A NOTCH1/LSD1/BMP2 co-regulatory network mediated by miR-137 negatively regulates osteogenesis of human adipose-derived stem cells

Cong Fan,Yunsong Liu,Yuan Zhu,Xiaohan Ma,Longwei Lv,Yuejun Wang,Hao Liu

doi:10.1186/s13287-021-02495-3

Abstract

BackgroundMicroRNAs 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.MethodsDual-luciferase reporter assay was performed to validate the binding to the 3′ untranslated region (3′ UTR) of NOTCH1 by miR-137. To further identify the role of NOTCH1 in miR-137-modulated osteogenesis, tangeretin (an inhibitor of NOTCH1) was applied to treat hASCs which were transfected with miR-137 knockdown lentiviruses, then together with negative control (NC), miR-137 overexpression and miR-137 knockdown groups, the osteogenic capacity and possible downstream signals were examined. Interrelationships between signaling pathways of NOTCH1-hairy and enhancer of split 1 (HES1), LSD1 and BMP2-SMADs were thoroughly investigated with separate knockdown of NOTCH1, LSD1, BMP2, and HES1.ResultsWe confirmed that miR-137 directly targeted the 3′ UTR of NOTCH1 while positively regulated HES1. Tangeretin reversed the effects of miR-137 knockdown on osteogenic promotion and downstream genes expression. After knocking down NOTCH1 or BMP2 individually, we found that these two signals formed a positive feedback loop as well as activated LSD1 and HES1. In addition, LSD1 knockdown induced NOTCH1 expression while suppressed HES1.ConclusionsCollectively, 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.

Highlights

MicroRNAs have been recognized as critical regulators for the osteoblastic lineage differentiation of human adipose-derived stem cells
This study identified the interactions of miR-137 and its downstream genes and revealed that the coregulatory signaling network of NOTCH1/lysine-specific histone demethylase 1 (LSD1)/bone morphogenetic protein 2 (BMP2) mediated by miR-137 negatively modulated the osteogenesis of human adipose-derived stem cells (hASCs), suggesting that miR-137 might be applied as a promising therapeutic target for bone regeneration
After transfecting hASCs with lentiviruses of negative control (NC), miR-137 overexpression, and miR-137 knockdown (Additional file 1: Fig. S1a), we evaluated the transfection rate was over 90% by computing the percentage of green fluorescent protein (GFP)-tagged cells (Additional file 1: Fig. S1b)

Summary

Introduction

MicroRNAs have been recognized as critical regulators for the osteoblastic lineage differentiation of human adipose-derived stem cells (hASCs). 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). This study aimed to further elucidate the epigenetic mechanisms of miR-137 on the osteogenic differentiation of hASCs. As we know, human adipose-derived stem cells (hASCs) have multilineage differentiation potentials and good application prospects for bone regeneration [1,2,3] because of their abundant sources and easy access for clinical uses [4]. During the process of hASCs differentiating into osteoblastic lineage, the function and epigenetic mechanisms of miR-137 have not been investigated except for our previous study [32], in which we disclose part of the mechanisms as the coordination between lysine-specific histone demethylase 1 (LSD1) and BMP2-mothers against decapentaplegic homolog 4 (SMAD4) pathway. Considering that the relationships of osteogenesis-associated signals are complex and diverse, and massive molecules participating in the LSD1/BMP2/ SMAD4 network remain unascertained, we need to further clarify the regulatory mechanisms of miR-137 on the osteogenesis

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