M6A Demethylase Inhibits Osteogenesis of Dental Follicle Stem Cells via Regulating miR-7974/FKBP15 Pathway.

N6-methyladenosine (m6A) is the most abundant RNA modification, regulating gene expression in physiological processes. However, its effect on the osteogenic differentiation of dental follicle stem cells (DFSCs) remains unknown. Here, m6A demethylases, the fat mass and obesity-associated protein (FTO), and alkB homolog 5 (ALKBH5) were overexpressed in DFSCs, followed by osteogenesis assay and transcriptome sequencing to explore potential mechanisms. The overexpression of FTO or ALKBH5 inhibited the osteogenesis of DFSCs, evidenced by the fact that RUNX2 independently decreased calcium deposition and by the downregulation of the osteogenic genes OCN and OPN. MiRNA profiling revealed that miR-7974 was the top differentially regulated gene, and the overexpression of m6A demethylases significantly accelerated miR-7974 degradation in DFSCs. The miR-7974 inhibitor decreased the osteogenesis of DFSCs, and its mimic attenuated the inhibitory effects of FTO overexpression. Bioinformatic prediction and RNA sequencing analysis suggested that FK506-binding protein 15 (FKBP15) was the most likely target downstream of miR-7974. The overexpression of FKBP15 significantly inhibited the osteogenesis of DFSCs via the restriction of actin cytoskeleton organization. This study provided a data resource of differentially expressed miRNA and mRNA after the overexpression of m6A demethylases in DFSCs. We unmasked the RUNX2-independent effects of m6A demethylase, miR-7974, and FKBP15 on the osteogenesis of DFSCs. Moreover, the FTO/miR-7974/FKBP15 axis and its effects on actin cytoskeleton organization were identified in DFSCs.

Ultrasound-Targeted Microbubble Destruction (UTMD) Combined with Liposome Increases the Effectiveness of Suppressing Proliferation, Migration, Invasion, and Epithelial- Mesenchymal Transition (EMT) via Targeting Metadherin (MTDH) by ShRNA.

BACKGROUND Reports show that ultrasound-targeted microbubble destruction (UTMD) is a promising method of gene therapy, and metadherin (MTDH) is related to the development of breast cancer. Thus, we investigated the role of MTDH in breast cancer and compared the effect of suppressing MTDH by shRNA using liposome, UTMD, or the combination of these 2 methods. MATERIAL AND METHODS Graphing of survival curves of MTDH was analyzed by bioinformatics. UTMD was conducted using an ultrasonic therapeutic apparatus. Cell counting kit-8 (CCK-8) assay was used to measure cell viability. Migration and invasion rates were measured by wound healing test and Transwell invasion assay, respectively. The expression of MTDH, E-cadherin, metastasis-associated protein-1 (MTA-1), matrix metalloproteinase (MMP)-2, and MMP-9 were measured by Western blot and qPCR. RESULTS The prognosis of breast cancer can be decreased by the high expression of MTDH, and elevated expression of MTDH was discovered in MCF-7, MCF-10A, and T47D cell lines. UTMD combined with liposome is most efficient in transfecting shRNA, clearly suppressing the expression of MTDH and thereby decreasing cell viability, migration, invasion rate, and epithelial- mesenchymal transition (EMT) processes in the MCF-7 cell line. CONCLUSIONS UTMD combined with liposome could be used as a more efficient way to transfect shRNA into cells to suppress the expression of MTDH and thus lead to the downregulation of proliferation, migration, and EMT processes of the MCF-7 cell line, showing the potential for use in gene therapy.

Bleomycin: A novel osteogenesis inhibitor of dental follicle cells via a TGF-ß1/SMAD7/RUNX2 pathway.

