Craniosynostosis is a common birth defect affecting 1 of the 2200 live births causing severe skull and cognitive defects, due to premature cranial suture fusion. The current surgical treatments require invasive calvaria vault remodeling and cranial bone resection in the baby. We demonstrate that inhibition of miR-200a in PMIS-miR-200a mice results in coronal suture fusion (craniosynostosis). Therefore, we use overexpression of miR-200a to prevent suture fusion in Twist1 mutant mice, a well-known model for craniosynostosis. We developed a PEGylated-peptide nanoparticle system to deliver plasmid DNA expressing miR-200a directly to the sutures of postnatal day 4 (P4) Twist1 mutant mice before suture fusion. Injection of the miR-200a nanoparticles under the scalp before suture fusion at P7 to P10 inhibited suture fusion. Treatments increased Gli1- and Six2-positive suture stem cells and the thickness of the periosteum layer. The treated Twist1+/- mice increased body weight and were alert and active. We demonstrate an effective noninvasive gene therapy treatment for craniosynostosis.

microRNAs (miRs) have been reported over the decades as important regulators in bone development and bone regeneration. They play important roles in maintaining the stem cell signature as well as regulating stem cell fate decisions. Thus, delivering miRs and miR inhibitors to the defect site is a potential treatment towards craniofacial bone defects. However, there are challenges in translation of basic research to clinics, including the efficiency, specificity, and efficacy of miR manipulation methods and the safety of miR delivery systems. In this review, we will compare miR oligonucleotides, mimics and antagomirs as therapeutic reagents to treat disease and regenerate tissues. Newer technology will be discussed as well as the efficiency and efficacy of using these technologies to express or inhibit miRs in treating and repairing oral tissues. Delivery of these molecules using extracellular vesicles and nanoparticles can achieve different results and depending on their composition will elicit specific effects. We will highlight the specificity, toxicity, stability, and effectiveness of several miR systems in regenerative medicine.

Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate, hypodontia, and taurodontism. WHS candidate 1 (WHSC1) gene is a H3K36-specific methyltransferase that is deleted in every reported case of WHS. Mutation in this gene also results in tooth anomalies in patients. However, the correlation between genetic abnormalities and the tooth anomalies has remained controversial. In our study, we aimed to clarify the role of WHSC1 in tooth development. We profiled the Whsc1 expression pattern during mouse incisor and molar development by immunofluorescence staining and found Whsc1 expression is reduced as tooth development proceeds. Using real-time quantitative reverse transcription PCR, Western blot, chromatin immunoprecipitation, and luciferase assays, we determined that Whsc1 and Pitx2, the initial transcription factor involved in tooth development, positively and reciprocally regulate each other through their gene promoters. miRNAs are known to regulate gene expression posttranscriptionally during development. We previously reported miR-23a/b and miR-24-1/2 were highly expressed in the mature tooth germ. Interestingly, we demonstrate here that these two miRs directly target Whsc1 and repress its expression. Additionally, this miR cluster is also negatively regulated by Pitx2. We show the expression of these two miRs and Whsc1 are inversely correlated during mouse mandibular development. Taken together, our results provide new insights into the potential role of Whsc1 in regulating tooth development and a possible molecular mechanism underlying the dental defects in WHS.

This new approach has identified several new scientific advancements in stem cell niche biology. 1) miR-200 family members are required for in vivo epithelial stem cell differentiation and stem cell niche maintenance; 2) The miR-200 family regulates genes required for generating the different dental epithelial cell lineages; 3) miR-200expression may be a common mechanism for defining the boundaries of adult epithelial stem cell niches; 4) By inhibiting the miRs that become active when a progenitor cell becomes fated to differentiate, the in vivo cell differentiation programs are determined assuming their terminal fates; 5) Dental epithelial stem cells and suture stem cells are regulated by members of the miR-200 family to yield the differentiated progeny and determine cell fate.

