miR-377-3p regulates adipogenic differentiation of human bone marrow mesenchymal stem cells by regulating LIFR

Molecular and Cellular Biochemistry - MicroRNAs are members of the family of non-coding small RNAs that regulate gene expression either by inhibiting mRNA translation or by promoting mRNA...     

miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have self-renewal and differentiation capabilities but the regulatory mechanisms of MSC fate determination remain poorly understood. Here, we aimed to identify microRNAs enriched in hMSCs that modulate differentiation commitments. Microarray analysis revealed that miR-140-5p is commonly enriched in undifferentiated hMSCs from various tissue sources. Moreover, bioinformatic analysis and luciferase reporter assay validated that miR-140-5p directly represses bone morphogenic protein 2 (BMP2). Furthermore, blocking miR-140-5p in hMSCs increased the expression of BMP signaling components and critical regulators of osteogenic differentiation. We propose that miR-140-5p functionally inhibits osteogenic lineage commitment in undifferentiated hMSCs.     

MicroRNA-100 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells by targeting BMPR2

Elucidation of the molecular mechanisms governing human adipose-derived mesenchymal stem cells (hASCs) osteogenic differentiation is of great importance for improving the treatment of bone-related diseases. In this study, we examined the role of microRNA (miR)-100 on the osteogenesis of hASCs. Overexpression of miR-100 inhibited osteogenic differentiation of hASCs in vitro, whereas downregulation of miR-100 enhanced the process. Target prediction analysis and dual luciferase report assay confirmed that bone morphogenetic protein receptor type II (BMPR2) was a direct target of miR-100. Furthermore, knockdown of BMPR2 by RNA interference inhibited osteogenic differentiation of hASCs, similar as the effect of upregulation miR-100. Taken together, our findings imply that miR-100 plays a negative role in osteogenic differentiation and might act through targeting BMPR2.     

MiRNA-1271-5p regulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting forkhead box O1 (FOXO1)

Forkhead box O1 (FOXO1) is a key regulator of osteogenesis. The aim of this study was to identify the mechanisms of microRNAs (miRNAs) targeting FOXO1 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). Three miRNA target prediction programs were used to search for potential miRNAs that target FOXO1. Quantitative real-time polymerase chain reaction was conducted to detect the expression of miR-1271-5p and FOXO1 during osteogenic differentiation. Target gene prediction and screening, luciferase reporter assay was used to verify the downstream target gene of miR-1271-5p. The expression levels of FOXO1 and Runx2 were detected by RT-qPCR and Western blot analysis. Alkaline phosphatase (ALP) activity and matrix mineralization were detected by biochemical methods. The expression levels of Runx2, ALP, and osteocalcin were detected by RT-qPCR. Our results showed that miR-1271-5p was downregulated during osteogenic induction. And the expression levels of miR-1271-5p were higher in osteoporotic tissues than that in adjacent nonosteoporotic tissues. The expression levels of FOXO1 were lower in osteoporotic tissues than that in adjacent nonosteoporotic tissues. And a negative correlation was found between miR-1271-5p and FOXO1 in osteoporotic tissues. Overexpression of miR-1271-5p downregulated FOXO1 and inhibited osteogenic differentiation in hMSCs. Overexpression of miR-1271-5p downregulated the expression of osteogenic markers and reduced ALP activity. In addition, ectopic expression of FOXO1 reversed the effect of miR-1271-5p on osteogenic differentiation. In conclusion, miR-1271-5p functioned as a therapeutic target of osteogenic differentiation in hMSCs by inhibiting FOXO1, which provides valuable insights into the use of miR-1271-5p as a target in the treatment of osteoporosis and other bone metabolic diseases.     

NT-3 promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells by regulating the Akt pathway

Objectives:To investigate the effect of neurotrophin-3 (NT-3) on osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods:Osteogenic differentiation was detected by alkaline phosphatase (ALP) staining and alizarin red staining (ARS). Adipogenic differentiation was detected by oil red O (ORO) staining. The expression of bone-related genes (Runx2, Osterix, OCN, ALP) and lipogenic genes (FABP4, PPAR, CEBP, LPL) was detected by real-time quantitative polymerase chain reaction (real-time qPCR). The expression of p-Akt and Akt protein was detected by Western blot assay.Results:ALP staining and ARS staining showed that the overexpression of NT-3 could promote the differentiation into osteoblasts, while knockdown of NT-3 could inhibit that. Real-time qPCR showed that the overexpression of NT-3 could increase the expression of osteoblast genes, while knockdown of NT-3 could inhibit that. ORO staining showed that the overexpression of NT-3 could inhibit the differentiation into adipogenesis, while knockdown of NT-3 can promote that. Real-time qPCR showed that the overexpression of NT-3 could reduce the expression of lipogenic genes. while knockdown NT-3 could increase that. In addition, the overexpression of NT-3 increased p-Akt/Akt levels significantly, while knockdown NT-3 reduced that significantly.Conclusion:NT-3 could promote the differentiation of mouse BMSCs into osteoblasts and inhibit their differentiation into adipogenesis.     

