MZB1 targeted by miR-185-5p inhibits the migration of human periodontal ligament cells through NF-κB signaling and promotes alveolar bone loss.

OBJECTIVE: To explore the role of Marginal Zone B and B-1 Cell-Specific Protein (MZB1), a novel molecule associated with periodontitis, in migration of human periodontal ligament cells (hPDLCs) and alveolar bone orchestration. BACKGROUND: MZB1 is an ER-localized protein and its upregulation has been found to be associated with a variety of human diseases. However, few studies have investigated the effect and mechanism of MZB1 on hPDLCs in periodontitis. METHODS: Gene expression profiles in human gingival tissues were acquired from the Gene Expression Omnibus (GEO) database, and candidate molecules were then selected through bioinformatic analysis. Subsequently, we identified the localization and expression of MZB1 in human gingival tissues, mice, and hPDLCs by immunofluorescence, RT-qPCR, and Western blot. Dual-luciferase reporter assay was applied to assess the binding of miR-185-5p to MZB1. Furthermore, the effects of MZB1 on cell migration, proliferation, and apoptosis in vitro were investigated by wound-healing assay, transwell assay, CCK-8 assay, and flow cytometry analysis. Finally, Micro-CT analysis and H&E staining were performed to examine the effects of MZB1 on alveolar bone loss in vivo. RESULTS: Bioinformatic analysis discovered that MZB1 was one of the most significantly increased genes in periodontitis patients. MZB1 was markedly increased in the gingival tissues of periodontitis patients, in the mouse models, and in the hPDLCs treated with lipopolysaccharide of Porphyromonas gingivalis (LPS-PG). Furthermore, in vitro experiments showed that MZB1, as a target gene of miR-185-5p, inhibited migration of hPDLCs. Overexpression of MZB1 specifically upregulated the phosphorylation of p65, while pretreatment of MZB1-overexpressed hPDLCs with PDTC (NF-κB inhibitor) notably reduced the p-p65 level and promoted cell migration. In addition, the mRNA expression levels of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx2) were inhibited in MZB1-overexpressed hPDLCs and miR-185-5p inhibitor treated hPDLCs, respectively. In vivo experiments showed that knockdown of MZB1 alleviated the loss of alveolar bone. CONCLUSION: As a target gene of miR-185-5p, MZB1 plays a crucial role in inhibiting the migration of hPDLCs through NF-κB signaling pathway and deteriorating alveolar bone loss.

TLR4 regulates ROS and autophagy to control neutrophil extracellular traps formation against Streptococcus pneumoniae in acute otitis media.

BACKGROUND: Otitis media (OM), a prevalent pediatric infectious disease, is mainly caused by Streptococcus pneumoniae (S.pn). Neutrophil extracellular traps (NETs), a novel antimicrobial strategy, were reported in 2004. We found that NETs formed in the middle ear with acute otitis media (AOM) induced by S.pn. However, the mechanisms of NETs formation are not entirely clear. METHODS: We stimulated neutrophils isolated from mouse bone marrow with S.pn clinical stain 19F in vitro, and established mouse model of AOM via transbullar injection with S.pn. NETs formation, reactive oxygen species (ROS) production, autophagy activation and bacterial load were analyzed in TLR4-/- and wild-type neutrophils stimulated in vitro with S.pn and in vivo during AOM. RESULTS: We found that autophagy and ROS were required for S.pn-induced NETs formation. Moreover, TLR4 partly mediated NETs formation in response to S.pn in vitro and in vivo during AOM. We also showed that attenuated NETs formation in TLR4-/- neutrophils correlated with an impaired ROS production and autophagy activation in vitro and in vivo. In addition, both the in vivo and in vitro-produced NETs were able to engulf and kill S.pn. CONCLUSIONS: TLR4 regulates ROS and autophagy to control NETs formation against S.pn in the course of AOM. IMPACT: S.pn can induce NETs formation in vitro and in vivo; TLR4 regulates NETs formation by ROS and autophagy; NETs contribute to the clearance of bacteria in acute otitis media. In this study, we firstly found that autophagy and ROS were required for S.pn-induced NETs formation in the model of acute otitis media (AOM). And to some extent, TLR4 mediated NETs formation during AOM. Our research might provide a potential strategy for the treatment of otitis media.

LncRNA-01126 inhibits the migration of human periodontal ligament cells through MEK/ERK signaling pathway.

BACKGROUND AND OBJECTIVE: Accumulating findings revealed that long noncoding RNAs (lncRNAs) are crucial regulator molecules in the progression of periodontitis. This study aimed to investigate the biological roles and mechanisms of lncRNA-01126 in the progression of periodontitis. MATERIALS AND METHODS: RT-qPCR was used to detect the levels of lncRNA-01126 in gingival tissues and human periodontal ligament cells (hPDLCs). Cell transfection experiments were performed to knock down or overexpress the level of lncRNA-01126 in hPDLCs. Cell Counting Kit-8, wound-healing assay, transwell assay, and flow cytometric analysis were used to evaluate the function of lncRNA-01126 in the progression of periodontitis. Finally, the signaling pathway was assessed by western blot. RESULTS: LncRNA microarray discovered that lncRNA-01126 was the most significantly increased lncRNA in periodontitis patients. LncRNA-01126 markedly increased in the gingival tissues of periodontitis mice and in the hPDLCs treated with lipopolysaccharide of Porphyromonas Gingivalis (LPS-PG). Furthermore, in vitro experiments showed that lncRNA-01126 dramatically suppressed the migration of hPDLCs through MEK/ERK signaling pathway. CONCLUSION: LncRNA-01126 plays a crucial role in inhibiting the migration of hPDLCs through MEK/ERK signaling pathway.