Stem cells are potent to self‐renew and differentiate which contribute to the regenerative process of various mature tissue types. It is important to understand how stem cell behaviors are regulated to give further insights to the application of stem cell therapies in tissue regeneration and repair. We use the developing murine lower incisor as a model to study stem cell maintenance and differentiation, which are housed in a niche and are fated to only a handful of differentiated cell types. Tooth development is a complex process involving rigid temporal and reciprocal regulation of transcription factors including Sox2 and Lef‐1, and signaling pathways including Wnt. We are particularly interested in the regulation between Lef‐1 and miR‐26b due to their inverse expression profiles during tooth development. Lef1 harbors a miR‐26b binding site in its 3’‐UTR and is negatively regulated by miR‐26b. In this study, we aim to understand the role of Lef‐1 and miR‐26b in dental epithelial stem cell (DESC) differentiation, determine the molecular mechanism of miR‐26b regulated Lef‐1 expression and identify genetic pathways for tooth development and regeneration.

microRNAs (miRs) are small RNA molecules that regulate many cellular and developmental processes. They control gene expression pathways during specific developmental time points and are required for tissue homeostasis and stem cell maintenance. miRs as therapeutic reagents in tissue regeneration and repair hold great promise and new technologies are currently being designed to facilitate their expression or inhibition. Due to the large amount of miR research in cells and cancer many cellular processes and gene networks have been delineated however, their in vivo response can be different in complex tissues and organs. Specifically, this report will discuss animal developmental models to understand the role of miRs as well as xenograft, disease, and injury models. We will discuss the role of miRs in clinical studies including their diagnostic function, as well as their potential ability to correct craniofacial diseases.

HMGN2 bound nonspecifically to the DNA probe as previously reported. Lef-1 protein (80 ng) bound to the DNA. HMGN2 titration (4, 10, 15, 20, and 40 ng) after Lef-1 bound to the probe revealed that HMGN2 inhibited Lef-1 binding in a dose-responsive manner. The EMSA experiments were analyzed in 8% native polyacrylamide gels. The free and bound forms of DNA were quantitated using the Molecular Dynamics STORM PhosphoIm-ager. The free probe and bound DNA are indicated. HMGN2, high mobility group protein N2. miR-23a-and miR-23b-binding elements (Fig. 5A). To deter-mine if miR-23a and miR-23b target Hmgn2 expression in oral epithelial cells, we cloned the Hmgn2 30UTR containing the miR-23a and miR-23b-binding site into a dual-luciferase re-porter and transfected this reporter into LS-8 cells alone or with the constructs to overexpress miR-23a and/or miR-23b. The luciferase activity of WT Hmgn2 30UTR was significantly repressed by the presence of either miR-23a or/and miR-23b (Fig. 5B). As controls, mutation of the conserved miR-23a+b binding site in the Hmgn2 30UTR abolished the repression by miR-23a+b (Fig. 5C). Overexpression of miR-23a and miR-23b separately and together leads to reduced endogenous Hmgn2 protein expression in LS-8 cells (Fig. 5D). miR-23a and miR-23b target and repress Hmgn2 expression. However, it is important to show that these miRs are coexpressed with Hmgn2 in the craniofacial/tooth regions of mice during development. miR-23a+b indirectly activate Pitx2 and amelogenin expression by repressing Hmgn2 expression in dental epithelial cells To further understand the regulation of Hmgn2 by miR-23a+b and the contribution to tooth organogenesis, we assayed for miR-23a and miR-23b expression in postnatal (P) P0 and P10 murine molars and incisors. We have previously

