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Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

ABSTRACT: Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

SUBMITTER: Chang H

PROVIDER: S-EPMC5870583 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

Chang Huaiguang H Wang Yue Y Liu Haochen H Nan Xu X Wong Singwai S Peng Saihui S Gu Yajuan Y Zhao Hongshan H Feng Hailan H

Cell death & disease 20171214 12

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY ...[more]

PMID: 29242628

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Project description:ObjectiveIntestinal ischemia-reperfusion (II/R) is a common pathological injury in clinic, and the systemic inflammatory response it causes will lead to multiple organ damage and functional failure. miR-185-5p has been reported to be a regulator of inflammatory response and autophagy, but whether it participates in the regulation of autophagy in II/R is still unclear. Therefore, we aimed to explore the mechanism of miR-185-5p regulating intestinal barrier injury in (II/R).MethodsCaco-2 cells was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to establish II/R model. The superior mesenteric artery of C57BL/6 mice was clamped for 45 min and then subjected to reperfusion for 4 h for the establishment of II/R mice model. miR-185-5p mimic, miR-185-5p inhibitor, pcDNA-autophagy-related 101 (ATG101) were respectively transfected into Caco-2 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess miR-185-5p expression. Western blot detected the level of ATG101 and tight junction-associated proteins ZO1, Occludin, E-cadherin, β-catenin, as well as autophagy markers ATG5, ATG12, LC3Ⅰ/Ⅱ, Beclin1 and SQSTM1. Transepithelial electrical resistance (TEER) values was detected by a resistance meter. FITC-Dextran was performed to measure cell permeability. 5-ethynyl-2'-deoxyuridine (EDU) staining measured cell proliferation. Transmission electron microscope was conducted to observe autophagosomes. Hematoxylin & eosin (H&E) staining observed the damage of mice intestinal. Immunohistochemistry (IHC) measured the percentage of ki67 positive cells. TdT-mediated dUTP nick-end labeling (TUNEL) assay assessed cell apoptosis in intestinal tissues of II/R. Dual-luciferase assay verified the targeting relationship between miR-185-5p and ATG101.Results miR-185-5p was overexpressed in OGD/R-induced Caco-2 cells and intestinal tissues of II/R mice. Knocking down miR-185-5p markedly promoted autophagy and TEER values, reduced cell permeability, and alleviated intestinal barrier damage. ATG101 was a target of miR-185-5p, and overexpression of ATG101 promoted autophagy and dampened OGD/R-induced intestinal barrier damage. Overexpression of miR-185-5p reversed the effect of overexpressed ATG101 on OGD/R-induced Caco-2 cells.ConclusionKnockdown of miR-185-5p enhanced autophagy and alleviated II/R intestinal barrier damage by targeting ATG101.

Dataset Information

Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

Publications

Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

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