aimed to further elucidate the epigenetic mechanisms of miR-137 on the osteogenic differentiation of hASCs.Methods: hASCs transfected with miR-137 overexpression or knockdown lentiviruses were used to assess the osteogenic capacity by testing the alkaline phosphatase activity, matrix mineralization degree, relative expression level of osteogenesis-associated genes and ectopic osteogenesis in nude mice. Dual-luciferase reporter assay was performed to examine the targeting of the 3' untranslated region (3' UTR) of NOTCH1 by miR-137. Interrelationships of signaling pathways of NOTCH1-hairy and enhancer of split 1 (HES1), LSD1, and BMP2-SMAD4 were thoroughly investigated by separate knockdown of NOTCH1, LSD1, and BMP2.Results: We confirmed that miR-137 directly targeted the 3' UTR of NOTCH1 while positively regulated HES1. After knocking down NOTCH1 or BMP2 individually, we found that these two signals formed a positive feedback loop and activated LSD1. In addition, LSD1 knockdown induced the expression of NOTCH1 while suppressed HES1.Conclusions: Collectively, we proposed a NOTCH1/LSD1/BMP2 co-regulatory signaling network to elucidate the modulation of miR-137 on the osteoblastic differentiation of hASCs, thus providing mechanism-based rationale for miRNA-targeted therapy of bone defect.

MiR-137-mediated negative relationship between LGR4 and RANKL modulated osteogenic differentiation of human adipose-derived mesenchymal stem cells

“…MiR-137 has been reported to participate in the progression of various cancers (Li et al, 2017;Liu et al, 2017;Qin et al, 2017;Luo et al, 2018;Zhang et al, 2018), development and maturation of neurons (Silber et al, 2008;Szulwach et al, 2010;Tarantino et al, 2010;Sun et al, 2011;Jiang et al, 2013) and bone metabolism (Zheng et al, 2019;Kong et al, 2020;Ma et al, 2020;Yu et al, 2020;Fan et al, 2021). But compared with the previous two research fields, miR-137-modulated osteogenic differentiation is still unclear.…”

“…The plasmids of negative control (NC), miR-137 overexpression (miR-137), miR-137 knockdown (anti-miR-137), LGR4 shRNA (anti-LGR4) and RANKL shRNA (anti-RANKL) were separately employed to generate the corresponding lentiviruses. The hASCs were transfected by respective lentiviruses as previously described (Ma et al, 2020;Fan et al, 2021). After being immersed in the viral supernatant (multiplicity of infection = 100) with 5 mg/mL polybrene (Sigma-Aldrich) for 24 h, the cells were then selected by 1 μg/ml puromycin (Sigma-Aldrich) and cultured in fresh PM.…”

“…The mutant-type reporter vectors of LGR4 (LGR4-MT) were formed by using a site-directed mutagenesis kit (SBS Genetech, Beijing, China). As mentioned before (Fan et al, 2016(Fan et al, , 2021Ma et al, 2020), the luciferase assay was performed by the co-transfection of LGR4-WT or LGR4-MT plasmids, NC or miR-137 mimics and lipofectamine 3000 (Invitrogen). At 48 h after transfection, the luciferase activity was examined by the dual-luciferase reporter assay system (Promega, Madison, WI) and normalized to Renilla luciferase activity.…”

“…As an ideal stem cell source, human adipose-derived mesenchymal stem cells (hASCs) are widely used in bone tissue engineering due to their accessibility, multipotency and low immunogenicity (Dinescu et al, 2021). MicroRNAs (miRNAs), a class of small non-coding RNAs ranging from 19-25 nucleotides in size, regulate protein-coding genes post-transcriptionally and play crucial roles in various biological activities, including the proliferation and osteogenic differentiation of hASCs (Ranganathan and Sivasankar, 2014;Fan et al, 2021). It has been demonstrated that miR-137 acts as a key regulator in various kinds of malignancies (Li et al, 2017;Liu et al, 2017;Qin et al, 2017;Luo et al, 2018;Zhang et al, 2018) and neural development (Silber et al, 2008;Szulwach et al, 2010;Tarantino et al, 2010;Sun et al, 2011;Jiang et al, 2013).…”

“…It has been demonstrated that miR-137 acts as a key regulator in various kinds of malignancies (Li et al, 2017;Liu et al, 2017;Qin et al, 2017;Luo et al, 2018;Zhang et al, 2018) and neural development (Silber et al, 2008;Szulwach et al, 2010;Tarantino et al, 2010;Sun et al, 2011;Jiang et al, 2013). But up to present, only a few studies investigated the effects of miR-137 on osteogenesis (Zheng et al, 2019;Kong et al, 2020;Ma et al, 2020;Yu et al, 2020;Fan et al, 2021), and the mechanisms of osteogenic modulation by miR-137 are still not fully understood, especially considering the diverse biological properties of different cell types.…”