The chromatin-associated high mobility group protein N2 (HMGN2) regulates transcription factor activity through both chromatin and protein interactions. Several homeodomain and HMG-box domain transcription factors regulate development and differentiation and are modulated by protein interactions and epigenetic factors. We demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down and co-immunoprecipitation assays show HMGN2 directly interacts with Lef-1 through its HMG-box domain. Furthermore, electrophoretic mobility shift assays demonstrate that HMGN2 inhibits Lef-1 DNA binding activity. Thus, HMGN2 inhibits the transcriptional activities of Lef-1, Dlx2, FoxJ1 as well as Pitx2. Pitx2 and Lef-1 activate the Hmgn2 promoter and HMGN2 expression inhibits their transcriptional activation. Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter demonstrating Hmgn2 association with open chromatin. Hmgn2 expression is developmentally regulated however, the post-transcriptional mechanisms that regulate Hmgn2 expression remain unclear. We demonstrate that miR-23a and miR-23b directly target Hmgn2, promoting transcriptional activation of several gene promoters including the amelogenin promoter, regulated by Pitx2. Hmgn2 expression decreases correlating with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Analyses of Hmgn2 LacZ expression demonstrate that Hmgn2 expression is gradually decreased during embryonic development. Ablation of Hmgn2 in mice results in increased amelogenin expression due to increased Pitx2, Dlx2, Lef-1 and FoxJ1 transcriptional activity. We demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational gene expression by Hmgn2 protein interactions. HMGN2 appears to regulate tooth development through its interaction with multiple transcription factors.

Wolf‐Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate (CLP), hypodontia and taurodontism. However, the causative factors and underlying mechanisms of these oral anomalies are relatively unknown. Wolf‐Hirschhorn syndrome candidate 1 (WHSC1) is a H3K36‐specific methyltransferase that is frequently deleted in WHS. This gene has been associated with craniofacial defects including CLP and defects in occipital ossification. In our study, we aim to understand the role of WHSC1 in tooth development.

The present study was conducted to characterize microRNA-200c (miR-200c) and its regulators in adipogenic differentiation, obesity, and periodontitis in obese subjects (PiOSs), and to determine the therapeutic efficacy of plasmid DNA encoding miR-200c as a treatment for PiOSs. We report that highly expressed miR-200c in gingival tissues was downregulated in diet-induced obese (DIO) mice and during adipogenic differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). Local injection of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) in the maxilla interdental gingiva of DIO mice reduced miR-200c in gingival and adipose tissues and induced periodontal inflammation associated with systemic elevation of interleukin-6 (IL-6) and impaired glucose tolerance. The inhibitory functions of Pg-LPS and IL-6 on miR-200c and their effectiveness on Zeb1 were confirmed in vitro. Injection of naked plasmid DNA encoding miR-200c into the gingiva effectively rescued miR-200c downregulation, prevented periodontal and systemic inflammation, and alleviated the impaired glucose metabolism in obese mice with LPS-induced periodontitis. Increased circulating exosomal miR-200c and its function on suppressing proinflammatory cytokines and adipogenesis explained the mechanism(s) of gingival application of miR-200c in attenuating systemic inflammation in PiOSs. These results demonstrated that miR-200c reduced by Pg-LPS and IL-6 in periodontitis and obesity might lead to the pathogenesis of PiOSs, and upregulation of miR-200c in the gingiva presents a therapeutic approach for PiOSs.

Anaplastic thyroid cancer (ATC) is an aggressive, highly metastatic cancer that expresses high levels of the microRNA (miR)-17-92 cluster. We employ an miR inhibitor system to study the function of the different miRs within the miR-17-92 cluster based on seed sequence homology in the ATC SW579 cell line. While three of the four miR-17-92 families were oncogenic, we uncovered a novel role for miR-17 as a tumor suppressor in vitro and in vivo. Surprisingly, miR-17 inhibition increased expression of the miR-17-92 cluster and significantly increased the levels of the miR-18a and miR-19a mature miRs. miR-17 inhibition increased expression of the cell cycle activator CCND2, associated with increased cell proliferation and tumor growth in transplanted SW579 cells in xenograft mice. miR-17 regulates MYCN and c-MYC expression in SW579 cells, and the inhibition of miR-17 increased MYCN and c-MYC expression, which increased pri-miR-17-92 transcripts. Thus, inhibition of miR-17 activated the expression of the oncogenic miRs, miR-18a and miR-19a. While many cancers express high levels of miR-17, linking it with tumorigenesis, we demonstrate that miR-17 inhibition does not inhibit thyroid tumor growth in SW579 and MDA-T32 ATC cells but increases expression of the other miR-17-92 family members and genes to induce cancer progression.

