LncRNA HOTAIRM1 promotes dental follicle stem cell-mediated bone regeneration by regulating HIF-1α/KDM6/EZH2/H3K27me3 axis.

Large bone defect reconstruction undergoes hypoxia and remains a major practical challenge. Bone tissue engineering with a more promising stem cell source facilitates the development of better therapeutic outcomes. Human dental follicle stem cells (hDFSCs) with superior multipotency, osteogenic capacity, and accessibility have been proven a promising cell source for bone regeneration. We previously identified a novel long noncoding RNA (lncRNA), HOTAIRM1, to be highly expressed in hDFSCs. Here we found that HOTAIRM1 overexpressed hDFSCs promoted bone regeneration in rat critical-size calvarial defect model. Mechanically, HOTAIRM1 was induced in hDFSCs under hypoxic conditions and activated HIF-1α. RNA-sequencing analysis indicated that HOTAIRM1 upregulated oxygen-sensing histone demethylases KDM6A/B and suppressed methyltransferase EZH2 via targeting HIF-1α. The osteogenic differentiation of hDFSCs was accompanied with demethylation of H3K27, and HOTAIRM1 overexpression decreased the distribution of H3K27me3 in osteogenic genes, including ALP, M-CSF, Wnt-3a, Wnt-5a, Wnt-7a, and ß-catenin, thus promoted their transcription. Our study provided evidence that HOTAIRM1 upregulated KDM6A/B and inhibited EZH2 in a HIF-1α dependent manner to enhance the osteogenesis of hDFSCs. HOTAIRM1-mediated hDFSCs may serve as a promising therapeutic approach to promote bone regeneration in clinical practice.

Three-dimensional evaluation of soft tissues in hyperdivergent skeletal class II females in Guangdong.

OBJECTIVES: To establish the three-dimensional facial soft tissue morphology of adolescent and adult females in the Guangdong population and to study the morphological characteristics of hyperdivergent skeletal class II females in Guangdong compared with that of normodivergent class I groups. MATERIALS AND METHODS: The 3dMDface system was used to capture face scans of 160 patients, including 45 normal and 35 hyperdivergent skeletal class II adolescents (aged 11-14 years old) and 45 normal and 35 hyperdivergent skeletal class II adults (aged 18-30 years old). Thirty-two soft tissue landmarks were mapped, and 21 linear, 10 angular and 17 ratio measurements were obtained by 3dMDvultus analysis software. Data were assessed with a t-test of two independent samples between the normal adolescent and adult groups and between the normal and hyperdivergent skeletal class II groups. RESULTS: The linear measurements of the Guangdong adult females were larger than those of the adolescents in both Class I and Class II groups. However, the angular and ratio measurements had no significant difference. The vertical linear measurements were higher and the sagittal and transverse linear measurements were smaller in the hyperdivergent class II group (p < 0.05). The soft tissue ANB angle, chin-lip angle, and mandibular angle were significantly larger and the soft tissue facial convexity angle and nasal convexity angle were significantly smaller in the hyperdivergent class II group (p < 0.05). Additionally, there were significant differences in the ratio measurements between the hyperdivergent class II groups and the control groups (p < 0.05). CONCLUSIONS: The three-dimensional facial morphology of Guangdong adolescent and adult females was acquired. The facial soft tissue measurements of the adults were higher in the three dimensions except for the facial convexity and proportional relationships which were similar, suggesting that the growth pattern remained the same. The three-dimensional facial soft tissue features of hyperdivergent skeletal class II were characterized by the terms "long, convex, and narrow". Three-dimensional facial measurements can reflect intrinsic hard tissue characteristics.

lncRNA HOTAIRM1 promotes osteogenesis of hDFSCs by epigenetically regulating HOXA2 via DNMT1 in vitro.

