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1.
Oncol Lett ; 9(1): 341-346, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25435989

RESUMO

The present study examined the role of the PRDI-BF1-RIZ (PR) domain of tumor suppressor retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) as an anticancer domain and its ability to induce apoptosis in esophageal squamous cell carcinoma (ESCC) cells. The TE13 ESCC cell line was transfected with pcDNA3.1(+) eukaryotic expression vectors bearing the open reading frames of either the human RIZ1 gene or the PR domain, and the mRNA and protein expression levels were then detected using quantitative reverse transcription polymerase chain reaction and western blotting, respectively. The rate of apoptosis was determined by flow cytometry and the cell invasion ability was determined by an invasion assay. RIZ1 and the PR domain induced apoptosis and reduced the cell invasion ability (P<0.01). These findings indicate that the RIZ1 gene possesses anticancer activity in the PR domain, which may be important in inhibiting the development of ESCC.

2.
Oncol Rep ; 32(3): 1064-70, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24993551

RESUMO

The present study aimed to investigate the expression and role of SET and MYND domain-containing protein 3 (SMYD3) in esophageal squamous cell carcinoma; to observe the proliferation of esophageal squamous cell carcinoma after suppression of SMYD3 expression; and to explore the effect of SMYD3 downregulation on the expression of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1). Tissues from 11 patients, including cancer and normal esophageal tissues, were obtained by surgery to observe the SMYD3 protein expression immunohistochemistry. Esophageal squamous cell carcinoma TE13 cells were transfected with four different SMYD3-shRNA plasmids, and SMYD3 mRNA expression levels were assessed to select the most efficient interfering plasmid. After SMYD3 downregulation in TE13 cells, mRNA and protein expression levels of SMYD3 and RIZ1 were determined using RT-PCR and western blotting, and cell proliferation was evaluated by the MTT method. In all 11 tissue paired samples, SMYD3 protein expression was higher in the cancer tissues (72.7%; 8/11), than that in the normal tissues (18.2%; 2/11) (Fisher's exact test, P=0.03). The mRNA expression levels of SMYD3 were significantly decreased by RNA interference (P<0.05), and plasmid SMYD3-shRNA-1242 was determined to be the most effective. Compared with the controls, transfection with the SMYD3-shRNA interfering plasmid significantly reduced the SMYD3 mRNA and protein expression levels in TE13 cells (P<0.05), whereas the expression levels of the anti-oncogene RIZ1 were increased (P<0.05). The MTT assay showed that ablation of SMYD3 expression significantly inhibited proliferation of TE13 cells (P<0.05). SMYD3 may participate in the biological activity of esophageal squamous cell carcinoma, as overexpression of SMYD3 correlates with its occurrence and its downregulation inhibits cancer cell proliferation. The shRNA efficiently downregulated SMYD3 in TE13 cells, which represents an SMYD3-interfered cell-test-model for future experiments. RNAi suppression of SMYD3 promoted the expression of RIZ1 in TE13 cells, suggesting a signal transduction pathway between SMYD3 and RIZ1. The SMYD3-RIZ1 pathway may represent a therapeutic target for esophageal squamous cell carcinoma.


Assuntos
Carcinoma de Células Escamosas/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias Esofágicas/patologia , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Humanos , Interferência de RNA , Transdução de Sinais
3.
World J Gastroenterol ; 19(37): 6170-7, 2013 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-24115813

RESUMO

AIM: To investigate the effect of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) upregulation in gene expression profile and oncogenicity of human esophageal squamous cell carcinoma (ESCC) cell line TE13. METHODS: TE13 cells were transfected with pcDNA3.1(+)/RIZ1 and pcDNA3.1(+). Changes in gene expression profile were screened and the microarray results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Nude mice were inoculated with TE13 cells to establish ESCC xenografts. After two weeks, the inoculated mice were randomly divided into three groups. Tumors were injected with normal saline, transfection reagent pcDNA3.1(+) and transfection reagent pcDNA3.1(+)/RIZ1, respectively. Tumor development was quantified, and changes in gene expression of RIZ1 transfected tumors were detected by RT-PCR and Western blotting. RESULTS: DNA microarray data showed that RIZ1 transfection induced widespread changes in gene expression profile of cell line TE13, with 960 genes upregulated and 1163 downregulated. Treatment of tumor xenografts with RIZ1 recombinant plasmid significantly inhibited tumor growth, decreased tumor size, and increased expression of RIZ1 mRNA compared to control groups. The changes in gene expression profile were also observed in vivo after RIZ1 transfection. Most of the differentially expressed genes were associated with cell development, supervision of viral replication, lymphocyte costimulatory and immune system development in esophageal cells. RIZ1 gene may be involved in multiple cancer pathways, such as cytokine receptor interaction and transforming growth factor beta signaling. CONCLUSION: The development and progression of esophageal cancer are related to the inactivation of RIZ1. Virus infection may also be an important factor.


Assuntos
Carcinoma de Células Escamosas/genética , Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/genética , Perfilação da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares/genética , Oncogenes , Fatores de Transcrição/genética , Animais , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transfecção , Carga Tumoral , Regulação para Cima
4.
Clin Lab ; 59(5-6): 647-53, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23865365

RESUMO

BACKGROUND: To study the expression of Syk (spleen tyrosine kinase) gene in human ESCC (esophageal squamous cell carcinoma). METHODS: We used immunohistochemical staining to detect Syk protein expression in esophageal carcinoma tissues and RT-PCR (semi-quantitative reverse transcription PCR), Real-time PCR (Real-time quantitative PCR) and western blotting technique to detect the expression of Syk mRNA and protein in specimens from 4 esophageal cell lines. RESULTS: Immunohistochemistry analyses showed that in the esophageal cancer tissues Syk protein was expressed inferior to the adjacent normal one, p < 0.05. Real-time PCR and Western blotting showed that low expression of the Syk gene was detected in the 4 esophageal cancer cell lines compared with positive and negative cell lines, p < 0.05. CONCLUSIONS: The low expression of the Syk gene may play an important role in tumorigenesis in esophageal cancer.


Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma de Células Escamosas/enzimologia , Neoplasias Esofágicas/enzimologia , Genes Supressores de Tumor , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas Tirosina Quinases/biossíntese , Biomarcadores Tumorais/genética , Western Blotting , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Quinase Syk
5.
Clin Lab ; 58(1-2): 41-51, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22372344

RESUMO

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/metabolismo
6.
World J Gastroenterol ; 18(6): 576-82, 2012 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-22363126

RESUMO

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.


Assuntos
Carcinoma de Células Escamosas/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Sequência de Bases , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Epigênese Genética , Neoplasias Esofágicas/patologia , Feminino , Inativação Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência de DNA
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