RESUMO
The Wnt/ß-catenin signaling pathway plays a key role in development and carcinogenesis. Although some target genes of this signaling have been identified in various tissues and neoplasms, the comprehensive understanding of the target genes and their roles in the development of human cancer, including hepatoma and colorectal cancer remain to be fully elucidated. In this study, we searched for genes regulated by the Wnt signaling in liver cancer using HuH-7 hepatoma cells. A comparison of the expression profiles between cells expressing an active form of mutant ß-catenin and cells expressing enhanced green fluorescent protein (EGFP) identified seven genes upregulated by the mutant ß-catenin gene (CTNNB1). Among the seven genes, we focused in this study on ODAM, odontogenic, ameloblast associated, as a novel target gene. Interestingly, its expression was frequently upregulated in hepatocellular carcinoma, colorectal adenocarcinoma, and hepatoblastoma. We additionally identified a distant enhancer region that was associated with the ß-catenin/TCF7L2 complex. Further analyses revealed that ODAM plays an important role in the regulation of the cell cycle, DNA synthesis, and cell proliferation. These data may be useful for clarification of the main molecular mechanism(s) underlying these cancers.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Via de Sinalização Wnt/genética , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , beta Catenina/genética , Ameloblastos/metabolismo , Ameloblastos/patologia , Neoplasias Hepáticas/patologia , Proliferação de Células/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Circular RNAs (circRNAs) are known to regulate tumorigenesis. In this study, circRNAs microarray was used to analyze the circRNA expression in lung adenocarcinoma (LUAD) tissues, and CircRNA zinc finger MYM-type containing 4(circZMYM4) was selected for further analysis. In this study, we detected circZMYM4 expression in LUAD specimens and cell lines using RT-PCR. The expression of circZMYM4 was further verified in the GEO datasets and TCGA datasets. Gain-of-function and loss-of-function experiments were used to analyze the effects of circZMYM4 on LUAD in vivo and in vitro. The relationship between miR-587 and circZMYM4 or ODAM was predicted by bioinformatics tools and confirmed using dual-luciferase reporter assays and RNA-pull down. We found that circZMYM4 was distinctly down-regulated in LUAD tissues and cell lines. Functional assays revealed that circZMYM4 overexpression suppressed LUAD cell proliferation, metastasis and suppressed apoptosis, while miR-587 overexpression could weaken these effects. Importantly, circZMYM4 upregulated ODAM expression via sponging miR-587 to suppress LUAD progression. ODAM knockdown could reverse the repressive effect of circZMYM4 overexpression on cell proliferation, migration and invasion abilities. Overall, circZMYM4 regulates the miR-587/ODAM axis to suppress LUAD progression, which may become a potential biomarker and therapeutic target.
Assuntos
Adenocarcinoma de Pulmão/genética , Amiloide/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , RNA Circular/genética , Adenocarcinoma de Pulmão/patologia , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica/genética , Metástase Neoplásica/patologiaRESUMO
OBJECTIVE: In this study, we investigated the potential and mechanism of odontogenic ameloblast-associated protein (ODAM) in the promoting junctional epithelium-related gene expression in an ameloblast-like cell line ALC. BACKGROUND: ODAM is expressed in ameloblasts and JE and acts as a component of the inner basal lamina (IBL) and intercellular matrix of JE. ODAM KO mice showed destruction of the integrity of the JE, which detaches from teeth. ODAM was confirmed to regulate the cytoskeleton through the ODAM-ARHGEF5-RhoA signaling pathway of the JE. Whether ODAM contributes to the regulation of ameloblast differentiation in JE remains unclear. After the formation of enamel, the ameloblast undergoes a series of morphological changes. Whether ODAM will