Dental follicle (DF) can develop into periodontal tissues including periodontal ligament, cementum, and alveolar bone. Possessing superior pluripotency and osteogenic capacity, dental follicle stem cells (DFSCs) have become a promising stem cell source for bone regeneration and periodontal engineering. However, the mechanisms underlying DFSCs-mediated osteogenesis remain elusive. Our previous long noncoding RNA (lncRNA) microarray revealed that lncRNA HOTAIRM1 was significantly higher expressed in human DFSCs (hDFSCs) compared with human periodontal ligament stem cells (hPDLSCs). lncRNA HOTAIRM1, an antisense transcript of the HOXA1/2 intergenic region, can epigenetically regulate proximal and distant HOXA genes through histone and DNA methylation. HOXA2, a target of HOTAIRM1, is crucial for cranial neural crest morphogenesis, branchial arches development, and osteogenesis. However, the roles of both HOTAIRM1 and HOXA2 in odontogenic stem cells remain unknown. Here, we investigated the functions and regulatory mechanisms of these two genes in hDFSCs. Both genes were confirmed highly expressed in hDFSCs compared with hPDLSCs, and they displayed similar expression patterns in the DF and surrounding periodontium during mice tooth morphogenesis. Knockdown of either HOTAIRM1 or HOXA2 inhibited osteogenic differentiation of hDFSCs, while overexpressed HOTAIRM1 inhibited hDFSCs proliferation and promoted osteogenesis. Furthermore, HOTAIRM1 inhibited both overall DNMT1 expression and DNMT1 enrichment on HOXA2 promoter, mechanically binding to the CpG islands of the HOXA2 promoter region, leading to hypomethylation and HOXA2 induction. These findings suggested that HOTAIRM1 promoted the osteogenesis of hDFSCs by epigenetically regulating HOXA2 via DNMT1. Taken together, HOTARIM1 and HOXA2 exerted pivotal functions in hDFSCs, and the regulatory mechanism of HOTARIM1 within the HOXA cluster was uncovered.

Down-regulated lncRNA MEG3 promotes osteogenic differentiation of human dental follicle stem cells by epigenetically regulating Wnt pathway.

Our previous long noncoding RNA (lncRNA) microarray results showed that lncRNA MEG3 (maternally expressed 3) was significantly downregulated in human dental follicle cells than human periodontal ligament cells. Latest studies show that MEG3 contributes to polycomb repressive complex 2 (PRC2) recruitment to silence gene expression. The enhancer of zeste homolog 2 (EZH2), a crucial catalytic subunit of PRC2, mediates gene silencing and participates in cell lineage determination via methyltransferase activity. In this study, we found that the expression of EZH2 and H3K27me3 (trimethylation on lysine 27 in histone H3) decreased during osteogenesis of human dental follicle stem cells (hDFSCs). Knockdown studies of MEG3 and EZH2 by siRNA showed that MEG3/EZH2 negatively regulated osteogenesis of hDFSCs. We investigated the role of Wnt signaling pathway during the osteogenesis of hDFSCs and its relationship with EZH2. Besides, we studied the key genes of the canonical/noncanonical Wnt signaling pathway which might be related to EZH2. ChIP (chromatin immunoprecipitation) analysis showed that these effects were due to the EZH2 regulation of H3K27me3 level on the Wnt genes promotors. We first demonstrated that the decrease of MEG3 or EZH2 activated the Wnt/ß-catenin signaling pathway via epigenetically regulating the H3K27me3 level on the Wnt genes promotors. Our research offers a new target for periodontal tissue engineering and osteogenic tissue regeneration.

Effect of microRNA-21 on hypoxia-inducible factor-1α in orthodontic tooth movement and human periodontal ligament cells under hypoxia.

Orthodontic tooth movement can lead to temporary hypoxia of periodontal tissues. Periodontal ligament cells (PDLCs) react to hypoxia, releasing various biological factors to promote periodontal tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) is one of the most sensitive factors involved in the response to hypoxia. HIF-1α has been identified to be involved in osteogenic and osteoclast differentiation in vitro; however, few studies have investigated the expression of HIF-1α in the periodontal ligament (PDL) during orthodontic movement in vivo. In a previous study, microRNA-21 (miR-21) was demonstrated to be highly expressed in a rat model of orthodontic tooth movement. Additionally, miR-21 can increase the expression of HIF-1α in certain tumor cell types and is involved in tumor bioactivities. In the present study, HIF-1α exhibited expression patterns in a similar way to miR-21 in PDL samples from a rat model of orthodontic tooth movement, with expression initially increased and followed by a decrease over time. Furthermore, human PDLCs were exposed to a hypoxic environment in vitro, which induced significant upregulation of HIF-1α and miR-21 expression. Furthermore, miR-21 mimics increased HIF-1α expression and promoted osteogenic differentiation, indicated by upregulated expression of the osteogenic markers osteopontin, runt-related gene-2 and alkaline phosphatase. miR-21 inhibitors suppressed HIF-1α expression and downregulated the osteogenic markers. In conclusion, the results revealed that miR-21 has a positive effect on HIF-1α expression in PDLCs under hypoxia and has important roles in osteogenic differentiation during orthodontic tooth movement. These findings provide a theoretical basis by which to promote tissue reconstruction during orthodontic tooth movement.