BACKGROUND AND PURPOSE: Tooth eruption is a complicated process regulated by the dental follicles (DF). Our recent study discovered that tooth eruption was inhibited upon injection of bleomycin into DF. However, the mechanisms were unknown. EXPERIMENTAL APPROACH: Human dental follicle cells (hDFCs) were treated by bleomycin or exogenous TGF-ß1 or transfected by plasmids loading SMAD7 or shRNA targeting SMAD7, followed by osteogenesis induction assay and signalling analysis. Human fresh DF tissues and Wistar rats were used to further confirm bleomycin function. KEY RESULTS: Bleomycin decreased expression of RUNX2 and osteogenic genes in hDFCs, reducing osteogenic capacity. TGF-ß1 expression was up-regulated in bleomycin-treated hDFCs. The effects of exogenous TGF-ß1 were similar to those of bleomycin in hDFCs. Additionally, compared to SMAD2/3, SMAD7 expression increased more in bleomycin- or TGF-ß1-treated hDFCs. Overexpression of SMAD7 likewise significantly decreased RUNX2 expression and osteogenic capacity of hDFCs. Knockdown of SMAD7 markedly attenuated the inhibitory effects of bleomycin and TGF-ß1 on osteogenic capacity and RUNX2 expression of hDFCs. Most importantly, changes in TGF-ß1, SMAD7, and RUNX2 expressions were similar in the DF of rats and humans treated with bleomycin. CONCLUSION AND IMPLICATIONS: SMAD7 was a negative regulator of osteogenic differentiation in DFCs through suppressing RUNX2 expression. Bleomycin or TGF-ß1 inhibited osteogenic differentiation of DFCs via a TGF-ß1/SMAD7/RUNX2 pathway. Our findings might be beneficial for enhancing the osteogenic activity of DFCs or inhibiting the eruption of undesirable teeth.

Effects of bleomycin on tooth eruption: a novel potential application.

There are many kinds of potentially undesirable teeth. At present, surgical extraction is the most efficient way to eliminate these teeth, but it's very complex and invasive. In this study, we investigated the effects of bleomycin (BLM) on dental follicle and tooth eruption as a potential conservative therapy for undesirable teeth. Our data showed that local injection of 0.2 U/kg BLM had no significant effects on tooth eruption compared to the control group in Wistar rats. With higher dose of BLM (0.5 or 2 U/kg), the eruption of treated teeth was interrupted and their root formation failed until 4 weeks postnatal without significant systemic toxicity. Additionally, those effects were not depending on the toxicity of overdose evidenced by TUNEL assay. In summary, injecting BLM into dental follicle at an early stage could interrupt tooth development and eruption, and may prevent the potentially clinical problems resulting from undesirable teeth instead of surgical removal.

Elevated ATF4 Expression in Odontogenic Keratocysts Epithelia: Potential Involvement in Tissue Hypoxia and Stromal M2 Macrophage Infiltration.

The aim of this study was to investigate the expression of the activating transcription factor 4 (ATF4) in odontogenic keratocysts (OKC), its association with hypoxia and M2-polarized macrophages infiltration, and its potential relationships with angiogenesis in OKC. The expression of ATF4, hypoxia-inducible factor 1α (HIF-1α), macrophage colony-stimulating factor (M-CSF), and receptor activator of nuclear factor κ-B ligand (RANKL) in OKC samples and normal oral mucosa (OM) was detected by immunohistochemistry. Meanwhile, microvessel density (MVD) was measured using antibody against CD31. M2-polarized macrophages were identified using double-staining for CD68+ and CD163+. The correlations of ATF4 with HIF-1α, M-CSF, and M2-polarized macrophages infiltration were determined by Spearman's rank correlation test and hierarchical clustering. Human immortalized oral epithelial cells (HIOECs) were used in in vitro experiments. Our data showed that the expression of HIF-1α, ATF4, and M-CSF was significantly upregulated in the epithelium of OKC when compared with the OM. The expression of ATF4 was positively correlated with that of HIF-1α, M-CSF, MVD, and M2-polarized macrophages infiltration. Elevated expression of ATF4 in the epithelial lining of OKC may facilitate the M2 macrophages infiltration in response to hypoxia, leading to the development of OKC.