affect the biological behavior of ameloblasts making them have the characteristics of JE is unclear. METHODS: A murine ameloblast-like cell line, ALC, was used to investigate the effects of ODAM on the JE-like changes of ALC cells in an epithelium-induced environment by generating ODAM overexpression and ODAM knockdown cells through a lentivirus transduction approach. The biomarkers of junctional epithelium CK19, SLPI, and ODAM and the potential regulatory gene WNT1 were investigated by real-time PCR, western blot, immunocytochemistry, immunostaining, luciferase reporter, and rescue assays. RESULTS: ODAM, CK19, and SLPI were significantly upregulated after epithelial induction. Overexpression of ODAM in ALC cells markedly increased CK19 and SLPI expression, while knockdown of ODAM in ALC cells clearly decreased CK19 and SLPI expression. A reporter luciferase assay showed that ODAM activated the WNT signaling pathway, especially through WNT1. Exogenous overexpression of ODAM upregulated WNT1 expression, while knockdown of ODAM reversed this effect. The WNT1 inhibition assay further confirmed the above results and showed that the WNT1 pathway was positively correlated with biomarkers of junctional epithelium CK19 and SLPI expression. Rescue studies showed that knocking down WNT1 in the ODAM-overexpressing ALC cells decreased the expression of CK19 and SLPI. Immunocytochemistry showed that ODAM colocalized with CK19, SLPI, and WNT1 in the cells. CONCLUSION: In conclusion, the research work showed that ODAM promotes junctional epithelium-related gene expression in ALC via the ODAM-WNT1 axis, which may provide new insight into the function of ODAM and JE formation.
Assuntos
Ameloblastos , Inserção Epitelial , Animais , Linhagem Celular , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de SinaisRESUMO
BACKGROUND: The gene for odontogenic ameloblast-associated (ODAM) is a member of the secretory calcium-binding phosphoprotein gene family. ODAM is primarily expressed in dental tissues including the enamel organ and the junctional epithelium, and may also have pleiotropic functions that are unrelated to teeth. Here, we leverage the power of natural selection to test competing hypotheses that ODAM is tooth-specific versus pleiotropic. Specifically, we compiled and screened complete protein-coding sequences, plus sequences for flanking intronic regions, for ODAM in 165 placental mammals to determine if this gene contains inactivating mutations in lineages that either lack teeth (baleen whales, pangolins, anteaters) or lack enamel on their teeth (aardvarks, sloths, armadillos), as would be expected if the only essential functions of ODAM are related to tooth development and the adhesion of the gingival junctional epithelium to the enamel tooth surface. RESULTS: We discovered inactivating mutations in all species of placental mammals that either lack teeth or lack enamel on their teeth. A surprising result is that ODAM is also inactivated in a few additional lineages including all toothed whales that were examined. We hypothesize that ODAM inactivation is related to the simplified outer enamel surface of toothed whales. An alternate hypothesis is that ODAM inactivation in toothed whales may be related to altered antimicrobial functions of the junctional epithelium in aquatic habitats. Selection analyses on ODAM sequences revealed that the composite dN/dS value for pseudogenic branches is close to 1.0 as expected for a neutrally evolving pseudogene. DN/dS values on transitional branches were used to estimate ODAM inactivation times. In the case of pangolins, ODAM was inactivated ~ 65 million years ago, which is older than the oldest pangolin fossil (Eomanis, 47 Ma) and suggests an even more ancient loss or simplification of teeth in this lineage. CONCLUSION: Our results validate the hypothesis that the only essential functions of ODAM that are maintained by natural selection are related to tooth development and/or the maintenance of a healthy junctional epithelium that attaches to the enamel surface of teeth.
