Mir24-2-5p suppresses the osteogenic differentiation with Gnai3 inhibition presenting a direct target via inactivating JNK-p38 MAPK signaling axis.

Background: Congenital anomalies are increasingly becoming a global pediatric health concern, which requires immediate attention to its early diagnosis, preventive strategies, and efficient treatments. Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 (Gnai3) gene mutation has been demonstrated to cause congenital small jaw deformity, but the functions of Gnai3 in the disease-specific microRNA (miRNA) upregulations and their downstream signaling pathways during osteogenesis have not yet been reported. Our previous studies found that the expression of Mir24-2-5p was significantly downregulated in the serum of young people with overgrowing mandibular, and bioinformatics analysis suggested possible binding sites of Mir24-2-5p in the Gnai3 3'UTR region. Therefore, this study was designed to investigate the mechanism of Mir24-2-5p-mediated regulation of Gnai3 gene expression and explore the possibility of potential treatment strategies for bone defects. Methods: Synthetic miRNA mimics and inhibitors were transduced into osteoblast precursor cells to regulate Mir24-2-5p expression. Dual-luciferase reporter assay was utilized to identify the direct binding of Gnai3 and its regulator Mir24-2-5p. Gnai3 levels in osteoblast precursor cells were downregulated by shRNA (shGnai3). Agomir, Morpholino Oligo (MO), and mRNA were microinjected into zebrafish embryos to control mir24-2-5p and gnai3 expression. Relevant expression levels were determined by the qRT-PCR and Western blotting. CCK-8 assay, flow cytometry, and transwell migration assays were performed to assess cell proliferation, apoptosis, and migration. ALP, ARS and Von Kossa staining were performed to observe osteogenic differentiation. Alcian blue staining and calcein immersions were performed to evaluate the embryonic development and calcification of zebrafish. Results: The expression of Mir24-2-5p was reduced throughout the mineralization process of osteoblast precursor cells. miRNA inhibitors and mimics were transfected into osteoblast precursor cells. Cell proliferation, migration, osteogenic differentiation, and mineralization processes were measured, which showed a reverse correlation with the expression of Mir24-2-5p. Dual-luciferase reporter gene detection assay confirmed the direct interaction between Mir24-2-5p and Gnai3 mRNA. Moreover, in osteoblast precursor cells treated with Mir24-2-5p inhibitor, the expression of Gnai3 gene was increased, suggesting that Mir24-2-5p negatively targeted Gnai3. Silencing of Gnai3 inhibited osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization. Promoting effects of osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization by low expression of Mir24-2-5p was partially rescued upon silencing of Gnai3. In vivo, mir24-2-5p Agomir microinjection into zebrafish embryo resulted in shorter body length, smaller and retruded mandible, decreased cartilage development, and vertebral calcification, which was partially rescued by microinjecting gnai3 mRNA. Notably, quite similar phenotypic outcomes were observed in gnai3 MO embryos, which were also partially rescued by mir24-2-5p MO. Besides, the expression of phospho-JNK (p-JNK) and p-p38 were increased upon Mir24-2-5p inhibitor treatment and decreased upon shGnai3-mediated Gnai3 downregulation in osteoblast precursor cells. Osteogenic differentiation and mineralization abilities of shGnai3-treated osteoblast precursor cells were promoted by p-JNK and p-p38 pathway activators, suggesting that Gnai3 might regulate the differentiation and mineralization processes in osteoblast precursor cells through the MAPK signaling pathway. Conclusions: In this study, we investigated the regulatory mechanism of Mir24-2-5p on Gnai3 expression regulation in osteoblast precursor cells and provided a new idea of improving the prevention and treatment strategies for congenital mandibular defects and mandibular protrusion.

New low viscous hydrophobic deep eutectic solvents in vortex-assisted liquid-liquid microextraction for the determination of phthalate esters from food-contacted plastics.