MicroRNA‐98 regulates osteogenic differentiation of human bone mesenchymal stromal cells by targeting BMP2

To study the effects of microRNA‐98 (miR‐98) on human bone mesenchymal stromal cells (hBMSCs). The patients undergoing hip arthroplasty were selected by inclusion/exclusion criteria for this study. The extracted hBMSCs were detected of osteogenic differentiation by alizarin red S staining, and of cell phenotype by flow cytometry. Bioinformatics, dual luciferase report, western blotting, RT‐PCR and immunoblotting were used in our study. The hBMSCs were divided into miR‐98 mimics, miR‐98 negative control (NC), miR‐98 inhibitors, Mock and miR‐98 inhibitors + siBMP2 groups. Human bone mesenchymal stromal cells were extracted and purified in vitro and had specific cytological morphology, surface markers and abilities of self‐renewal and differentiation. Compared with the NC group and Mock group, the miR‐98 mimics group showed increased miR‐98 level while the miR‐98 inhibitors group decreased miR‐98 level (both P < 0.01). Dual luciferase reporter showed BMP2 was the target gene of miR‐98. The levels of mRNA and protein expression of BMP2, protein expression of RUNX2, alkaline phosphatase activity and osteocalcin content significantly decreased in the miR‐98 mimics group while increased in the miR‐98 inhibitors group and showed no changes in the NC group and Mock group (all P < 0.05). The miR‐98 mimics group showed obviously declined stained red particles and the miR‐98 inhibitors group showed opposite result. After lowering the expression of miR‐98, osteogenic differentiation ability of hBMSCs rose, which was weakened by the transfection with siBMP2. miR‐98 may regulate osteogenic differentiation of hBMSCs by targeting BMP2.     

LncRNA TUG regulates osteogenic differentiation of bone marrow mesenchymal stem cells via miRNA-204/SIRT 1

Objective:To explore the regulation of LncRNA TUG /miRNA-204/SIRT1 pathway on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), so as to provide a new theoretical basis for the clinical treatment of osteoporosis.Methods:Detect changes of LncRNA and miRNA expression predicted in post-differentiation BMSCs with Western blot and qPCR tests. Verify the regulatory relationship between LncRNA and miRNA, miRNA and SIRT1 through the luciferase reporter assay. Transfect recombinant plasmids with LncRNA and their shRNA or transfected miRNA mimics and inhibitors.Results:According to the bioinformatic prediction, LncRNA TUG/miR-204 affected the regulation of SIRT1 on osteogenic differentiation of BMSCs, which were consistent with the results of luciferase reporter assay, namely, there are direct regulation targets between LncRNA TUG and miR-204, miR-204 and SIRT1. Overexpression and knockdown experiments revealed that LncRNA TUG overexpression/knockdown down/up-regulated miR-204 expression, which otherwise increased/decreased SIRT1 levels, and was positively correlated with osteogenic differentiation of BMSCs. Conversely, miR-204 was negatively correlated with LncRNA TUG and SIRT1, and negatively regulated osteogenic differentiation.Conclusion:This study found the direct regulatory relationship of LncRNA TUG/miR-204/SIRT1 during the osteogenic differentiation of BMSCs, and revealed that SIRT1 positively regulates the osteogenic differentiation of BMSCs, which provides a theoretical basis and potential therapeutic targets for a series of osteogenic differentiation-related diseases including osteoporosis.     

miR-4735-3p regulates phenotypic modulation of vascular smooth muscle cells by targeting HIF-1-mediated autophagy in intracranial aneurysm

Intracranial aneurysm (IA) is recognized as a lethal form of cerebrovascular disease mainly featured with a modulated phenotype of vascular smooth muscle cells (SMCs). It is generally believed that enhanced SMC proliferation and migration capabilities are the main characteristics in this process. In this study, we revealed that microRNA-4735 (miR-4735) participates in phenotypic modulation in a hypoxia-inducible factor-1 (HIF-1)-dependent manner of SMCs. miR-4735 targets the 3′-untranslated region of HIF-1. The downregulated expression of miR-4735 in IA tissues leads to elevated expression of HIF-1, which activates autophagy and promotes autophagy-mediated SMC proliferation and migration. Overexpression of miR-4735 suppressed HIF-1 expression and HIF-1-mediated autophagy, which led to impaired SMC proliferation and migration abilities. Forced expression of HIF-1 in miR-4735-overexpressed SMCs rescued the impaired SMC proliferation and migration abilities. In conclusion, miR-4735 plays an important role in phenotypic modulation in IA by regulating autophagy-promoted SMC proliferation and migration.     

Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells

Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assaying reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling.     

Exosomal miR-21-5p derived from bone marrow mesenchymal stem cells promote osteosarcoma cell proliferation and invasion by targeting PIK3R1

Mesenchymal stem cells (MSCs) are a class of pluripotent cells that can release a large number of exosomes which act as paracrine mediators in tumour-associated microenvironment. However, the role of MSC-derived exosomes in pathogenesis and progression of cancer cells especially osteosarcoma has not been thoroughly clarified until now. In this study, we established a co-culture model for human bone marrow-derived MSCs with osteosarcoma cells, then extraction of exosomes from induced MSCs and study the role of MSC-derived exosomes in the progression of osteosarcoma cell. The aim of this study was to address potential cell biological effects between MSCs and osteosarcoma cells. The results showed that MSC-derived exosomes can significantly promote osteosarcoma cells’ proliferation and invasion. We also found that miR-21-5p was significantly over-expressed in MSCs and MSC-derived exosomes by quantitative real-time polymerase chain reaction (qRT-PCR), compared with human foetal osteoblastic cells hFOB1.19. MSC-derived exosomes transfected with miR-21-5p could significantly enhance the proliferation and invasion of osteosarcoma cells in vitro and in vivo. Bioinformatics analysis and dual-luciferase reporter gene assays validated the targeted relationship between exosomal miR-21-5p and PIK3R1; we further demonstrated that miR-21-5p-abundant exosomes derived human bone marrow MSCs could activate PI3K/Akt/mTOR pathway by suppressing PIK3R1 expression in osteosarcoma cells. In summary, our study provides new insights into the interaction between human bone marrow MSCs and osteosarcoma cells in tumour-associated microenvironment.     

Vangl2 limits chaperone-mediated autophagy to balance osteogenic differentiation in mesenchymal stem cells

1. Download : Download high-res image (164KB)
2. Download : Download full-size image