IRF4 suppresses osteogenic differentiation of BM-MSCs by transcriptionally activating miR-636/DOCK9 axis.

OBJECTIVES: Osteoblasts are derived from Bone Marrow-derived Mesenchymal Stem Cells (BM-MSCs), which play an indispensable role in bone formation. In this study, the authors aim to investigate the role of IRF4 in the osteogenic differentiation of BM-MSCs and its potential molecular mechanism. METHODS: The authors used lentivirus infection to overexpress IRF4 in BM-MSCs. The expression of IRF4 and osteogenesis-related genes were detected by qRT-PCR and western blot analysis. The osteogenic differentiation of BM-MSCs was evaluated by Alkaline Phosphatase (ALP) activity, Alizarin red staining, and Alkaline Phosphatase (ALP) staining. Chromatin Immunoprecipitation (ChIP), Dual-Luciferase reporter assay and RNA Immunoprecipitation Assay were applied to confirm the regulatory mechanism between IRF4, miR-636 and DOCK9. RESULTS: The authors found IRF4 was down-regulated during the osteogenic differentiation of BM-MSCs, and IRF4 overexpression could decrease the osteogenic differentiation of BM-MSCs by specifically promoting the reduction of Alkaline Phosphatase (ALP) activity and down-regulating osteogenic indicators, including OCN, OPN, Runx2 and CollA1. Mechanistically, IRF4 activated microRNA-636 (miR-636) expression via binding to its promoter region, and Dedicator of Cytokinesis 9 (DOCK9) was identified as the target of miR-636 in BM-MSCs. Moreover, the damage in the capacity of osteogenic differentiation of BM-MSCs induced by IRF4 overexpression could be rescued by miR-636 inhibition. CONCLUSIONS: In summary, this paper proposed that IRF4/miR-636/DOCK9 may be considered as targets for the treatment of osteoporosis (OP).

IRF4 suppresses osteogenic differentiation of BM-MSCs by transcriptionally activating miR-636/DOCK9 axis

Abstract Objectives Osteoblasts are derived from Bone Marrow-derived Mesenchymal Stem Cells (BM-MSCs), which play an indispensable role in bone formation. In this study, the authors aim to investigate the role of IRF4 in the osteogenic differentiation of BM-MSCs and its potential molecular mechanism. Methods The authors used lentivirus infection to overexpress IRF4 in BM-MSCs. The expression of IRF4 and osteogenesis-related genes were detected by qRT-PCR and western blot analysis. The osteogenic differentiation of BM-MSCs was evaluated by Alkaline Phosphatase (ALP) activity, Alizarin red staining, and Alkaline Phosphatase (ALP) staining. Chromatin Immunoprecipitation (ChIP), Dual-Luciferase reporter assay and RNA Immunoprecipitation Assay were applied to confirm the regulatory mechanism between IRF4, miR-636 and DOCK9. Results The authors found IRF4 was down-regulated during the osteogenic differentiation of BM-MSCs, and IRF4 overexpression could decrease the osteogenic differentiation of BM-MSCs by specifically promoting the reduction of Alkaline Phosphatase (ALP) activity and down-regulating osteogenic indicators, including OCN, OPN, Runx2 and CollA1. Mechanistically, IRF4 activated microRNA-636 (miR-636) expression via binding to its promoter region, and Dedicator of Cytokinesis 9 (DOCK9) was identified as the target of miR-636 in BM-MSCs. Moreover, the damage in the capacity of osteogenic differentiation of BM-MSCs induced by IRF4 overexpression could be rescued by miR-636 inhibition. Conclusions In summary, this paper proposed that IRF4/miR-636/DOCK9 may be considered as targets for the treatment of osteoporosis (OP).

Enhanced Wnt signaling by methylation-mediated loss of SFRP2 promotes osteosarcoma cell invasion.