Assuntos
Ameloblastos/metabolismo , Esmalte Dentário/metabolismo , Eutérios/genética , Inativação Gênica , Odontogênese , Proteínas/genética , Baleias/genética , Animais , Sequência de Bases , Teorema de Bayes , Códon/genética , Feminino , Fósseis , Funções Verossimilhança , Mutação/genética , Filogenia , Gravidez , Proteínas/metabolismoRESUMO
Purpose/aim of the study: Odontogenic ameloblast-associated protein (ODAM) is predominantly expressed during the maturation stage of enamel formation and interacts strongly with amelotin (AMTN). AMTN is involved in enamel mineralization, but the effect of ODAM on mineralization has not been investigated. This study determined whether ODAM was able to induce hydroxyapatite (HA) mineralization in modified simulated body fluid (SBF) and in a collagen matrix in vitro. MATERIALS AND METHODS: To monitor the kinetics of calcium phosphate mineralization, recombinant human (rh) ODAM protein in SBF buffer was incubated at 37°C and a light-scattering assay was conducted at intervals. To investigate the nucleation of ODAM in collagen matrix, the ODAM-impregnated collagen hydrogel was incubated in SBF buffer for 24 hours. Bovine serum albumin (BSA) was used as negative control. Mineral deposits were visualized using electron microscopy. RESULTS: The presence of rh-ODAM protein in SBF resulted in higher light-scattering values after 18-24 hours. Calcium phosphate precipitates were observed on the surface of the ODAM-treated, but not BSA-treated collagen hydrogel after 24 hours in SBF. TEM and SAED analyses showed that these crystals consisted of needle-like HA. CONCLUSION: Similar to AMTN, ODAM is able to promote HA nucleation in a dose-dependent manner in SBF, and even outside of its biological context in vitro.
Assuntos
Calcinose , Proteínas de Transporte/química , Colágeno/química , Proteínas do Esmalte Dentário/química , Matriz Extracelular/química , Amiloide , Proteínas de Transporte/metabolismo , Colágeno/metabolismo , Proteínas do Esmalte Dentário/metabolismo , Matriz Extracelular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Neoplasias , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Odontogenic Ameloblast-Associated Protein (ODAM) in gingival crevicular fluid (GCF) can provide evidence of the detachment of junctional epithelium from the tooth surface by periodontitis. This study sought to investigate the ability of ODAM to reflect the severity of periodontitis at a site-specific level; thus whether there was a relationship between clinical diagnostic parameters and the value of ODAM in GCF was analyzed. METHODS: Eight periodontitis patients with various severities were enrolled, and the clinical parameters and samples of GCF were obtained from 44 to 60 sites of each subject. The ODAM concentration was quantified by enzyme-linked immunosorbent assay. Correlation analyses between clinical parameters and ODAM values and unadjusted and adjusted (linear) mixed model analyses were performed. The accuracy of ODAM to reflect sites having a probing depth (PD) ≥ 5 mm and a positive bleeding on probing (BOP) was evaluated by receiver-operating characteristic analysis. RESULTS: A total of 424 GCF samples were collected. The mean ODAM concentration from each patient varied from 0.2 to 1.52 ng/ml. Correlations between PD or clinical attachment level (CAL) and ODAM values were found (p < 0.0001). An adjusted linear mixed model showed that PD or CAL were associated with ODAM values (p < 0.05). The area under the curve of ODAM, which reflected sites with PD ≥ 5 mm and positive BOP, was 0.661 (p < 0.0001). CONCLUSION: This result shows the possibility of GCF ODAM as a site-specific biomarker for periodontal tissue destruction.
Assuntos
Proteínas de Transporte/metabolismo , Líquido do Sulco Gengival/química , Periodontite/diagnóstico , Adulto , Idoso , Amiloide , Biomarcadores/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias , Índice Periodontal , Periodontite/metabolismo , Projetos PilotoRESUMO
The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.
Assuntos
Infecções por Bacteroidaceae/metabolismo , Proteínas do Esmalte Dentário/metabolismo , Inserção Epitelial/metabolismo , Gengiva/metabolismo , Infecções por Pasteurellaceae/metabolismo , Periodontite/metabolismo , Proteínas/metabolismo , Aggregatibacter actinomycetemcomitans , Animais , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Porphyromonas gingivalisRESUMO
Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin ß3- and ß6-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.
