MiR-152-5p suppresses osteogenic differentiation of mandible mesenchymal stem cells by regulating ATG14-mediated autophagy.

BACKGROUND: Osteoporosis affects the mandible resulting in bone loss. Though impairments are not life threatening, they affect a person's quality-of-life particularly vulnerable elderly. MicroRNAs (miRNAs) are novel regulatory factors that play an important role in regulating bone metabolism. Autophagy is evolutionarily conserved intracellular self-degradation process and is vital in the maintenance of both miRNA and bone homeostasis. However, the role of autophagy in the pathogenesis of miRNA regulating osteoporosis remains unclear. METHODS: In the study, we established a rat osteoporosis model induced by ovariectomy (OVX) and isolated mesenchymal stem cells from mandible (MMSCs-M). Several miRNAs were identified to regulate osteoporosis in some studies. qRT-PCR was applied to examine the expression of miRNA, autophagy and osteogenic differentiation-related genes. Western blotting assays were performed to detect the expression of autophagy and osteogenic differentiation proteins. Immunofluorescence and transmission electron microscope were used to verify the autophagy activity. Transfecting technology was used to enhance or suppress the expression of miR-152-5p which enable us to observe the relationship between miR-152-5p, autophagy and osteogenic differentiation. Additionally, the measurement of reactive oxygen species was used to investigate the mechanism of autophagy affecting osteogenic differentiation. RESULTS: We found an upregulated expression of miR-152-5p in MMSCs-M in OVX group. Downregulated autophagy-related gene, proteins and autophagosome were detected in vitro of OVX group compared with sham group. Moreover, downregulation of miR-152-5p promoted osteogenic differentiation of MMSCs-M as well as enhanced autophagy-related proteins in OVX group. Conversely, overexpression of miR-152-5p showed opposite effect in sham group. Meanwhile, we found Atg14 (autophagy-related protein homolog 14) was identified to be a direct target of miR-152-5p theoretically and functionally. In other words, we confirmed inhibition of miR-152-5p promoted the osteogenic differentiation via promoting ATG14-mediated autophagy. Furthermore, miR-152-5p/ATG14-mediated autophagy regulated osteogenic differentiation by reducing the endogenous ROS accumulation and maintaining cellular redox homeostasis. CONCLUSION: Our data suggest that miR-152-5p is the first identified to regulate osteogenic differentiation by directly targeting autophagy-related protein ATG14 and regulating oxidative stress and therapeutic inhibition of miR-152-5p may be an efficient anabolic strategy for osteoporosis.

CHNQD-00603 Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by the miR-452-3p-Mediated Autophagy Pathway.

Background: Periodontitis is a chronic and progressive disease accompanied by bone loss. It is still a challenge to restore the bone structure. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) plays a decisive role in bone restoration and regeneration. Marine natural products (MNPs) have multiple biological activities, including anti-tumor and anti-inflammatory properties. However, the exploration of MNPs in osteogenesis is far from sufficient. Methods: We obtained a series of derivatives through structural optimization from 4-phenyl-3,4-dihydroquinolin-2(1H)-one alkaloid isolated from Scopulariopsis sp. Some preliminary cytological experiments showed that CHNQD-00603, obtained by adding a methoxy group to the position C3 and a hydroxyl group to the position C4 of 4-phenyl-3,4-dihydroquinolin-2(1H)-one, might promote the osteogenic differentiation of BMSCs. To further investigate the effects of CHNQD-00603 on BMSCs, we performed a CCK-8 assay and qRT-PCR, alkaline phosphatase staining (ALP), and alizarin red S staining to assess the cytotoxicity and the ability of osteogenic differentiation of CHNQD-00603. The autophagy level was assessed and validated by WB, qRT-PCR, and transmission electron microscopy. Then, 3-methyladenine (3-MA) was added to further examine the role of autophagy. Based on the expression of autophagy-related genes, we predicted and examined the potential miRNAs by bioinformatics. Results: CCK-8 assay showed that CHNQD-00603 at 1 µg/ml did not influence BMSCs activity. However, the proliferation rate decreased from the seventh day. qRT-PCR, ALP staining, ALP activity assay, and Alizarin red S staining showed that the best concentration of CHNQD-00603 to promote osteogenic differentiation was 1 µg/ml. Further investigations indicated that CHNQD-00603 activated autophagy, and the inhibition of autophagy by 3-MA attenuated CHNQD-00603-enhanced osteogenic differentiation. Subsequently, the findings from bioinformatics and qRT-PCR indicated that miR-452-3p might be a regulator of autophagy and osteogenesis. Furthermore, we transfected BMSCs with miR-452-3p NC and mimics separately to further determine the function of miR-452-3p. The data showed that the overexpression of miR-452-3p moderated the level of autophagy and osteogenic differentiation of CHNQD-00603-treated BMSCs. Conclusion: Our data suggested that CHNQD-00603 promoted the osteogenic differentiation of BMSCs by enhancing autophagy. Meanwhile, miR-452-3p played a regulatory role in this process.

A chimeric peptide of intestinal trefoil factor containing cholesteryl ester transfer protein B cell epitope significantly inhibits atherosclerosis in rabbits after oral administration.

Vaccination against cholesteryl ester transfer protein (CETP) is proven to be effective for inhibiting atherosclerosis in animal models. In this study, the proteases-resistant intestinal trefoil factor (TFF3) was used as a molecular vehicle to construct chimeric TFF3 (cTFF3) containing CETP B cell epitope and tetanus toxin helper T cell epitope. It was found that cTFF3 still preserved a trefoil structure, and can resist proteases digestion in vitro. After oral immunization with cTFF3, the CETP-specific IgA and IgG could be found in intestine lavage fluid and serum, and the anti-CETP antibodies could inhibit partial CETP activity to increase high-density lipoprotein cholesterol, decrease low-density lipoprotein cholesterol, and inhibit atherosclerosis in animals. Therefore, TFF3 is a potential molecular vehicle for developing oral peptide vaccines. Our research highlights a novel strategy for developing oral peptide vaccines in the future.