RESUMO
Family with sequence similarity 20-member C (FAM20C) is a kinase specific to most of the secreted phosphoproteome. FAM20C has been identified as the causative gene of Raine syndrome, initially characterized by lethal osteosclerosis bone dysplasia. However, since the identification of the cases of nonlethal Raine syndrome characterized by hypophosphatemia rickets, the previous definition of Raine syndrome has become debatable and raised a question about the role of mutations of FAM20C in controversial skeletal manifestation in the two forms of the disease. In this study, we aimed to investigate the influence of FAM20C mutations on skeletogenesis. We developed transgenic mice expressing Fam20c mutations mimicking those associated with human lethal and nonlethal Raine syndrome. The results revealed that transgenic mice expressing the mutant Fam20c found in the lethal (KO;G374R) and nonlethal (KO;D446N) Raine syndrome exhibited osteomalacia without osteosclerotic features. Additionally, both mutants significantly increased the expression of the Fgf23, indicating that Fam20c deficiency in skeletal compartments causes hypophosphatemia rickets. Furthermore, as FAM20C kinase activity catalyzes the phosphorylation of secreted proteomes other than those in the skeletal system, global FAM20C deficiency may trigger alterations in other systems resulting in osteosclerosis secondary to hypophosphatemia rickets. Together, the findings of this study suggest that FAM20C deficiency primarily causes hypophosphatemia rickets or osteomalacia; however, the heterogeneous skeletal manifestation in Raine syndrome was not determined solely by specific mutations of FAM20C. The findings also implicated that rickets or osteomalacia caused by FAM20C deficiency would deteriorate into osteosclerosis by the defects from other systems or environmental impacts.
Assuntos
Hipofosfatemia , Osteomalacia , Osteosclerose , Raquitismo , Camundongos , Animais , Humanos , Osteomalacia/complicações , Osteomalacia/genética , Osteosclerose/genética , Osteosclerose/complicações , Mutação/genética , Raquitismo/complicações , Camundongos Transgênicos , Hipofosfatemia/genética , Hipofosfatemia/complicações , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Ligação ao Cálcio/genéticaRESUMO
OBJECTIVE: This study focused on investigating relation between biglycan (BGN) and decorin (DCN) expression and prognostic outcome for oral squamous cell carcinoma (OSCC) cases. MATERIAL AND METHODS: BGN and DCN mRNA and protein expression was detected by qRT-PCR and Western-blotting (WB) assays from 31 OSCC samples as well as healthy samples. This work harvested 101 paraffin-embedded OSCC together with 30 healthy samples, and conducted immunohistochemical (IHC) staining for assessing pathological changes. Association of DCN with BGN within OSCC was explored by Spearman's analysis. Survival rate was explored by Kaplan-Meier (KM) approach. Multivariate analysis was conducted by Cox regression. RESULTS: WB and qRT-PCR results showed BGN up-regulation (p < 0.001, p < 0.0001) whereas DCN down-regulation (p < 0.0001, p < 0.0001) with fresh OSCC tissues; the expression of BGN and DCN associated with the OSCC histopathological grade. IHC results suggested elevated BGN level (p < 0.0001) whereas DCN down-regulation (p < 0.0001) with paraffin embedded OSCC tissues. The expression of BGN and DCN associated with histopathologic grades and tumor stage of OSCC. The result of Spearman's analysis demonstrated significant association between the expression of BGN and DCN in OSCC. Survival analysis revealed that patients with higher BGN/lower DCN level showed poor overall survival (OS) as well as tumor-specific survival (TSS). Multivariate analysis proved that BGN and DCN independently predicted the prognosis of OS and TSS. CONCLUSION: BGN and DCN expression levels can be adopted for predicting OSCC prognostic outcome.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Biglicano/genética , Decorina/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Neoplasias Bucais/genética , PrognósticoRESUMO
OBJECTIVE: The current study was aimed to investigate the association of nudix hydrolase 1 (NUDT1) levels with the prognosis of oral squamous cell carcinoma (OSCC) patients. MATERIAL AND METHODS: Western immunoblotting and qRT-PCR were used to detect the protein and mRNA levels of NUDT1 in 31 cases of OSCC and normal tissues. The paraffin-embedded 62 cases of OSCC and 18 normal tissues were collected, and the pathological alterations were assessed by immunohistochemistry. The prognosis of all patients was followed up. Kaplan-Meier method was used to analyze the survival rate, and Cox regression was used for multivariate analysis. RESULTS: Both the protein and gene levels of NUDT1 were statistically increased (P = .0007 and P < .0001) in the OSCC tissue and had a significant association with the histopathologic grades of OSCC (P < .0001 and P = .0223). Immunohistochemistry detection of NUDT1 in 62 human OSCC tissues and 18 normal control tissues showed that NUDT1 expression was significantly increased in OSCC tissue and showed a strong association with histopathologic grades (P < .0001) and tumor stage (P = .005). Patients with high NUDT1 expression exhibited poorer overall survival rate (OS) and tumor-specific survival rate (TSS) than those with low NUDT1 expression (P < .0001 and P = .0008), and NUDT1 was independent prognostic factors for OS and TSS (P < .0001 and P < .001). CONCLUSION: The expression level of NUDT1 might be used to predict the prognosis of OSCC patients.