Assuntos
Proteínas de Transporte/metabolismo , Inserção Epitelial/metabolismo , Periodontite/metabolismo , Proteínas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Dente/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Amiloide , Animais , Proteínas de Transporte/análise , Linhagem Celular , Inserção Epitelial/patologia , Fibronectinas/análise , Fibronectinas/metabolismo , Humanos , Integrinas/análise , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Laminina/análise , Laminina/metabolismo , Camundongos , Proteínas de Neoplasias , Periodontite/patologia , Proteínas/análise , Fatores de Troca de Nucleotídeo Guanina Rho/análise , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/análiseRESUMO
BACKGROUND: Approximately 15% of gastrointestinal stromal tumors (GISTs) will not respond to tyrosine kinase inhibitors and drug resistance can develop over time. For refractory tumors, additional therapies are needed. Odontogenic ameloblast-associated protein (ODAM) is expressed in some epithelial malignancies and can correlate with clinical outcomes. This study evaluated ODAM and its relationship to phosphatase and tensin homolog on chromosome 10 (PTEN) and phosphorylation of AKT to an activated state (pAKT) in GISTs. MATERIALS AND METHODS: Ninety-five distinct tumor specimens from 79 patients were identified. Morphologic features and clinical data were recorded for all tumors. Risk of recurrence was calculated using the Memorial Sloan-Kettering nomogram. Immunohistochemistry was performed using antibodies to ODAM, PTEN, and pAKT. Immunoreactivity was assessed for both cytoplasmic and nuclear expression. Staining patterns were correlated with clinical outcomes. RESULTS: Increasing cytoplasmic ODAM staining correlated with a lower recurrence score (P = 0.002), a lower mitotic rate (P = 0.0001), and smaller tumor size (P = 0.038). Increasing pAKT cytoplasmic staining correlated with a higher recurrence score (P = 0.037) and a higher mitotic rate (P = 0.036). ODAM and pAKT expression in the nucleus was associated with tumor origin. PTEN nuclear expression increased with increasing mitotic rate. pAKT expression increased in the cytoplasm and nucleus in high-risk tumors. CONCLUSIONS: Risk of recurrence correlated with cytoplasmic expression of ODAM and pAKT, whereas nuclear expression did not predict recurrence. The staining pattern for ODAM and pAKT in the cytoplasm may further clarify the risk of recurrence beyond the available nomograms. The increased expression of pAKT in the cytoplasm and nucleus of high-risk tumors suggests a potential target for systemic therapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Gastrointestinais/metabolismo , Tumores do Estroma Gastrointestinal/metabolismo , Recidiva Local de Neoplasia/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Amiloide , Feminino , Seguimentos , Neoplasias Gastrointestinais/mortalidade , Tumores do Estroma Gastrointestinal/mortalidade , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Logísticos , Masculino , Proteínas de Neoplasias , Recidiva Local de Neoplasia/mortalidade , Fosforilação , Prognóstico , Sistema de Registros , Estudos Retrospectivos , Análise de SobrevidaRESUMO
Odontogenic ameloblast-associated protein (ODAM) contributes to cell adhesion. In human cancer, ODAM is down-regulated, and the overexpression of ODAM results in a favourable prognosis; however, the molecular mechanisms underlying ODAM-mediated inhibition of cancer invasion and metastasis remain unclear. Here, we identify a critical role for ODAM in inducing cancer cell adhesion. ODAM induced RhoA activity and the expression of downstream factors, including Rho-associated kinase (ROCK). ODAM-mediated RhoA signalling resulted in actin filament rearrangement by activating PTEN and inhibiting the phosphorylation of AKT. When ODAM is overexpressed in MCF7 breast cancer cells and AGS gastric cancer cells that activate RhoA at high levels, it decreases motility, increases adhesion and inhibits the metastasis of MCF7 cells. Conversely, depletion of ODAM in cancer cells inhibits Rho GTPase activation, resulting in increased cancer migration and invasion. These results suggest that ODAM expression in cells maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells. SIGNIFICANCE Breast cancer represents the first most frequent cancer, and the ratio of mortality is high in women. Of utmost importance for reducing risk by breast cancer are their anti-invasion mechanisms, particularly in the non-invasive cancer cells because metastasis is the principal cause of death among cancer patients. ODAM induced RhoA activity. ODAM-mediated RhoA signalling resulted in actin filament rearrangement, increased cell adhesion and inhibited the migration/invasion of MCF7 cells. These results suggest that ODAM expression maintains their adhesion, resulting in the prevention of their metastasis via the regulation of RhoA signalling in breast cancer cells.
Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Adesão Celular , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Adenocarcinoma/metabolismo , Amiloide , Animais , Neoplasias da Mama/metabolismo , Carcinogênese , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Proteínas de Neoplasias , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Severe periodontitis affects nearly 1 billion individuals worldwide, highlighting the need for early diagnosis. Here, an integrated system consisting of a microfluidic chip and a portable point-of-care (POC) diagnostic device is developed using a polymethyl methacrylate (PMMA) chip fabrication and a three-dimensional printing technique, which is automatically controlled by a custom-designed smartphone application to routinely assess the presence of a specific periodontitis biomarker, odontogenic ameloblast-associated protein (ODAM). A sandwich-type fluorescence aptasensor is developed on a microfluidic chip, utilizing aptamer pair (MB@OD64 and OD35@FAM) selectively binding to target ODAM. Then this microfluidic chip is integrated into an automated Internet of Things (IoT)-based POC device, where fluorescence intensity, as a signal, from the secondary aptamer binding to ODAM in a sandwich-type binding reaction on the microfluidic chip is measured by a complementary metal oxide semiconductor (CMOS) camera with a 488 nm light-emitting diode (LED) excitation source. Obtained signals are processed by a microprocessor and visualized on a wirelessly connected smartphone application. This integrated biosensor system allows the rapid and accurate detection of ODAM within 30 min with a remarkable limit of detection (LOD) of 0.011 nM under buffer conditions. Clinical application is demonstrated by successfully distinguishing between low-risk and high-risk individuals with 100 % specificity. A strong potential in the translation of this fluorescence-based microfluidic aptasensor integrated with an IoT-based POC system is expected to be employed for non-invasive, on-site, rapid, and accurate ODAM detection, facilitating periodontitis diagnosis.
Assuntos
Técnicas Biossensoriais , Internet das Coisas , Doenças Periodontais , Periodontite , Humanos , Doenças Periodontais/diagnóstico , Periodontite/metabolismo , ProteínasRESUMO
OBJECTIVES: Junctional epithelium (JE) connects the tooth surface and gingival epithelium and adheres directly to the tooth enamel. JE plays an important role as a barrier preventing the invasion of exogenous bacteria and substances. However, the cellular characteristics of this epithelium have not been adequately described, because no useful in vitro experimental model exists for JE. METHODS: We generated a novel JE cell line, mHAT-JE01, using naturally immortalized dental epithelium derived from incisor labial cervical cells and by selecting cells that adhered to apatite. mHAT-JE01 was characterized by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction and compared with the gingival epithelial cell line, mOE-PE01. RESULTS: The mHAT-JE01 cells had a higher capacity for producing JE-specific markers than oral mucous epithelial cells. In addition, the presence of lipopolysaccharides from Porphyromonas gingivalis downregulated the expression of JE protein markers in mHAT-JE01 cells. CONCLUSIONS: This cell line is stable and presents the opportunity to characterize JE efficiently, which is essential for the prevention and treatment of periodontal disease.
