RESUMO
Age-related organ and tissue-specific cell kinetic and morphological alterations are associated with the incidence of numerous diseases in old age. Salivary dysfunction frequently appears in a wide range of older people and thus is a physiological and biological aspect of aging. The transcription factor Dec1 (differentiated embryo chondrocyte expressed gene 1) is essential for the regulation of cellular senescence. Here, we explored the morphological and physiological abnormalities and the microRNA (miRNA) expression profiles in the submandibular glands (SMGs) of young (3-month-old) and of aged (24-month-old) wild-type (WT) and Dec1KO mice. Hematoxylin-eosin (H-E) staining, Masson's Trichrome staining, immunohistochemistry, immunofluorescence, and quantitative real time PCR were employed. MicroRNA (miRNA) expression profiles were examined using an Agilent system with a Mouse 8x60K array. Immunohistochemical analysis revealed an increased oxidative stress response (8-OHdG), increased expression levels of type I collagen in the fibrotic tissues with substantial amounts of fibroblasts and collagen fibers, the presence of CCl-22-positive lymphocytes infiltrating the SMGs of aged WT mice and a subsequently enhanced expression of fibrosis-associated gene (MMP-2) in the aged SMGs. The water channel protein aquaporin-5 (AQP5) was expressed in the basal cytoplasmic regions of acini in young SMGs but showed a decreased expression in aged SMGs. Myoepithelial cell markers (p63 immunoreactivity and a-SMA immunofluorescence staining) were also decreased in aged SMGs. Quantitative real-time PCR revealed decreased mRNA expression levels of AQP5 and increased mRNA expression levels of Dec1 in aged WT mice. All those characteristics were attenuated in aged Dec1KO mice. There were no apparent differences between young WT and Dec1KO mice. Of the miRNAs analyzed, miR-181c-5p, miR-141-3p, miR-374c-5p and miR-466i-3p are proposed regulatory targets of Dec1 and AQP5 genes that are involved in SMG dysfunction in aged mice. We suggest that a Dec1 deficiency might alleviate the aging-induced hypofunction of SMGs and relevant alterations of Dec1 would be useful to keep SMGs healthy. This study provides clues for determining unique microRNAs concerned with SMG dysfunction. Subsequent activation of such diversely expressed miRNAs be of great value in clarifying the nature of age-related alterations in SMGs.
Assuntos
Envelhecimento/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Homeodomínio/genética , MicroRNAs , Glândula Submandibular , Animais , Aquaporina 5/genética , Senescência Celular , Fibroblastos , Camundongos , MicroRNAs/genética , Glândula Submandibular/fisiopatologiaRESUMO
Fibroblast injury and autophagy dysfunction have been shown to contribute to the persistence of oral wounds. Recently, microRNAs have emerged as vital regulators and fine tuners of various pathophysiological cellular processes that influence the wound healing process. This study explored the biological function and regulatory mechanism of miRNA-21 (miR-21) in the healing of oral wounds by interfering with autophagy. Healthy gingival cells derived from wild-type (WT) and from miR-21KO mice were characterized by immunocytofluorescence, and changes in wound healing were subsequently assessed using an in vitro scratch wound healing assay. The roles of critical proteins required for autophagy, autophagy related 5 (ATG5) and Bcl-2 interacting coiled-coil protein 1 (Beclin1) were evaluated by immunohistochemistry. Human gingival fibroblasts (HGFs) were transfected with a miR-21 mimic and a miR-negative control, and the relative expression of miR-21, ATG5, Beclin1 and LC3-I/II was characterized by qRT-PCR and Western blot. Pathological changes were observed in a palatal wound healing model using WT and miR-21KO mice. Immunohistochemistry was used to examine extracellular matrix (ECM) proteins and autophagy markers. Cell migration was delayed in gingiva-derived mesenchymal stem cells (GMSCs) from miR-21KO mice compared with WT mice. The expression of ATG5 and Beclin1 was significantly up-regulated in miR-21KO gingiva. Transfection of a miR-21 mimic into HGFs inhibited autophagy and up-regulated miR-21 expression. Knockdown of miR-21 suppressed the expression of fibronectin and CTGF, enhanced the autophagy effect of fibroblasts, suggesting that autophagy is involved in miR-21 regulated palatal wound healing. Taken together, these results suggest that miR-21 promotes oral wound healing by increasing ECM production through the inhibition of autophagy and facilitates clinical management of wound healing.