Assuntos
Carcinoma de Células Escamosas/diagnóstico , Enzimas Reparadoras do DNA/genética , Neoplasias Bucais/diagnóstico , Monoéster Fosfórico Hidrolases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Prognóstico , RNA Mensageiro , Taxa de SobrevidaRESUMO
Autophagy inhibition is emerging as a new paradigm for efficient cancer therapy by overcoming multidrug resistance (MDR). Here, we developed an effective chemotherapeutic system for oral squamous cell carcinoma (OSCC) based on polymeric nanomicelles for codelivery of the anticancer drug doxorubicin (DOX) and the autophagy inhibitor LY294002 (LY). The hydrophobic DOX was conjugated onto a hydrophilic and pH-responsive hyperbranched polyacylhydrazone (HPAH), forming the DOX-conjugated HPAH (HPAH-DOX). Due to its amphiphilicity, HPAH-DOX self-assembled into nanomicelles in an aqueous solution and the autophagy inhibitor LY could be loaded into the HPAH-DOX micelles. The release of DOX and LY from the LY-loaded HPAH-DOX micelles was pH-dependent, whereas LY was released significantly faster than DOX at a mildly acidic condition. The in vitro evaluation demonstrated that the LY-loaded HPAH-DOX micelles could rapidly enter cancer cells and then release LY and DOX in response to an intracellular acidic environment. Compared to the HPAH-DOX micelles and the physical mixture of HPAH-DOX and LY, the LY-loaded HPAH-DOX micelles induced a higher proliferation inhibition of tumor cells, illustrating a synergistic effect of LY and DOX. The preferentially released LY inhibited the autophagy of tumor cells and made them more sensitive to the subsequent liberation of DOX. The polymeric codelivery system for programmable release of the chemotherapy drug and the autophagy inhibitor provides a new platform for combination of traditional chemotherapy and autophagy inhibition.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/química , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Micelas , Polímeros/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Humanos , Neoplasias Bucais/metabolismoRESUMO
Short root defects are prone to cause various periodontal diseases and lead to tooth loss in some serious cases. Studies about the mechanisms governing the development of the root are needed for a better understanding of the pathogenesis of short root defects. The protein family with sequence similarity 20 group C (FAM20C) is a Golgi casein kinase that has been well studied in the development of tooth crown formation. However, whether FAM20C plays a role in the development of tooth root is still unknown. Thus, we generated Sox2-Cre;Fam20cfl/fl (cKO) mice, in which Fam20c was ablated in both the dental epithelium and dental mesenchyme, and found that the cKO mice showed severe short root defects mainly by inhibiting the development of dental mesenchyme in the root region. In this investigation, we found morphological changes and differentiation defects, with reduced expression of dentin sialophosphoprotein (DSPP) in odontoblasts of the root region in cKO mice. Furthermore, the proliferation rate of apical papillary cells was reduced in the root of cKO mice. In addition, the levels of bone morphogenetic protein 4 (BMP4) and phospho-Smad1/5/8, and that of Osterix and Krüppel-like factor 4 (KLF4), two downstream target molecules of the BMP signaling pathway, were significantly reduced in the root of cKO mice. These results indicate that FAM20C plays an essential role in the development of the root by regulating the BMP signaling pathway.