Micropeptides (microproteins) encoded by transcripts previously annotated as long noncoding RNAs (lncRNAs) are emerging as important mediators of fundamental biological processes in health and disease. Here, we applied two computational tools to identify putative micropeptides encoded by lncRNAs that are expressed in the human pancreas. We experimentally verified one such micropeptide encoded by a β cell- and neural cell-enriched lncRNA TCL1 Upstream Neural Differentiation-Associated RNA (TUNAR, also known as TUNA, HI-LNC78, or LINC00617). We named this highly conserved 48-amino-acid micropeptide beta cell- and neural cell-regulin (BNLN). BNLN contains a single-pass transmembrane domain and localizes at the endoplasmic reticulum (ER) in pancreatic β cells. Overexpression of BNLN lowered ER calcium levels, maintained ER homeostasis, and elevated glucose-stimulated insulin secretion in pancreatic β cells. We further assessed the BNLN expression in islets from mice fed a high-fat diet and a regular diet and found that BNLN is suppressed by diet-induced obesity (DIO). Conversely, overexpression of BNLN enhanced insulin secretion in islets from lean and obese mice as well as from humans. Taken together, our study provides the first evidence that lncRNA-encoded micropeptides play a critical role in pancreatic β cell functions and provides a foundation for future comprehensive analyses of micropeptide function and pathophysiological impact on diabetes.

The murine lower incisor ectodermal organ contains a single epithelial stem cell (SC) niche that provides epithelial progenitor cells to the continuously growing rodent incisor. The dental stem cell niche gives rise to several cell types and we demonstrate that the miR-200 family regulates these cell fates. The miR-200 family is highly enriched in the differentiated dental epithelium and absent in the stem cell niche. In this study, we inhibited the miR-200 family in developing murine embryos using new technology, resulting in an expanded epithelial stem cell niche and lack of cell differentiation. Inhibition of individual miRs within the miR-200 cluster resulted in differential developmental and cell morphology defects. miR-200 inhibition increased the expression of dental epithelial stem cell markers, expanded the stem cell niche and decreased progenitor cell differentiation. RNA-seq. identified miR-200 regulatory pathways involved in cell differentiation and compartmentalization of the stem cell niche. The miR-200 family regulates signaling pathways required for cell differentiation and cell cycle progression. The inhibition of miR-200 decreased the size of the lower incisor due to increased autophagy and cell death. New miR-200 targets demonstrate gene networks and pathways controlling cell differentiation and maintenance of the stem cell niche. This is the first report demonstrating how the miR-200 family is required for in vivo progenitor cell proliferation and differentiation.

Currently, chronic hepatitis B virus (HBV) infection remains a serious public health problem in the world. Recombinant HBV vaccine, as a preventive strategy against HBV infection, generates high antibody level, but it is not effective to activate innate and cellular immunity for chronic HBV infection therapy. Lectins from mushroom are natural and active proteins which have been shown important biological functions. However, little is known about the immunological mechanism engaged by mushroom lectins. Here we report that, lectin from Pleurotus ostreatus (POL) stimulated innate response by activating Toll-like receptor 6 signal pathway of dendritic cells. Subsequently POL enhanced HBV specific antibody level and follicular helper T cells response which overcame HBV tolerance in transgenic mice. This study suggests a novel mechanism for POL acting on immune response and a therapeutic approach to break HBV tolerance.

Background: Physical function is a cornerstone of geriatric medicine.Impairment in physical function may threaten one's ability to live independently and is associated with multiple negative health outcomes.Although Chinese people is one of the fastest growing populations in the world, there is a limited understanding of the epidemiology of physical function impairment in the global Chinese aging population.Methods: Researchers used the PRISMA statement and performed a comprehensive online search to highlight the global epidemiology of physical function impairment of Chinese older adults in terms of prevalence, incidence, risk and protective factors, and health consequences.Search items include Chinese, older adult, Asian elderly,