Assuntos
Autofagia/fisiologia , MicroRNAs/genética , Palato/metabolismo , Cicatrização/fisiologia , Animais , Autofagia/genética , Movimento Celular/genética , Movimento Celular/fisiologia , Células Cultivadas , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/patologia , Gengiva/citologia , Gengiva/patologia , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Palato/lesões , Ratos , Regulação para Cima , Cicatrização/genéticaRESUMO
Autophagy is a highly conserved intracellular digestion process that degrades damaged proteins and organelles but the biological roles of autophagy in pathological aspects of oral tissues remain largely unknown. We sought to elucidate the function of autophagy, especially its interplay with apoptosis and oxidative stress, in the oral toxicity induced by exposure to 5 mM sodium fluoride (NaF). Human cementoblasts (HCEM-2) in culture were exposed to 5 mM NaF for 5 min, after which cell viability and cell apoptosis were assessed using the MTS assay and an Annexin V-FITC/PI apoptosis detection kit, respectively. Quantitative RT-PCR and Western blotting were performed to characterize the expression levels of markers for autophagy, apoptosis, and oxidative stress. Senescence-resistant (SAMR1) mice were exposed to 5 mM NaF in their drinking water from 12 to 58 weeks. Micro-computed tomography was used to measure changes in their alveolar bone while immunohistochemistry and immunofluorescence staining was used to evaluate protein expression levels. HCEM-2 cells exposed to 5 mM NaF had decreased levels of autophagy, as shown by reduced expression levels of ATG5, Beclin-1 and LC3-II, elicited apoptosis, which in turn induced oxidative stress and inflammation, as manifested by elevated levels of Bax, cleaved caspase-3, SOD1 and phospho NF-κB. Treatment of mice with 5 mM NaF resulted in histological abnormalities in periodontal tissues, induced excessive oxidative stress and apoptosis, and reduced autophagy. Micro-computed tomography analysis demonstrated that 5 mM NaF caused a decrease in bone areas of mice compared with controls. Exposure to 5 mM NaF induced RANKL (receptor activator of nuclear factor κB ligand) and cathepsin K expression in periodontal tissues, while ATG5 and Beclin-1 expression was abrogated by 5 mM NaF. Taken together, our findings suggest that 5 mM NaF elicits oral toxicity that contributes to excessive apoptosis, oxidative stress, and defective autophagy, which aggravates periodontal tissue damage.
Assuntos
Autofagia/efeitos dos fármacos , Cemento Dentário/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Proteína 5 Relacionada à Autofagia/fisiologia , Reabsorção Óssea/induzido quimicamente , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Fluoreto de Sódio/toxicidade , Microtomografia por Raio-XRESUMO
BACKGROUND AND OBJECTIVES: Disruption of transcriptional regulation is a confounding factor associated with a wide range of human inflammatory diseases. To investigate mechanistic links between transcription factor DEC1 and pathways underlying inflammation, wild-type and DEC1 knockout (KO) C57BL/6 mice were treated with Porphyromonas gingivalis (or carboxymethyl cellulose as a control) to induce periodontal inflammation. It provoked an inflammatory response within the oral environment, which showed robust variation in alveolar bone resorption and expression of inflammatory cytokines. MATERIAL AND METHODS: Male DEC1KO mice and their wild-type littermates were used for the experimental periodontitis model. Measurement of alveolar bone resorption, micro-computed tomography, isolation of gingival mononuclear cells (GMCs), flow cytometry and immunohistochemical analysis were used in this study. Human gingival fibroblast cells (HGF-1) were used for DEC1 over-expression and short interference RNA (siRNA) studies and quantitative real-time polymerase chain reaction and western blot analysis were performed. RESULTS: Micro-computed tomography analysis demonstrated that P. gingivalis caused a decrease in bone area of wild-type mice compared with DEC1KO mice. Expression of inflammatory and immune markers in GMCs was significantly decreased in DEC1KO mice after treatment with P. gingivalis. Conversely, interleukin (IL)-4 and IL-10 mRNAs were significantly increased in GMCs isolated from DEC1KO mice. The results show that treatment of DEC1KO mice with P. gingivalis decreased the numbers of CD11b+ F4/80+ and CD4+ RANKL+ T cells. Moreover, expression of CD4, F4/80, RANKL and cathepsin K in inflammatory cell infiltrates was significantly reduced in DEC1KO mice treated with P. gingivalis compared with controls. Furthermore, over-expression of DEC1 in HGF-1 cells increased the expression of IL-1ß and tumor necrosis factor-α mRNAs and their expression levels reached a maximum in response to treatment with lipopolysaccharide. Inhibition of DEC1 by short interference RNA interference suppressed the P. gingivalis-derived lipopolysaccharide-induced expression of IL-1ß, tumor necrosis factor-α and toll-like receptor4. CONCLUSION: These results suggest that transcription factor DEC1 can modulate P. gingivalis-induced periodontitis in the oral mucosa.