RESUMO
BACKGROUND: This investigation examined the effects of the microRNA miR-34c-5p on the proliferation, migration, and invasion of oral squamous cell carcinoma (OSCC) and the mechanisms involved. METHODS: The Gene Expression Omnibus (GEO) database was used to filter the chips, and the GEO2R software (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to analyze the microarray data (GSE28100 and GSE45238). Gene set enrichment analysis (GSEA) was used to study the relationship between the expression of miR-34c-5p and the distant metastasis and pathological grade of OSCC. The correlation between TRIM29 (tripartite motif containing 29) expression and the malignant clinical phenotype of OSCC was also examined. The mRNA and protein expression levels of miR-34c-5p and TRIM29 were measured by real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot analysis. The proliferation, migration, invasion and apoptosis of the human oral squamous carcinoma cell lines CAL-27 and Tca8113 was assessed by performing cell-counting kit-8 (CCK-8) assays, colony formation assays, transwell tests, wound scratch tests and flow cytometry. Luciferase reporter assays were used to predict the relationship between miR-34c-5p and TRIM29. A xenograft nude model was established and used to evaluate the effect of miR-34c-5p on tumor growth in female BALB/c mice. RESULTS: The expression of miR-34c-5p was significantly correlated with the proliferation, migration, and metastasis of OSCC. Overexpression of miR-34c-5p promoted the proliferation, migration, and invasion of CAL-27 and Tca8113 cells, and suppressed their apoptosis. Inversely, low expression of miR-34c-5p suppressed the proliferation, migration, and invasion of CAL-27 and Tca8113 cells, and promoted their apoptosis. Overexpression of miR-34c-5p promoted tumor growth in the xenograft nude mice model. The expression of TRIM29 was related to malignant clinical phenotype of OSCC. Overexpression of TRIM29 inhibited the proliferation, migration and invasion of CAL-27 and Tca8113 cell, and induced their apoptosis. TRIM29 knockout had just the opposite effect. Importantly, miR-34c-5p binds to TRIM29 and inhibited TRIM29 expression. CONCLUSIONS: MiR-34c-5p regulates the proliferation, migration, invasion, and apoptosis of OSCC through targeted binding of TRIM29. This may represent a novel therapeutic target for the treatment of patients with OSCC.
RESUMO
BACKGROUND: Amelogenesis imperfecta (AI) is a type of hereditary diseases that manifest defects in the formation or mineralization of enamel. Recently, it is reported that inactivation of FAM20C, a well-known Golgi casein kinase, caused AI. However, the mechanism of it is still unknown. The aim of this study was to explore the molecular mechanism of AI, which caused by ablation of FAM20C. RESULTS: In the Sox2-Cre;Fam20Cfl/fl (cKO) mouse, we found abnormal differentiation of ameloblasts, improper formation and mineralization of enamel, and downregulation of both mRNA and protein level of enamel matrix proteins, including amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). The levels of BMP2, BMP4 and BMP7, the ligands of BMP signaling pathway, and phosphorylation of Smad1/5/8, the key regulators of BMP signaling pathway, were all decreased in the enamel matrix and the ameloblast of the cKO mice, respectively. The expression of cyclin-dependent kinase inhibitor (P21), muscle segment homeobox genes 2 (Msx2), which are the target genes of the BMP signaling pathway, and laminin 3, the downstream factor of Msx2, were all significantly decreased in the ameloblasts of the cKO mice compared to the control mice. CONCLUSION: the results of our study suggest that ablation of FAM20C leads to AI through inhibiting the Smad dependent BMP signaling pathway in the process of amelogenesis.
Assuntos
Amelogênese Imperfeita/genética , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas do Esmalte Dentário/genética , Proteínas da Matriz Extracelular/genética , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/genética , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Esmalte Dentário/metabolismo , Regulação para Baixo , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Camundongos Knockout , Proteínas Smad Reguladas por Receptor/metabolismoRESUMO
Intervertebral disc (IVD) disorder is often caused by the defect of annulus fibrosus (AF), especially that of the outer AF. Studies about the mechanisms governing the development of the outer AF are needed for a better understanding of pathogenesis of IVD defects. Glycosaminoglycans (GAGs) are essential components of extracellular matrix (ECM) in AF. FAM20B is a newly identified xylose kinase that catalyzes the biosynthesis of GAGs. In this study, we created Fam20B conditional knockout (cKO) mice in which FAM20B was inactivated in type I collagen-expressing cells, the main type of cells in the outer AF of IVD. The cKO mice showed severe spine deformity and remarkable IVD defects associated with AF malformation. The AF of cKO mice had a lower level of chondroitin sulfate and heparan sulfate, and the outer AF cells lost their normal fibroblast-like morphology and acquired chondrocyte phenotypes, expressing a higher level of Sox 9 and type II collagen along with a reduced level of type I collagen. The level of phospho-Smad 2 and phospho-Smad 3, and that of scleraxis, a downstream target molecule of canonical TGF-ß signaling pathway were significantly lower in the AF of cKO mice. The AF in cKO mice also manifested altered levels in the molecules associated with the activations of MAPK pathway; the changes included the increase of phospho-P38 and phospho-ERK and a decrease of phospho-JNK. These results indicate that FAM20B plays an essential role in the development of AF by regulating the TGF-ß signaling and MAPK signaling pathways.