Assuntos
Células Epiteliais , Incisivo , Incisivo/química , Incisivo/metabolismo , Células Epiteliais/química , Células Epiteliais/metabolismo , Epitélio/química , Epitélio/metabolismo , Proteínas/análise , Proteínas/metabolismo , Linhagem CelularRESUMO
Recently, point-of-care tests (POCT) have gained much attention due to their convenient, fast, simple, and easy characteristics. For POCT, portability is an essential feature. In this study, we have successfully fabricated a portable mini-potentiostat. Using chronoamperometry, electrical signals of this portable mini-potentiostat were measured, and the analytical performance of electrochemical aptasensors was compared with a benchtop potentiostat. The electrochemical signals measured by mini-potentiostat can be displayed on the screen of a smartphone. To verify the analytical performance of this portable electrochemical aptasensor platform with a mini-potentiostat, two well-known model protein biomarkers, vaspin, a type 2 diabetes biomarker, and thrombin, a biomarker for pulmonary metastasis and cardiovascular disease, were confirmed to be detected by using corresponding aptamer duo. After solid verification of this portable electrochemical aptasensor platform, we have successfully implemented this portable mini-potentiostat system to develop a portable sandwich-type binding pair of aptamers-based electrochemical biosensor, which can diagnose periodontal disease by measuring ODAM biomarker. The linear range of this ODAM biosensor was 0 to 15 nM with a detection limit of 0.02 nM and 1 nM in buffer and saliva, respectively. The sensitivity of this biosensor has been greatly enhanced, compared to previously developed surface plasmon resonance (SPR) or lateral flow assay (LFA) based aptasensors. This study showed that this new portable aptamer duo-based biosensor is expected to diagnose the early stage of periodontal diseases from real samples, such as saliva or gingival crevicular fluid in a short time as a point-of-care (POC) testing.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Diabetes Mellitus Tipo 2 , Doenças Periodontais , Diagnóstico Precoce , Técnicas Eletroquímicas , Humanos , Limite de Detecção , Doenças Periodontais/diagnósticoRESUMO
This research aims to develop biosensors which could diagnose periodontal diseases in early phases and predict the illness stage of patients, in order to give them adequate treatment timely. Human odontogenic ameloblast-associated protein (ODAM) is considered to be a potential biomarker for periodontal diseases, based on high correlation between the level of ODAM in gingival crevicular fluid (GCF) and the degree of periodontitis. Many aptamers, including a cognate pair of aptamers which can bind to the different sites of ODAM, were successfully screened in a very stringent condition employing saliva as a counter target through the graphene oxide-based systemic evolution of ligands by exponential enrichment (GO-SELEX). For the characterization of the aptamer candidates, GO-based fluorescence resonance energy transfer (GO-FRET) and surface plasmon resonance (SPR) assays were conducted. The sandwich-type binding of a cognate pair of aptamers to ODAM was additionally confirmed by employing circular dichroism (CD) and magnetic beads-based fluorescence imaging methods. The resulting cognate pair of aptamers, OD64 and OD35, were found to have their dissociation constant (Kd), 47.71â¯nM and 51.36â¯nM, respectively. The minimum detectable concentrations of a sandwich-type SPR biosensor were found to be 0.24â¯nM and 1.63â¯nM, respectively, for both buffered and saliva samples. Finally, using this cognate pair of aptamers, a sandwich-type lateral flow strip biosensor was successfully realized. This research shows the potential for implementation of a cognate pair of aptamers on point-of-care biosensors which enables simple, rapid, and non-invasive saliva-based diagnosis of periodontal-related diseases that can overcome current diagnostic methods and improve health care system.
Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas de Transporte/análise , Doenças Periodontais/diagnóstico , Saliva/química , Ressonância de Plasmônio de Superfície/métodos , Amiloide , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Limite de Detecção , Proteínas de Neoplasias , Sistemas Automatizados de Assistência Junto ao Leito , Técnica de Seleção de Aptâmeros/métodosRESUMO
OBJECTIVE: Odontogenic Ameloblast-Associated Protein (ODAM) is encoded by a secretory calcium-binding phosphoprotein cluster gene, which generally plays an important role for mineralization. Dental follicle (DF) is essential in regulating bone formation for tooth eruption. This study aims to reveal ODAM expression in the DFs of developing and erupting molars, and to determine the possible role of ODAM. DESIGN: DFs were collected from human third molars and rat mandibular molars for gene expression assessment and for establishment of cell cultures. RT-PCR and western blot were conducted to determine ODAM expression. Over- or silencing expression of ODAM in the dental follicle stem cells (DFSCs) was done by transfecting the cells with ODAM plasmid or siRNA to evaluate ODAM effects on osteogenesis. RESULTS: Rat DFs weakly expressed ODAM at early-postnatal days, but a chronological increment of ODAM expression from days 1 to 11 was observed. Differences in expression of ODAM were seen in the human DFs of different individuals. In vitro, ODAM was expressed in DFSCs, but almost no expression in DF-derived fibroblast-like cells. Forcing the DFSCs to overexpress ODAM accelerated osteogenesis, whereas continuously silencing the ODAM in the DFSCs reduced osteogenesis only at 2 weeks of osteogenic induction. CONCLUSIONS: ODAM is differentially expressed in the DFs of different age molars. Its expression is coincident with the increased bone formation of tooth crypt during tooth eruption in rat DFs. Increase of ODAM expression may accelerate osteogenic differentiation of DFSCs. Thus, ODAM expression in the DF may regulate bone formation for timely tooth eruption.
