Periodontal ligament stem cells (PDLSCs) are considered as candidate cells for the regeneration of periodontal and alveolar bone tissues. Antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), which is a newly discovered circular RNA (circRNA), has been reported to act as an miR-7 sponge and to be involved in many biological processes. Here, we investigated the potential roles of CDR1as and miR-7 in the osteogenic differentiation of PDLSCs. The expression pattern of CDR1as and miR-7 in PDLSCs during osteogenesis was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then we overexpressed or knocked down CDR1as or miR-7 to confirm whether they were involved in the regulation of osteoblast differentiation in PDLSCs. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were used to detect the activity of osteoblasts and mineral deposition. Furthermore, a dual luciferase reporter assay was conducted to analyze the binding of miR-7 to growth differentiation factor (GDF)5. To further verify the role of CDR1as in osteoblast differentiation, we conducted animal experiments in vivo. New bone formation in specimens was analyzed by microcomputed tomography (micro-CT), hematoxylin and eosin staining, and immunofluorescence staining. We observed that CDR1as was significantly upregulated during the osteogenic differentiation, whereas miR-7 was significantly downregulated. Moreover, knockdown of CDR1as and overexpression of miR-7 inhibited the ALP activity, ARS staining, and expression of osteogenic genes. Overexpression of miR-7 significantly reduced the activity of luciferase reporter vectors containing the wild-type, but not the mutant, 3’ untranslated region (UTR) sequence of GDF5. Furthermore, knockdown of GDF5 partially reversed the effects of miR-7 inhibitor on osteoblast differentiation. Downregulation of CDR1as or GDF5 subsequently inhibited phosphorylation of Smad1/5/8 and p38 mitogen-activated protein kinases (MAPK), while upregulation of miR-7 decreased the level of phosphorylated Smad1/5/8 and p38 MAPK. In vivo, CDR1as knockdown lead to less bone formation compared with the control group as revealed by micro-CT and the histological analysis. Our results demonstrated that CDR1as acts as a miR-7 inhibitor, triggering the upregulation of GDF5 and subsequent Smad1/5/8 and p38 MAPK phosphorylation to promote osteogenic differentiation of PDLSCs. This study provides a novel understanding of the mechanisms of osteogenic differentiation, and suggests a potential method for promoting bone formation.

Background: Alveolar bone defects, particularly palatal bone dehiscence (PBD) and labial bone fenestration (LBF), occur frequently as a result of retraction of the maxillary anterior teeth. The study aims to explore the long-term bone remodeling of maxillary anterior teeth in adult patients with post-orthodontic treatment PBD and LBF. Materials and methods: The study includes 24 adult patients with maxillary protrusion (8 males, 16 females) who were treated with extraction of four first premolars and had alveolar bone defects (PBD or LBF) in maxillary anterior teeth following orthodontic treatment. Cone-beam computed tomography imaging measurements were obtained before (T1), after (T2) orthodontic treatment, and after at least 1-year removable thermoplastic retainer retention (T3). The maxillary anterior teeth with PBD or LBF at T2 were divided into the PBD or LBF groups, respectively. The labial and palatal alveolar bone height (ABH), alveolar bone thickness (ABT), and movement of maxillary anterior teeth were measured during retraction (T2–T1) and retention (T3–T2) periods. Results: The incidence of PBD and LBF in maxillary anterior teeth significantly increased after orthodontic treatment and decreased during the retention period. In the PBD group, the palatal ABH of all maxillary anterior teeth significantly increased from T1 to T2 but decreased from T2 to T3. The ABT of the maxillary central incisor and canine significantly increased on the palatal side and decreased on the labial side during the retention period. In the LBF group, the labial ABT of the maxillary central incisor at the apical level showed a significant decrease from T1 to T2, followed by an increase from T2 to T3. In both groups, the maxillary central incisor showed significant labial movement, with a relative intrusion during the retention period. Conclusion: For adult patients with maxillary protrusion, the alveolar bone defect of maxillary anterior teeth caused by orthodontic retraction significantly improved during the retention period, indicating good long-term bone remodeling. Our findings suggest that a combination of spontaneous reorientation of maxillary anterior teeth and bone remodeling contributed to alveolar bone covering in these patients.

Purpose: Periodontitis is the leading cause of tooth loss. The role of long non-coding RNA (lncRNA) in periodontal inflammation remains unclear. The aim of this study was to investigate the role of lncRNA H19 in periodontitis and its possible regulation of autophagy in periodontitis. Material and Methods: Inflammation level was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) in periodontal ligament cells (PDLCs). Western blotting, flow cytometric analysis, and immunofluorescence staining were used to detect the autophagy flux. Overexpression or knockdown of H19 was used to confirm its function. Ligature-induced periodontitis model in mice and periodontitis-affected human gingival tissue were used in vivo. RNA sequencing was performed to determine the differentially expressed genes. Results: Autophagy was significantly increased in PDLCs after inflammatory stimulation as well as in a ligature-induced periodontitis model in mice and periodontitis-affected human gingival tissue. During the inflammatory process, H19 expression was also significantly upregulated. Further, the levels of autophagic markers were significantly upregulated after overexpressing H19 in PDLCs, and the increased autophagic activity induced by inflammatory stimulation was reversed by H19 knockdown. RNA sequencing showed that the expression profiles of mRNAs were significantly altered after H19 overexpression, and the differentially expressed genes were enriched in the PI3K/AKT signaling pathway, which was confirmed by the decreased p-AKT protein expression in the H19 overexpression group. Conclusion: Periodontal inflammation activates autophagy flux, and H19 mediates the activation of autophagy via AKT pathway in periodontitis. This study expands our understanding of molecular regulation in periodontitis.

Oxidative stress was shown to promote the translocation of Ataxia-telangiectasia mutated (ATM) to cytoplasm and trigger the LKB1-AMPK-tuberin pathway leading to a down-regulation of mTOR and subsequently inducing the programmed cell death II (autophagy). Cisplatin was previously found to induce the ATM-dependent phosphorylation of ΔNp63α in squamous cell carcinoma (SCC) cells. In this study, phosphorylated (p)-ΔNp63α was shown to bind the ATM promoter, to increase the ATM promoter activity and to enhance the ATM cytoplasmic accumulation. P-ΔNp63α protein was further shown to interact with the Rpn13 protein leading to a proteasome-dependent degradation of p-ΔNp63α and thereby protecting LKB1 from the degradation. In SCC cells (with an altered ability to support the ATM-dependent ΔNp63α phosphorylation), the non-phosphorylated ΔNp63α protein failed to form protein complexes with the Rpn13 protein and thereby allowing the latter to bind and target LKB1 into a proteasome-dependent degradation pathway thereby modulating a cisplatin-induced autophagy. We thus suggest that SCC cells sensitive to cisplatin-induced cell death are likely to display a greater ratio of p-ΔNp63α/non-phosphorylated ΔNp63α than cells with the innate resistant/impaired response to a cisplatin-induced cell death. Our data also suggest that the choice made by Rpn13 between p-ΔNp63α or LKB1 to be targeted for degradation is critical for cell death decision made by cancer cells in response to chemotherapy.