Assuntos
Anel Fibroso/patologia , Degeneração do Disco Intervertebral/patologia , Deslocamento do Disco Intervertebral/patologia , Sistema de Sinalização das MAP Quinases , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Anel Fibroso/anormalidades , Anel Fibroso/metabolismo , Feminino , Disco Intervertebral/anormalidades , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/genética , Deslocamento do Disco Intervertebral/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transdução de SinaisRESUMO
Mutations in the gene encoding family with sequence similarity 20, member A (FAM20A) caused amelogenesis imperfecta (AI), in humans. However, the roles of FAM20A in amelogenesis and dentinogenesis are poorly understood. In this study, we generated a Fam20a knockout (Sox2-Cre;Fam20afl/fl) mouse model by crossing Fam20afl/fl mice with Sox2-Cre transgenic mice, in which Fam20a was ablated in both dental epithelium and dental mesenchyme. We found that these mice developed an enamel phenotype that resembles human AI associated with FAM20A mutations, but did not have apparent dentin defects. The secretory stage ameloblasts in the mandibular incisors from the Sox2-Cre;Fam20afl/fl mice were shorter and detached from the enamel matrix, and subsequently lost their polarity, became disorganized and formed numerous spherical extracellular matrices in place of normal enamel. At the molecular level, the Sox2-Cre;Fam20afl/fl mice displayed dramatically reduced expression levels of the genes encoding the enamel matrix proteins, but unaltered levels of the genes encoding the dentin matrix proteins. Moreover, Fam20a ablation resulted in a great decrease in FAM20C protein level, but it did not alter the intracellular localization of FAM20C protein in ameloblasts and odontoblasts. These results indicate that FAM20A is essential for amelogenesis, but is dispensable for dentinogenesis.
Assuntos
Amelogênese Imperfeita/genética , Amelogênese/genética , Proteínas do Esmalte Dentário/genética , Dentinogênese/genética , Ameloblastos/metabolismo , Amelogênese Imperfeita/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Esmalte Dentário/metabolismo , Proteínas do Esmalte Dentário/metabolismo , Dentina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Odontoblastos/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
BACKGROUND: Interleukin-24(IL-24), also referred to as melanoma differentiation-associated gene-7(mda-7), is a unique member of the IL-10 gene family, which displays nearly ubiquitous cancer-specific toxicity. The most notable feature of IL-24 is selectively induced growth suppression and apoptosis in various cancer cells, with no harmful effects toward normal cells. Autophagy is a self-protective mechanism in many kinds of tumor cells that respond to anticancer treatment. It is reported that autophagy inhibition could enhance the effects of many kinds of anticancer treatments, including gene therapy. However, whether IL-24 is effective to treat oral squamous cell carcinomas (OSCC) and if autophagy inhibition could improve the anticancer effect of IL-24 towards OSCC is has not been detected. METHODS: MTT assays were carried out to determine the cell proliferation; Transfection was used to gene transfer; Western Blot was performed to detect the protein level of LC3II, P62, Beclin 1, Cleaved caspase-3, ß-Tubulin and ß-actin; Apoptosis rates and cell cycle alteration were analyzed using flow cytometry; Autophagy induction was confirmed by MDC staining, GFP-LC3 staining and transmission electron microscopy. Amount of IL-24 in the culture medium was quantified by ELISA. Apoptosis in vivo was analyzed by TUNEL assay. HE staining was used to observe the morphology of the samples. RESULTS: In the present study, we proved that IL-24 have a novel anticancer effect towards KB cells and that autophagy inhibition could improve the anticancer effect of IL-24. IL-24 treated cells showed autophagy characteristics and autophagy inhibition by 3-methyladenine (3-MA) significantly enhanced IL-24-induced apoptosis. Similar results were obtained in the KB cells xenograft tumor model. CONCLUSIONS: These results suggest that the combination of autophagy inhibitors and IL-24 based on the AdLTR2EF1α-mediated gene transfer could be a promising way to cure OSCC.