Long noncoding RNAs (lncRNAs) are emerging as important regulatory molecules at the transcriptional and post-transcriptional levels and may play essential roles in the differentiation of human bone marrow mesenchymal stem cell (hMSC). However, their roles and functions remain unclear. Here, we showed that lncRNA H19 was significantly upregulated after the induction of osteoblast differentiation. Overexpression of H19 promoted osteogenic differentiation of hMSCs in vitro and enhanced heterotopic bone formation in vivo, whereas knockdown of H19 inhibited these effects. Subsequently, we found that miR-675, encoded by exon1 of H19, promoted osteoblast differentiation of hMSCs and was partially responsible for the pro-osteogenic effect of H19. Investigating the underlying mechanism, we demonstrated that H19/miR-675 inhibited mRNA and protein expression of transforming growth factor-β1 (TGF-β1). The downregulation of TGF-β1 subsequently inhibited phosphorylation of Smad3. Meanwhile, H19/miR-675 downregulated the mRNA and protein levels of histone deacetylase (HDAC) 4/5, and thus increased osteoblast marker gene expression. Taken together, our results demonstrated that the novel pathway H19/miR-675/TGF-β1/Smad3/HDAC regulates osteogenic differentiation of hMSCs and may serve as a potential target for enhancing bone formation in vivo. Stem Cells 2015;33:3481–3492

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.

Saliva, which contains biological information, is considered a valuable diagnostic tool for local and systemic diseases and conditions because, similar to blood, it contains important molecules like DNA, RNA, and proteins. Exosomes are cell-derived vesicles 30-100 nm in diameter with substantial biological functions, including intracellular communication and signalling. These vesicles, which are present in bodily fluids, including saliva, are released upon fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. Salivary diagnosis has notable advantages, which include noninvasiveness, ease of collection, absence of coagulation, and a similar content as plasma, as well as increased patient compliance compared to other diagnostic approaches. However, investigation of the roles of salivary exosomes is still in its early years. In this review, we first describe the characteristics of endocytosis and secretion of salivary exosomes, as well as database and bioinformatics analysis of exosomes. Then, we describe strategies for the isolation of exosomes from human saliva and the emerging role of salivary exosomes as potential biomarkers of oral and other systemic diseases. Given the ever-growing role of salivary exosomes, defining their functions and understanding their specific mechanisms will provide novel insights into possible applications of salivary exosomes in the diagnosis and treatment of systemic diseases.

Bone marrow mesenchymal stem cells (BMSCs) exhibit an increased propensity toward adipocyte differentiation accompanied by a reduction in osteogenesis in osteoporotic bone marrow. However, limited knowledge is available concerning the role of long non-coding RNAs (lncRNAs) in the differentiation of BMSCs into adipocytes. In this study, we demonstrated that lncRNA H19 and microRNA-675 (miR-675) derived from H19 were significantly downregulated in BMSCs that were differentiating into adipocytes. Overexpression of H19 and miR-675 inhibited adipogenesis, while knockdown of their endogenous expression accelerated adipogenic differentiation. Mechanistically, we found that miR-675 targeted the 3′ untranslated regions of the histone deacetylase (HDAC) 4–6 transcripts and resulted in deregulation of HDACs 4–6, essential molecules in adipogenesis. In turn, trichostatin A, an HDAC inhibitor, significantly reduced CCCTC-binding factor (CTCF) occupancy in the imprinting control region upstream of the H19 gene locus and subsequently downregulated the expression of H19. These results show that the CTCF/H19/miR-675/HDAC regulatory pathway plays an important role in the commitment of BMSCs into adipocytes.

Orthodontic treatment was found to have an impact on the quantity and constitution of subgingival microbiota. However, contradictory findings regarding the effects of fixed appliances on microbial changes were reported. The aim of this systematic review was to investigate the microbial changes in subgingival plaques of orthodontic patients. The PubMed, Cochrane Library, and EMBASE databases were searched up to November 20, 2016. Longitudinal studies observing microbial changes in subgingival plaques at different time points of orthodontic treatment are included. The methodological quality of the included studies was assessed by Methodological index for non-randomized studies (MINORS). The studies that reported the frequency of subgingival periodontopathogens were used for quantitative analysis. Other studies were analysed qualitatively to describe the microbial changes during orthodontic treatment. Thirteen studies were selected, including two controlled clinical trials, three cohort studies and eight self-controlled studies. Four periodontopathogens, including Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Tannerella forsythia (Tf), were analysed. Following orthodontic appliance placement, the frequencies of Pg and Aa showed no significant change (P = 0.97 and P = 0.77), whereas the frequency of Tf significantly increased (P < 0.01) during short-term observation (0–3 months). The frequency of Pi showed a tooth-specific difference, as it presented no significant difference (P = 0.25) at the site of the first molar but was significantly increased (P = 0.01) at the incisor. During long-term observation (> = 6 months), two studies reported that the levels of subgingival periodontopathogens exhibited a transient increase but decreased to the pretreatment levels afterwards. After removal of the orthodontic appliance, the four periodontopathogens showed no significant difference compared with before removal. The levels of subgingival pathogens presented temporary increases after orthodontic appliance placement, and appeared to return to pretreatment levels several months later. This indicates that orthodontic treatment might not permanently induce periodontal disease by affecting the level of subgingival periodontal pathogen levels. Further studies of high methodological quality are required to provide more reliable evidence regarding this issue.