Assuntos
Infecções por Bacteroidaceae , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Periodontite/genética , Periodontite/microbiologia , Porphyromonas gingivalis , Perda do Osso Alveolar , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Gengiva/citologia , Gengiva/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Periodontite/metabolismo , Periodontite/patologia , RNA Interferente PequenoRESUMO
BACKGROUND: MicroRNA (miRNA)-126 (miR-126) is an endothelial-specific miRNA located within intron 7 of epidermal growth factor-like domain 7 (EGFL7). However, the role of miR-126 in cancer is controversial. METHODS: We examined the function of miR-126 in oral squamous cell carcinoma (OSCC) cells. Furthermore, a series of 118 cases with OSCC were evaluated for the expression levels of miR-126. RESULTS: MicroRNA-126 (miR-126) was associated with cell growth and regulation of vascular endothelial growth factor-A activity, and demethylation treatment increased expression levels of miR-126 and EGFL7 in OSCC cells. A significant association was found between miR-126 expression and tumour progression, nodal metastasis, vessel density, or poor prognosis in OSCC cases. In the multivariate analysis, decreased miR-126 expression was strongly correlated with disease-free survival. CONCLUSION: The present results suggest that miR-126 might be a useful diagnostic and therapeutic target in OSCC.
Assuntos
Carcinoma de Células Escamosas/genética , Linfangiogênese/genética , MicroRNAs/metabolismo , Neoplasias Bucais/genética , Neovascularização Patológica/genética , Fator A de Crescimento do Endotélio Vascular/genética , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação ao Cálcio , Linhagem Celular Tumoral , Intervalo Livre de Doença , Regulação para Baixo , Família de Proteínas EGF , Fatores de Crescimento Endotelial/genética , Feminino , Humanos , Masculino , MicroRNAs/antagonistas & inibidores , Pessoa de Meia-Idade , Ativação TranscricionalRESUMO
Beta-tricalcium phosphate (ß-TCP) is widely used in clinical orthopedic surgery due to its high biodegradability, osteoconductivity, easy manipulation and lack of histotoxicity. However, little is known about the molecular mechanisms responsible for the beneficial effects of ß-TCP in bone formation. In this study, ß-TCP was implanted in dog mandibles, after which the gene expression profiles and signaling pathways were monitored using microarray and Ingenuity Pathways Analysis (IPA). Following the extraction of premolars and subsequent bone healing, ß-TCP was implanted into the artificial osseous defect. Histological evaluation (H-E staining) was carried out 4, 7 and 14 days after implantation. In addition, total RNA was isolated from bone tissues and gene expression profiles were examined using microarray analysis coupled with Ingenuity Pathways Analysis (IPA). Finally, real-time PCR was used to confirm mRNA levels. It was found that ß-TCP implantation led to a two-fold change in 3409 genes on day 4, 3956 genes on day 7, and 6899 genes on day 14. Among them, the expression of collagen type I α1 (COL1A1), alkaline phosphatase (ALP) and transforming growth factor (TGF)-ß2 was increased on day 4, the expression of receptor activator of NF-kappaB ligand (RANKL) and interferon-γ (IFN-γ) was decreased on day 7, and the expression of osteoprotegerin (OPG) was decreased on day 14, affecting the bone morphogenetic protein (BMP), Wnt/ß-catenin and nuclear factor-kappaB (NF-κB) signaling pathways in osteoblasts and osteoclasts. Simultaneously, vascular cell adhension molecule (VCAM)-1 expression was increased on day 4 and stromal cell-derived factor (SDF)-1 expression was increased on days 4 and 14. Taken together, these findings shed light on some of the cellular events associated with bone formation, bioresorption, regeneration and healing of ß-TCP following its implantation. The results suggest that ß-TCP enhances bone healing processes and stimulates the coordinated actions of osteoblasts and osteoclasts, leading to bone regeneration.
