Chanyuan Jin

Long Non-coding RNA H19 Inhibits Adipocyte Differentiation of Bone Marrow Mesenchymal Stem Cells through Epigenetic Modulation of Histone Deacetylases.

Bone marrow mesenchymal stem cells (BMSCs) exhibit an increased propensity toward adipocyte differentiation accompanied by a reduction in osteogenesis in osteoporotic bone marrow. However, limited knowledge is available concerning the role of long non-coding RNAs (lncRNAs) in the differentiation of BMSCs into adipocytes. In this study, we demonstrated that lncRNA H19 and microRNA-675 (miR-675) derived from H19 were significantly downregulated in BMSCs that were differentiating into adipocytes. Overexpression of H19 and miR-675 inhibited adipogenesis, while knockdown of their endogenous expression accelerated adipogenic differentiation. Mechanistically, we found that miR-675 targeted the 3′ untranslated regions of the histone deacetylase (HDAC) 4–6 transcripts and resulted in deregulation of HDACs 4–6, essential molecules in adipogenesis. In turn, trichostatin A, an HDAC inhibitor, significantly reduced CCCTC-binding factor (CTCF) occupancy in the imprinting control region upstream of the H19 gene locus and subsequently downregulated the expression of H19. These results show that the CTCF/H19/miR-675/HDAC regulatory pathway plays an important role in the commitment of BMSCs into adipocytes.

The epigenetic promotion of osteogenic differentiation of human adipose-derived stem cells by the genetic and chemical blockade of histone demethylase LSD1.

Human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering. It has become increasingly clear that chromatin regulators play an important role in cell fate determination. However, how osteogenic differentiation of hASCs is controlled by epigenetic mechanisms is not fully understood. Here we use genetic tools and chemical inhibitors to modify the epigenetic program of hASCs and identify lysine-specific demethylase 1 (LSD1), a histone demethylase that specifically catalyzes demethylation of di- and mono- methyl histone H3 lysine 4 (H3K4me2/1), as a key regulator in osteogenic differentiation of hASCs. Specifically, we demonstrated that genetic depletion of LSD1 with lentiviral strategy for gene knockdown promoted osteogenic differentiation of hASCs by cell studies and xenograft assays. At the molecular level, we found that LSD1 regulates osteogenesis-associated genes expression through its histone demethylase activity. Significantly, we demonstrated LSD1 demethylase inhibitors could efficiently block its catalytic activity and epigenetically boost osteogenic differentiation of hASCs. Altogether, our study defined the functional and biological roles of LSD1 and extensively explored the effects of its enzymatic activity in osteogenic differentiation of hASCs. A better understanding of how LSD1 influences on osteogenesis associated epigenetic events will provide new insights into the modulation of hASCs based cell therapy and improve the development of bone tissue engineering with epigenetic intervention.

Inhibition of lncRNA MIR31HG Promotes Osteogenic Differentiation of Human Adipose‐Derived Stem Cells

Osteogenic differentiation and bone formation is suppressed under condition of inflammation induced by proinflammation cytokines. A number of studies indicate miRNAs play a significant role in tumor necrosis factor‐α‐induced inhibition of bone formation, but whether long non‐coding RNAs are also involved in this process remains unknown. In this study, we evaluated the role of MIR31HG in osteogenesis of human adipose‐derived stem cells (hASCs) in vitro and in vivo. The results suggested that knockdown of MIR31HG not only significantly promoted osteogenic differentiation, but also dramatically overcame the inflammation‐induced inhibition of osteogenesis in hASCs. Mechanistically, we found MIR31HG regulated bone formation and inflammation via interacting with NF‐κB. The p65 subunit bound to the MIR31HG promoter and promoted MIR31HG expression. In turn, MIR31HG directly interacted with IκBα and participated in NF‐κB activation, which builds a regulatory circuitry with NF‐κB. Targeting this MIR31HG–NF‐κB regulatory loop may be helpful to improve the osteogenic capacity of hASCs under inflammatory microenvironment in bone tissue engineering. Stem Cells 2016;34:2707–2720

Long non-coding RNA MEG3 inhibits adipogenesis and promotes osteogenesis of human adipose-derived mesenchymal stem cells via miR-140-5p

AbstractlncRNAs are an emerging class of regulators involved in multiple biological processes. MEG3, an lncRNA, acts as a tumor suppressor, has been reported to be linked with osteogenic differentiation of MSCs. However, limited knowledge is available concerning the roles of MEG3 in the multilineage differentiation of hASCs. The current study demonstrated that MEG3 was downregulated during adipogenesis and upregulated during osteogenesis of hASCs. Further functional analysis showed that knockdown of MEG3 promoted adipogenic differentiation, whereas inhibited osteogenic differentiation of hASCs. Mechanically, MEG3 may execute its role via regulating miR-140-5p. Moreover, miR-140-5p was upregulated during adipogenesis and downregulated during osteogenesis in hASCs, which was negatively correlated with MEG3. In conclusion, MEG3 participated in the balance of adipogenic and osteogenic differentiation of hASCs, and the mechanism may be through regulating miR-140-5p.

Histone Acetyltransferase GCN5 Regulates Osteogenic Differentiation of Mesenchymal Stem Cells by Inhibiting NF-κB.