In this work, a novel class of eight low viscosity hydrophobic deep eutectic solvents (DESs) has been designed and prepared from two kinds of quaternary ammonium salts and six kinds of water-insoluble fatty alcohols/acids. Several physical properties of these DESs, such as melting point, density and viscosity, have been determined to investigate their adaptability as solvents. Then, such DESs are used as extraction solvents in a vortex-assisted liquid-liquid microextraction for the extraction and preconcentration of phthalate esters (PEs) from water samples, and the content of PEs from food-contacted plastics is then determined by using gas chromatography. It is found that the analytic method developed in this work exhibits wide linear range of 5-1000 µg L-1, and the limit of detections for target analytes is as low as 1 µg L-1. Finally, the proposed method has been successfully used for the determination of PEs from food-contacted plastics.

Platelet-rich plasma inhibits RANKL-induced osteoclast differentiation through activation of Wnt pathway during bone remodeling.

Platelet-rich plasma (PRP) is used in the clinic as an autologous blood product to stimulate bone regeneration and chondrogenesis. Numerous studies have demonstrated that PRP affects bone remodeling by accelerating osteoblast formation. With the research perspective focusing on osteoclasts, the present study established a mouse model of mandibular advancement to examine the effect of PRP on osteoclast differentiation induced by modification of the dynamics of the temporomandibular joint (TMJ). The lower incisors of the mice were trimmed by 1 mm and the resultant change in mandibular position during the process of eating induced condylar adaptation to this change. PRP significantly increased the bone mass and decreased osteoclastic activity, in vitro as well as in vivo. Mechanistically, the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL)­induced differentiation marker genes, including nuclear factor of activated T-cells, cytoplasmic 1, c-fos and tartrate-resistant acid phosphatase, and that of the resorptive activity marker genes such as cathepsin k, carbonic anhydrase 2 and matrix metalloproteinase 9, indicated that PRP suppresses RANKL-induced osteoclast differentiation. A microarray analysis revealed that several genes associated with the Wnt pathway were differentially expressed, which indicated the involvement of this pathway in osteoclast differentiation. Furthermore, the activation of the Wnt pathway was verified by reverse transcription-quantitative polymerase chain reaction and immunoblot analysis of Dickkopf-related protein 1 and ß-catenin. The results of the present study indicated that PRP inhibits osteoclast differentiation through activation of the Wnt pathway.

Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae.

During tooth root development, stem cells from apical papillae (SCAPs) are indispensable, and their abilities of proliferation, migration and odontoblast differentiation are linked to root formation. Leucine-rich repeat-containing GPCR 4 (LGR4) modulates the biological processes of proliferation and differentiation in multiple stem cells. In this study, we showed that LGR4 is expressed in all odontoblast cell lineage cells and Hertwig's epithelial root sheath (HERS) during the mouse root formation in vivo. In vitro we determined that LGR4 is involved in the Wnt/ß-catenin signaling pathway regulating proliferation and odonto/osteogenic differentiation of SCAPs. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that LGR4 is expressed during odontogenic differentiation of SCAPs. CCK8 assays and in vitro scratch tests, together with cell cycle flow cytometric analysis, demonstrated that downregulation of LGR4 inhibited SCAPs proliferation, delayed migration and arrested cell cycle progression at the S and G2/M phases. ALP staining revealed that blockade of LGR4 decreased ALP activity. QRT-PCR and Western blot analysis demonstrated that LGR4 silencing reduced the expression of odonto/osteogenic markers (RUNX2, OSX, OPN, OCN and DSPP). Further Western blot and immunofluorescence studies clarified that inhibition of LGR4 disrupted ß-catenin stabilization. Taken together, downregulation of LGR4 gene expression inhibited SCAPs proliferation, migration and odonto/osteogenic differentiation by blocking the Wnt/ß-catenin signaling pathway. These results indicate that LGR4 might play a vital role in SCAPs proliferation and odontoblastic differentiation.