RESUMO
Cataracts are the most common eye disease to cause blindness in patients. The abnormal deposition of laminins (LMs) in the lens capsule and the disruption of capsular epithelium contribute to cataract development, although the mechanism by which this occurs is currently unclear. The present study aimed to reproduce HLE B3 basement membranes (BMs) using HLE B3 cells and to analyze the similarities of LM expression between HLE B3 BMs and human anterior lens capsule (ALC). Immunohistochemistry (IHC), ELISA, western blot analysis and immunoprecipitation (IP)western blot analysis were used to detect total LMs, LM trimers and 11 LM subunits in HLE B3 cells, HLE B3 BMs and human ALCs. In IHC staining, HLE B3 cells and human ALCs were positive for LMs. In LM ELISA, all samples analyzed were positive for LMs. Western blot analysis detected all LM subunits except for LMγ3 in HLE B3 cell lysate, 4 subunits (LMα4, LMα2, LMα1 and LMγ1) in HLE B3 cell culture supernatant, 5 subunits (LMα4, LMα2, LMα1, LMß3 and LMγ1) in HLE B3 BMs, and 3 subunits (LMα4, LMγ2 and LMγ1) in human ALCs. The results of IPwestern blot analysis revealed that the LM411 trimer was detected in HLE B3 cell culture supernatant. These results indicated that HLE B3 BMs were similar to human ALCs in terms of LM expression. Therefore, HLE B3 BMs could be used as an in vitro ALC model to determine the role of LMs in ALC in the pathogenesis of cataracts and to select potential anticataract drugs.
Assuntos
Células Epiteliais/metabolismo , Laminina/metabolismo , Cristalino/metabolismo , Adulto , Idoso , Membrana Basal/metabolismo , Biomarcadores , Linhagem Celular , Feminino , Humanos , Imuno-Histoquímica , Laminina/química , Masculino , Pessoa de Meia-Idade , Multimerização ProteicaRESUMO
Oral squamous cell carcinoma (OSCC) is a common human malignancy with a high incidence rate and poor prognosis. Although astrocyte elevated gene 1 (AEG-1) expression is up-regulated in various human cancers and plays an important role in carcinogenesis and tumour progression, the impact of AEG-1 on the development and progression of OSCC remains unclear. Accordingly, this study aims to clarify the biological significance of AEG-1 in OSCC. We found AEG-1 to be overexpressed in OSCC tissues compared to normal oral mucosa. Knockdown or overexpression of AEG-1 in OSCC cell lines showed that AEG-1 is important for tumour growth, apoptosis, drug tolerance, and maintaining epithelial-mesenchymal transition (EMT)-mediated cell migration and invasion in vitro. Moreover, in a xenograft-mouse model generated by AEG-1-overexpressing SCC15 cells, we found that higher expression of AEG-1 promoted tumour growth, angiogenesis, and EMT in vivo. These findings provide mechanistic insight into the role of AEG-1 in regulating OSCC tumour growth, apoptosis, drug tolerance, and invasion, as well as AEG-1-induced activation of p38 and NF-κB signalling, suggesting that AEG-1 is an important prognostic factor and therapeutic target for OSCC.
Assuntos
Moléculas de Adesão Celular/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Animais , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Proteínas de Membrana , Camundongos , Camundongos Nus , Mucosa Bucal/patologia , Neoplasias Bucais/genética , Invasividade Neoplásica/patologia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Prolonged glucocorticoids (GCs) treatment may lead to the formation of posterior subcapsular cataracts. The present study aimed to investigate differential gene expression in lens epithelial cells (LECs) in response to GCs using DNA microarray profiling. The gene expression profile of GSE13040 was downloaded from the Gene Expression Omnibus database, which includes 12 human LECs treated with vehicle or dexamethasone (Dex) for 4 or 16 h with six samples at each time period, of which three samples were treated with vehicle (control group) and three samples were treated with Dex (Dex group) at each time point. The differentially expressed genes (DEGs) were identified between the control group and the Dex group at each time period with the thresholds of P<0.05 and |logFC|>1. The DEGs were further analyzed using bioinformatics methods. Firstly, DEGs were subject to a hierarchical cluster analysis. Subsequently, the functional enrichment analysis was performed for the common DEGs between the two time periods. Finally, the transcription factors and binding sites of DEGs associated with response to GC stimulus were analyzed. A total of 696 and 949 DEGs were identified at 4 h and 16 h, respectively. Hierarchical cluster analysis revealed that DEG expression was higher in the Dex group than in the control group (P<0.05). A total of 13 significant functions were enriched for the 72 common DEGs at the two time periods. Chemokine (C-C motif) ligand 2 (CCL2), dual-specificity phosphatase-1 (DUSP1) and FAS were associated with the response to GC stimulus and the transcription factor cJun bound to promoter regulation regions of CCL2, DUSP1 and FAS. In conclusion, the transcription factors and binding sites of DEGs associated with the response of LECs to GCs may provide potential gene targets for designing and developing drugs to protect against GC-induced cataract formation.
Assuntos
Cristalinas/genética , Dexametasona/farmacologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Cristalino/efeitos dos fármacos , Fatores de Transcrição/genética , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Cristalino/citologia , Cristalino/metabolismoRESUMO
An Arg-Gly-Asp (RGD) motif in the fourth FAS1 domain of the human BIGH3 (transforming growth factor-ß1-inducible gene-h3) protein has been reported to play an important role in mediating tumor angiogenesis. The aim of this study was to investigate the inhibitory effect of a modified C-terminal fragment BIGH3 protein with an RGDRGD motif on corneal neovascularization in vitro and in vivo. Recombinant C-terminal fragment BIGH3 protein with wild-type sequence and modified C-terminal fragment BIGH3 protein containing an RGDRGD motif were successfully expressed and purified. We demonstrated that both proteins significantly inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) adhesion, migration, and tube formation and induced cell apoptosis but failed to inhibit HUVEC proliferation. We determined that the mechanism underlying this activity was an interaction between BIGH3 and αvß3 integrin, which blocked the phosphorylation of PI3K/Akt and ERK signaling pathways. The inhibitory effects of wild-type and modified C-terminal fragment BIGH3 proteins on angiogenesis were confirmed by a rabbit corneal neovascularization assay. More importantly, we provided evidence that the modified C-terminal fragment BIGH3 protein with an RGDRGD motif inhibited angiogenic activity far more effectively than did wild-type C-terminal fragment BIGH3. Collectively, our data show that a C-terminal fragment BIGH3 protein containing an RGDRGD motif might be promising as an effective drug in treating corneal neovascularization.