RESUMO
Long noncoding RNA taurine-upregulated gene 1 (lncRNA TUG1) has been reported to play a key role in the progression of diabetic nephropathy (DN). However, the role of lncRNA TUG1 in the regulation of diabetic nephropathy remains largely unknown. The aim of the present study is to identify the regulation of lncRNA TUG1 on extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ, and investigate the underlying mechanisms in progression of DN. Microarray was performed to screen differentially expressed miRNAs in db/db DN mice. Afterwards, computational prediction programs (TargetScan, miRanda, PicTar and miRGen) was applied to predict the target gene of miRNAs. The complementary binding of miRNA and lncRNA was assessed by luciferase assays. Protein and mRNA expression were detected by western blot and real time quantitate PCR. MiRNA-377 was screened by miRNA microarray and differentially up-regulated in db/db DN mice. PPARγ was predicted to be the target of miR-377 and the prediction was verified by luciferase assays. Expression of miR-377 was up-regulated in mesangial cell treated with high glucose (25 mM), and overexpression of miR-377 inhibited PPARγ expression and promoted PAI-1 and TGF-ß1 expression. The expression of TUG1 antagonized the effect of miR-377 on the downregulation of its target PPARγ and inhibited extracellular matrix accumulation, including PAI-1, TGF-ß1, fibronectin (FN) and collagen IV (Col IV), induced by high glucose. LncRNA TUG1 acts as an endogenous sponge of miR-377 and downregulates miR-377 expression levels, and thereby relieving the inhibition of its target gene PPARγ and alleviates extracellular matrix accumulation of mesangial cells, which provides a novel insight of diabetic nephropathy pathogenesis.
Assuntos
Nefropatias Diabéticas/metabolismo , Matriz Extracelular/patologia , Células Mesangiais/patologia , MicroRNAs/metabolismo , PPAR gama/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Células Cultivadas , Nefropatias Diabéticas/patologia , Matriz Extracelular/metabolismo , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: To study the expression of the RIZ1 (Retinoblastoma protein-interacting zinc-finger gene 1) gene and investigate the promoter region methylation status of RIZ1 gene in the human esophageal squamous cell carcinoma (ESCC) cell lines of KYSE150, KYSE510, TE13, EC9706, CaEsl7, and EC109. To investigate the influence of DNMT (DNA methyltransferase) 5-aza-CdR(5-aza-2'-deoxycytidine) on the transcription of the RIZ1 gene in one cell line whose RIZ1 gene promoter region methylation was detected, and to investigate its influence on the cell proliferation. METHODS: Real-time PCR (Real-time quantitative PCR) and an immunohistochemistry technique was used to get the expression of RIZ1 in specimens from 6 human ESCC cell lines and 28 ESCC patients (tumor tissues and adjacent non-cancerous tissues). MSP (Methylation-specific PCR) was used to investigate the promoter region methylation status of the RIZ1 gene in the 6 ESCC cell lines. One cell line, whose RIZ1 gene promoter region methylation was detected, was chosen for the next studies in which it was treated it by with 5-aza-CdR. Real-time PCR was used to investigate its influence on the transcription of RIZ1 gene and MTT (methyl thiazolyl tetrazolium) was used to detect if 5-aza-CdR inhibits the proliferation of the cell line. RESULTS: In the 28 ESCC patient samples, RIZ1 expression was significantly lower in the tumor tissues than that in their adjacent non-cancerous tissues (p < 0.05). Consistently, immunohistochemistry analyses of RIZ1 protein expression showed that in the ESCC tissues RIZ1 protein expression was also significantly lower than in the adjacent tissues. In the human ESCC tissues the rate of expression accounts for 0% (0/12), and in the adjacent noncancerous tissues the rate of expression was 66.7% (8/12), the correlation was highly significant (chi2 = 12.000, p < 0.05). Promoter methylation of the RIZ1 gene was detected in TE13, CaEsl7, EC109. The cell line TE13 was chosen for the next studies. The expression of RIZ1 mRNA in TE-13 was up-regulated after having been treated with 5-aza-CdR. 5-aza-CdR inhibited cell proliferation of TE-13 in a time and concentration-dependent manner. CONCLUSIONS: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression. Methylation of the RIZ1 promoter and loss of RIZ1 expression in human ESCC are independent biomarkers. Their determination may offer guidance for selecting appropriate diagnoses and treatments. RIZ1 may be a potential tumor suppressor in human ESCC.
Assuntos
Carcinoma de Células Escamosas/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Decitabina , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
AIMS: High glucose (HG)-induced pancreatic ß-cell apoptosis may be a major contributor to the progression of diabetes mellitus (DM). NADPH oxidase (NOX2) has been considered a crucial regulator in ß-cell apoptosis. This study was designed to evaluate the impact of GLP-1 receptor agonist (GLP-1Ra) liraglutide on pancreatic ß-cell apoptosis in diabetes and the underlying mechanisms involved. METHODS: The diabetic rat models induced by streptozotocin (STZ) and a high fat diet (HFD) received 12â¯weeks of liraglutide treatment. Hyperglycemic clamp test was carried out to evaluate ß-cell function in vivo. Flow cytometry analysis was used to measure apoptosis rates in vitro. DCFH-DA method was used to detected ROS level in vivo and in vitro. RESULTS: Liraglutide significantly improved islet function and morphology in diabetic rats and decreased cell apoptosis rates. Thr183/Thr185 p-JNK1/2 and NOX2 levels reduced in diabetic rats and HG-induced INS-1 cell following liraglutide treatment. In addition, liraglutide upregulated the phosphorylation of AMPKα (p-AMPKα), which prevented NOX2 activation and alleviated HG-induced ß-cell apoptosis. CONCLUSION: The p-AMPKα/NOX2/JNK1/2 pathway is essential for liraglutide to attenuate HG-induced ß-cell apoptosis, which further proves that GLP-1Ras may become promising therapeutics for diabetes mellitus.
Assuntos
Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/patologia , Células Secretoras de Insulina/efeitos dos fármacos , Liraglutida/farmacologia , NADPH Oxidase 2/metabolismo , Animais , Células Cultivadas , Citoproteção/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Liraglutida/uso terapêutico , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , EstreptozocinaRESUMO
AIM: To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) in the human esophageal squamous cell carcinoma (ESCC) cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogenesis, tumor progression and metastasis etc of ESCC. METHODS: Methylation-specific polymerase chain reaction (MSP) was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines. One cell line where RIZ1 promoter region methylation was detected was selected for the next study, where the cell line was treated with 5-aza-CdR. Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1. Experiments using frozen pathological specimens from 47 ESCC patients were performed using the same MSP methodology. RESULTS: Promoter methylation of RIZ1 gene was detected in TE13, CaEs17 and EC109 cell lines and the cell line TE13 was chosen for further study. The expression of RIZ1 mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR. The rate of methylation in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue, and the deviation of data was statistically significant (χ(2) = 24.136, P < 0.01). Analysis of the gender, age familial history, tumour deviation, tumour saturation, lymph gland displacement and clinical staging of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant. CONCLUSION: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human ESCC. RIZ1 is considered to be a potential tumor suppressor gene and may be a biological parameter for testing early stage human ESCC.