Assuntos
Ameloblastos/citologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Saco Dentário/citologia , Odontogênese/fisiologia , Osteogênese/fisiologia , Células-Tronco/citologia , Amiloide , Animais , Western Blotting , Células Cultivadas , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Neoplasias , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo , TransfecçãoRESUMO
Odontogenic ameloblast associated protein (ODAM) is a protein contributed to cell adhesion and has been shown to express in normal prostate tissue, but the expression and significance of ODAM in prostate cancer remain unknown. In this study, we detected the protein expressions of ODAM in 88 prostate cancer tissues with immunohistochemical staining, and found that 53 cases (60.2%) was high expression of ODAM, which was shown in the cytoplasm and paranuclear regions. Furthermore, low expression of ODAM was significantly correlated with lymph node metastasis, preoperative PSA and Gleason score, but not with mean age, follow-up duration, PSM rate and distribution of pathological T stage. Additionally, our results of multivariate analysis showed that low ODAM expression was an independent predictor of biomedical recurrence, while the positive lymph node metastasis, Gleason score, and preoperative PSA were not the independent risks for biomedical recurrence. Overexpression of ODAM did not inhibit the growth of prostate cancer cells PC3, but significant suppressed their invasion and migration with decrease of the protein levels of MMP-2. These results suggest that ODAM is a predictor for biomedical recurrence and inhibits the migration and invasion of prostate cancer.
RESUMO
Odontogenic ameloblast-associated protein (ODAM), an acidic matricellular protein, has been implicated in several epithelial neoplasms. However, its biological functions and molecular mechanisms in cancer progression, particular colorectal carcinoma (CRC), remain unknown. Here we demonstrated that ODAM was significantly down-regulated in CRC tissues compared with their normal counterparts. Then, we established that ODAM expression level was closely correlated with CRC development and patient prognosis. The abnormal expression of ODAM dramatically affected CRC cell growth in vitro and in vivo. We further revealed that the inhibitory effects of ODAM on CRC cell growth were associated with PTEN elevation and PI3K/AKT signaling inactivation. Furthermore, we determined that silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing CRC cells. Our study suggests matricellular protein ODAM may serve as a novel prognostic marker and act as a CRC growth suppressor.
Assuntos
Proteínas de Transporte/metabolismo , Proliferação de Células , Neoplasias Colorretais/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Idoso , Amiloide , Animais , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Estimativa de Kaplan-Meier , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas de Neoplasias , Transplante de Neoplasias , PTEN Fosfo-Hidrolase/genética , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
In mammals, the casein locus consists of stretches of non-coding DNA, the functions of most of which are unknown. These regions are believed to harbour elements responsible for spatio-temporally regulated expression of genes in this locus and so far, only a few such elements have been identified. In this study, we report a novel regulatory element in the casein locus. Comparative analysis of genomic DNA sequences of casein loci from different mammals identified a 147bp long evolutionarily conserved region (ECR) upstream of Odam, a gene in this locus. The ECR was found in close proximity of Odam gene in all the mammals examined. In-silico analysis predicted the ECR as a potential regulatory element. Functional analysis in different cell lines identified it as a unidirectional repressor element. From our findings we speculate that the ECR may be involved in the repression of the Odam expression in the mammary gland during lactation.