Comparison of long non­coding RNA expression profiles in human dental follicle cells and human periodontal ligament cells.

The dental follicle develops into the periodontal ligament, cementum and alveolar bone. Human dental follicle cells (hDFCs) are the precursor cells of periodontal development. Long non­coding RNAs (lncRNAs) have been revealed to be crucial factors that regulate a variety of biological processes; however, whether lncRNAs serve a role in human periodontal development remains unknown. Therefore, the present study used microarrays to detect the differentially expressed lncRNAs and mRNAs between hDFCs and human periodontal ligament cells (hPDLCs). A total of 845 lncRNAs and 1,012 mRNAs were identified to be differentially expressed in hDFCs and hPDLCs (fold change >2.0 or <­2.0; P<0.05). Microarray data were validated by reverse transcription­quantitative polymerase chain reaction. Bioinformatics analyses, including gene ontology, pathway analysis and coding­non­coding gene co­expression network analysis, were performed to determine the functions of the differentially expressed lncRNAs and mRNAs. Bioinformatics analysis identified that a number of pathways may be associated with periodontal development, including the p53 and calcium signaling pathways. This analysis also revealed a number of lncRNAs, including NR_033932, T152410, ENST00000512129, ENST00000540293, uc021sxs.1 and ENST00000609146, which may serve important roles in the biological process of hDFCs. In addition, the lncRNA termed maternally expressed 3 (MEG3) was identified to be differentially expressed in hDFCs by reverse transcription­quantitative polymerase chain reaction. The knockdown of MEG3 was associated with a reduction of pluripotency makers in hDFCs. In conclusion, for the first time, to the best of our knowledge, the current study determined the different expression profiles of lncRNAs and mRNAs between hDFCs and hPDLCs. The observations made may provide a solid foundation for further research into the molecular mechanisms of lncRNAs in human periodontal development.

Response of hPDLSCs on 3D printed PCL/PLGA composite scaffolds in vitro.

Three­dimensional printed (3DP) scaffolds have become an excellent resource in alveolar bone regeneration. However, selecting suitable printable materials remains a challenge. In the present study, 3DP scaffolds were fabricated using three different ratios of poly (ε­caprolactone) (PCL) and poly­lactic­co­glycolic acid (PLGA), which were 0.1PCL/0.9PLGA, 0.5PCL/0.5PLGA and 0.9PCL/0.1PLGA. The surface characteristics and degradative properties of the scaffolds, and the response of human periodontal ligament stem cells (hPDLSCs) on the scaffolds, were assessed to examine the preferable ratio of PCL and PLGA for alveolar bone regeneration. The results demonstrated that the increased proportion of PLGA markedly accelerated the degradation, smoothed the surface and increased the wettability of the hybrid scaffold. Furthermore, the flow cytometry and Cell Counting Kit­8 assay revealed that the adhesion and proliferation of hPDLSCs were markedlyincreased on the 0.5PCL/0.5PLGA and 0.1PCL/0.9PLGA scaffolds. Additionally, the alkaline phosphatase activity detection and reverse­transcription quantitative polymerase chain reaction demonstrated that the hPDLSCs on the 0.5PCL/0.5PLGA scaffold exhibited the best osteogenic capacity. Consequently, PCL/PLGA composite scaffolds may represent a candidate focus for future bone regeneration studies, and the 0.5PCL/0.5PLGA scaffold demonstrated the best bio­response from the hPDLSCs.