Periodontitis induced by orthodontic wire ligature drives oral microflora dysbiosis and aggravates alveolar bone loss in an improved murine model.

Aim: To assess the contribution of polymicrobial disruption of host homeostasis to periodontitis progression in orthodontic wire ligation murine model. Methods: Orthodontic wire rings were inserted between the first and second molars of mice for 18 days for the orthodontic wire ligation mouse model, and Pg injection model and Pg-LPS injection model were used as controls. Alveolar bone loss and periodontal inflammation were analyzed by micro-CT, histological staining and qRT-PCR. Further, pyrosequencing of 16S rRNA gene amplicon was used to analyze the development of oral microorganism dysbiosis in the mice. Results: Micro-CT, TRAP staining and qRT-PCR showed that orthodontic wire ligation model led to more severe alveolar bone loss than Pg and Pg-LPS models.H&E staining and qRT-PCR demonstrated that stronger inflammatory response was induced by the orthodontic wire treatment compared to the other models. In addition, pyrosequencing of 16S rRNA gene amplicons revealed that the composition of oral microbiota presented a transition as the disease progressed and significant differences emerged in oral microbiota communities between orthodontic ligature mice and healthy controls. Furthermore, antibiotic treatment decreased both inflammation and alveolar bone loss in response to microbial community dysbiosis. However, no significant difference in bacterial community composition was observed in Pg and Pg-LPS models. Conclusions: Orthodontic wire ligation drove oral microbial community transitions that mimicked polymicrobial communities characterized by polymicrobial synergy and dysbiosis. Our improved model is suitable for further study of pathogenesis of periodontitis and exploration of corresponding treatment strategies.

Identification and Exploration of Serine Peptidase Inhibitor Kazal Type I (SPINK1) as a Potential Biomarker Correlated with the Progression of Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Although significant advances have been achieved in the treatment of NSCLC during the past two decades, the 5-year survival rate of patients with NSCLC remains <20%. Thus, there is an urgent requirement to further understand the molecular mechanisms that promote NSCLC development and to identify novel therapeutic targets. In the present study, the gene expression profiles of patients with NSCLC from The Cancer Genome Atlas database were carefully analyzed and SPINK1 was identified as a tumor-inducing factor. SPINK1 expression level was found to be increased in both NSCLC tissues and cell lines. Moreover, SPINK1 promoted cell proliferation in A549 and H1299 cells. Knockdown of SPINK1 could activate cell autophagy and apoptosis. Mechanistically, SPINK1 was demonstrated to induce the proliferation of NSCLC via activating the MEK/ERK signaling pathway. In conclusion, these findings suggested that SPINK1 may serve as a potential biomarker in NSCLC.

TMEM100 induces cell death in non­small cell lung cancer via the activation of autophagy and apoptosis.

Lung cancer is one of the most malignant type of tumors worldwide. Non­small cell lung cancer (NSCLC), which is the most common type of lung cancer, is defined as a distinct disease that exhibits both genetic and cellular heterogeneity. Although in the past two decades significant advances in the treatment of NSCLC have besen performed, the 5­year survival rate of patients with NSCLC remains <20%. Thus, there is an urgent requirement to gain an in­depth understanding of the molecular mechanisms that promote NSCLC development and to identify novel therapeutic targets. In the present study, the gene expression profiles of patients with NSCLC from The Cancer Genome Atlas database were analyzed to determine potential therapeutic targets, and transmembrane protein 100 (TMEM100) was identified as a candidate tumor suppressor. TMEM100 expression level was discovered to be decreased in both NSCLC tissues and cell lines, and it was observed to be negatively associated with the TNM stage and positively associated with prognosis. Moreover, TMEM100 inhibited tumor growth and promoted cell apoptosis in A549 and H460 cells. Mechanistically, TMEM100 was demonstrated to induce autophagy in A549 cells via inhibiting the PI3K/AKT signaling pathway, whereas inhibiting autophagy using bafilomycin A1 significantly enhanced TMEM100­induced apoptosis to compensate for the cell death. In conclusion, these findings suggested that TMEM100 may serve as a tumor suppressor in NSCLC and promote autophagy via inhibiting the PI3K/AKT signaling pathway.

Glycolytic Metabolism Is Critical for the Innate Antibacterial Defense in Acute Streptococcus pneumoniae Otitis Media.

Objective: Streptococcus pneumoniae (S.pn) is a common respiratory pathogen and a frequent cause of acute otitis media (AOM) in children. However, little is known about the immunometabolism during AOM. This study was to assess the presence of glucose metabolic reprogramming during AOM and its underlying mechanism affecting inflammatory response and middle ear injury. Methods: The levels of glycolytic metabolism were evaluated by measuring the expression of glycolysis-related genes and the production of metabolites. HE stain, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and Western blot were performed to measure the effect of glucose metabolic reprogramming on inflammatory response, pneumococcal clearance, hypoxia-inducible factor 1 alpha (HIF-1α) expression and cytokine secretion during AOM, respectively. Results: The analysis of microarray revealed an increase of the expression of glycolysis-related genes during S.pn-induced AOM, which was verified by real-time PCR. Increased glycolysis promoted the production of IL-1ß and TNF-α and facilitated the clearance of S.pn by enhancing phagocytosis and killing capability of neutrophils, but also aggravated the middle ear injury. Furthermore, these pathogenic effects could be reversed after glycolytic inhibitor 2DG treatment. Additionally, HIF-1α was observed to involve in glycolytic metabolism during AOM. Conclusion: S.pn infection induced increased glycolysis conversion during AOM, which promoted inflammatory responses and bacterial clearance, but also aggravated tissue damage.