As the most well‐studied histone acetyltransferase (HAT) in yeast and mammals, general control nonderepressible 5 (GCN5) was documented to play essential roles in various developmental processes. However, little is known about its role in osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we detected the critical function of GCN5 in osteogenic commitment of MSCs. In this role, the HAT activity of GCN5 was not required. Mechanistically, GCN5 repressed nuclear factor kappa B (NF‐κB)‐dependent transcription and inhibited the NF‐κB signaling pathway. The impaired osteogenic differentiation by GCN5 knockdown was blocked by inhibition of NF‐κB. Most importantly, the expression of GCN5 was decreased significantly in the bone tissue sections of ovariectomized mice or aged mice. Collectively, these results may point to the GCN5‐NF‐κB pathway as a novel potential molecular target for stem cell mediated regenerative medicine and the treatment of metabolic bone diseases such as osteoporosis.

Long non-coding RNA MIAT knockdown promotes osteogenic differentiation of human adipose derived stem cells†

Recently, long non‐coding RNAs (lncRNAs) have emerged as critical players in gene regulation for multiple biological processes. However, their roles and functions in human adipose‐derived stem cells (hASCs) differentiation remain unclear. In the present study, we investigated the role of lncRNA myocardial infarction‐associated transcript (MIAT) in the osteogenic differentiation of hASCs. We found that the expression of MIAT was downregulated in a time‐dependent manner during hASCs osteoinduction. MIAT knockdown promoted osteogenic differentiation of hASCs both in vitro and in vivo. Moreover, MIAT expression was increased upon tumor necrosis factor‐α treatment and MIAT knockdown reversed the negative effects of inflammation on osteoblastic differentiation. This study improves our knowledge of lncRNAs in governing the osteogenic differentiation of hASCs and may provide novel therapeutic strategies for treating bone diseases.

MiR-34a Promotes Osteogenic Differentiation of Human Adipose-Derived Stem Cells via the RBP2/NOTCH1/CYCLIN D1 Coregulatory Network

Summary MiR-34a was demonstrated to be upregulated during the osteogenic differentiation of human adipose-derived stem cells (hASCs). Overexpression of miR-34a significantly increased alkaline phosphatase activity, mineralization capacity, and the expression of osteogenesis-associated genes in hASCs in vitro. Enhanced heterotopic bone formation in vivo was also observed upon overexpression of miR-34a in hASCs. Mechanistic investigations revealed that miR-34a inhibited the expression of retinoblastoma binding protein 2 (RBP2) and reduced the luciferase activity of reporter gene construct comprising putative miR-34a binding sites in the 3′ UTR of RBP2. Moreover, miR-34a downregulated the expression of NOTCH1 and CYCLIN D1 and upregulated the expression of RUNX2 by targeting RBP2, NOTCH1, and CYCLIN D1. Taken together, our results suggested that miR-34a promotes the osteogenic differentiation of hASCs via the RBP2/NOTCH1/CYCLIN D1 coregulatory network, indicating that miR-34a-targeted therapy could be a valuable approach to promote bone regeneration.

Histone H3K9 Acetyltransferase PCAF is Essential for Osteogenic Differentiation through BMP Signaling and May Be Involved in Osteoporosis

Human mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into osteoblasts, chondrocytes, and adipocytes. The importance of epigenetic regulation for osteogenic differentiation of MSCs is widely accepted. However, the molecular mechanisms are poorly understood. Here, we show that histone H3K9 acetyltransferase PCAF plays a critical role in osteogenic differentiation of MSCs. Knockdown of PCAF significantly reduced the bone formation both in vitro and in vivo. Mechanistically, PCAF controls BMP signaling genes expression by increasing H3K9 acetylation. Most importantly, PCAF expression is significantly decreased in bone sections of ovariectomized or aged mice. Histone modification enzyme is chemically modifiable; therefore, PCAF may represent a novel therapeutic target for stem cell‐mediated regenerative medicine and the treatment of osteoporosis. Stem Cells 2016;34:2332–2341

Histone H3K9 Acetyltransferase PCAF Is Essential for Osteogenic Differentiation Through Bone Morphogenetic Protein Signaling and May Be Involved in Osteoporosis

Human mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into osteoblasts, chondrocytes, and adipocytes. The importance of epigenetic regulation for osteogenic differentiation of MSCs is widely accepted. However, the molecular mechanisms are poorly understood. Here, we show that histone H3K9 acetyltransferase PCAF plays a critical role in osteogenic differentiation of MSCs. Knockdown of PCAF significantly reduced the bone formation both in vitro and in vivo. Mechanistically, PCAF controls BMP signaling genes expression by increasing H3K9 acetylation. Most importantly, PCAF expression is significantly decreased in bone sections of ovariectomized or aged mice. Histone modification enzyme is chemically modifiable; therefore, PCAF may represent a novel therapeutic target for stem cell‐mediated regenerative medicine and the treatment of osteoporosis. Stem Cells 2016;34:2332–2341

Knockdown of lncRNA MIR31HG inhibits adipocyte differentiation of human adipose-derived stem cells via histone modification of FABP4

Adipogenesis plays an important role in the regulation of whole-body energy homeostasis and is inextricably related to obesity. Several studies have highlighted the relevance of microRNAs in adipocyte differentiation, but the contributions of long non-coding RNAs (lncRNAs) are still largely uncharacterized. Here, we determined that lncRNA MIR31HG is related to adipocyte lineage commitment. We demonstrated that knockdown of MIR31HG inhibited adipocyte differentiation, whereas overexpression of MIR31HG promoted adipogenesis in vitro and in vivo. Furthermore, inhibition of MIR31HG reduced the enrichment of active histone markers, histone H3 lysine 4 trimethylation (H3K4me3) and acetylation (AcH3), in the promoter of the adipogenic-related gene, fatty acid binding protein 4 (FABP4), leading to suppression of its expression and adipogenesis. These results provide new insights into the molecular mechanisms of MIR31HG in terms of adipogenesis and may have implications for obesity and associated disorders.