Wnt signaling is essential for the initiation and progression of osteosarcoma (OS) tumors and is suppressed by the secreted frizzled-related proteins (SFRPs). The methylation-induced protein degradation reduces the activity of SFRPs and subsequently increases the activity of Wnt signaling. However, whether the methylation of SFRP2, a member of SFRPs, may be involved in the pathogenesis of OS is not known. Here, we investigated the expression levels of SFRP2 in OS specimens. We found that SFRP2 mRNA was significantly decreased and methylation of SFRP2 gene was significantly increased in malignant OS tumors as compared to the paired adjacent non-tumor tissue. Moreover, SFRP2 expression was significantly decreased in the malignant OS cell lines, SAOS2, MG63, and U2OS, but not in the primary osteoblast cells. The demethylation of SFRP2 gene by 5'-aza-deoxycytidine (5-aza-dCyd) in OS cell lines restored SFRP2 expression, at both mRNA and protein levels, and suppressed cell invasion. Furthermore, the demethylation of SFRP2 gene appeared to inhibit nuclear retention of a key Wnt signaling factor, ß-catenin, in OS cell lines. Together, these data suggest that SFRP2 may function as an OS invasion suppressor by interfering with Wnt signaling, and the methylation of SFRP2 gene may promote pathogenesis of OS.

Experimental Research on Differentiation-Inducing Growth of Nerve Lateral Bud by HUC-MSCs Chitosan Composite Conduit.

This study is intended to explore the role of human umbilical-cord-derived mesenchymal stem cells (HUC-MSCs) in nerve end-to-side anastomosis, as well as in the induction and promotion of growth of nerve lateral bud. The chitosan nerve conduit was prepared based on the biological characteristics of chitosan, and the nerve conduit was filled with HUC-MSCs, and was used to bridge the nerve end-to-side anastomotic stoma. The experimental animals were randomly assigned into three groups (10 in each group), and the nerve end-to-side anastomosis was conducted: (1) group A (control group): traditional tibial nerve-common peroneal nerve end-to-side anastomosis; (2) group B (experimental group 1): tibial nerve-common peroneal nerve end-to-side anastomotic stoma bridged with chitosan nerve conduit; (3) group C (experimental group 2): tibial nerve-common peroneal nerve end-to-side anastomotic stoma bridged by chitosan nerve conduit filled with HUC-MSCs. General morphological observation, nerve electrophysiology, and anti-S-100 immunohistochemistry were performed. All experimental animals survived, and no infections were found at operative incisions. The nerve continuity was in good condition through visual observation when sampling, which is mild adhesion to the surrounding tissue and easy to be separated. 12 W HUC-MSCs chitosan composite nerve conduits were degraded completely after operation. Electrophysiological test showed that the nerve conduction velocity (NCV) in group C was significantly higher than that in group A or group B (p < 0.01). There were no significant differences between NCVs of group A and group B. Toluidine blue staining and transmission electron microscope showed that the number of the medullated fibers and the myelin sheath thickness in group C were larger than those in group A or B. There were no significant differences between the numbers of the medullated fibers and between the myelin sheath thicknesses of groups A and B. By means of anti-S-100 immunohistochemistry, the arrangement of a large number of brown-red proliferating schwann cells around the regenerated nerve fibers in group C could be found, while fewer and sparse brown-red matters and very poor growth of schwann cells could be observed in groups A and B. Slightly more favorable situation could be observed in group B compared with group A. HUC-MSCs play obviously an important role in promoting nerve regeneration during the nerve end-to-side anastomosis, which induces the growth of axis bud, accelerates the growth velocity of regenerated fiber, and promotes the growth and maturity of schwann cells.

Inhibition of macrophage polarization prohibits growth of human osteosarcoma.

Osteosarcoma is the most malignant bone tumor characterized by high local aggressiveness and poor therapeutic outcome. Tumor-associated macrophages (TAM) have been shown to participate in the development and progress of many types of cancer cells. However, whether TAM may play a role in the pathogenesis of osteosarcoma is largely unknown. In a mouse model of human osteosarcoma implantation, we showed that the recruited macrophages at the site of the implanted tumor were polarized to an M2 subtype (same as TAM) during the development and growth of the osteosarcoma. In a loss-of-function experiment, we deleted these TAM with a specific macrophage-eliminating liposome, which resulted in decreased tumor growth. Moreover, when the epidermal growth factor receptor (EGFR) in the implanted cancer cells was inhibited by shRNA, the tumor failed to grow in response to the recruited macrophages. Taken together, for the first time, we show that the growth of an osteosarcoma is EGFR signaling-dependent and TAM-mediated. Our data suggest that TAM and EGFR may be good targets for treating human osteosarcoma.