Assuntos
Fosfatos de Cálcio/metabolismo , Perfilação da Expressão Gênica , Mandíbula/metabolismo , Animais , Cães , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
AIMS: Regenerating islet-derived family, member 4 (Reg IV) is associated with the progression of various cancers. The aim was to examine Reg IV expression in adenoid cystic carcinomas (ACCs) in salivary glands. METHODS AND RESULTS: Reg IV expression was detected by immunohistochemistry and compared with clinicopathological parameters. Expression of phosphorylated epidermal growth factor receptor (pEGFR), phosphorylated AKT (pAKT) and MUC2 was examined by immunohistochemistry. Reg IV function was assessed with Reg IV antisense S-oligodeoxynucleotides (AS) in ACC3 human ACC cells. Reg IV was expressed by salivary duct epithelia and acinus myoepithelia, but not in squamous epithelia. Reg IV expression was found in 41% (17/41) of ACCs, but in none of 40 oral squamous cell carcinomas (OSCCs) and was associated with nodal metastasis (P = 0.047) and poor prognosis (P = 0.012) in ACCs. Reg IV expression was associated with pEGFR (14/17, 82%) in Reg IV+ ACCs, but had no relationship with pAKT or MUC2 expression in ACCs. Cell growth was inhibited by AS treatment in Reg IV+ ACC3 cells, but not in HSC-4 OSCC cells, whereas in vitro invasion of neither cell types was affected by AS treatment. CONCLUSIONS: These results suggest that Reg IV might accelerate cell growth and disease progression of ACCs.
Assuntos
Carcinoma Adenoide Cístico/patologia , Lectinas Tipo C/metabolismo , Neoplasias das Glândulas Salivares/patologia , Glândulas Salivares/patologia , Idoso , Carcinoma Adenoide Cístico/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Intervalo Livre de Doença , Receptores ErbB/metabolismo , Humanos , Imuno-Histoquímica , Mucina-2/metabolismo , Proteínas Associadas a Pancreatite , Fosforilação , Prognóstico , Neoplasias das Glândulas Salivares/metabolismoRESUMO
AIMS: Receptor for advanced glycation end products (RAGE) has recently been recognized as a cancer-associated protein responsible for cancer progression and metastasis in gastrointestinal cancers. The aim was to examine the role of RAGE in oral squamous cell carcinoma (OSCC). METHODS AND RESULTS: RAGE expression was examined by immunohistochemistry in 74 OSCC patients and evaluated with a grading based on Allred's score. RAGE expression was compared with clinicopathological parameters including clinical stage, invasive depth, nodal metastasis, disease recurrence and disease-free survival. High-grade expression of RAGE (RAGE-H) was observed in 30 (40.5%) of 74 OSCCs. RAGE-H was associated with depth of invasion (P < 0.0001) and local recurrence (P < 0.0001), but not with histological differentiation, clinical stage or nodal metastasis. Disease-free survival in patients with RAGE-H was significantly worse than in those with low-level RAGE expression. Multivariate analysis showed RAGE-H to be an independent prognostic factor for disease-free survival in OSCC patients (P = 0.0022). CONCLUSION: RAGE is a relevant factor in predicting disease recurrence and patients' prognosis in OSCC.