RESUMO
Increasing evidence demonstrates that long noncoding RNAs (lncRNAs) regulate gene and protein expression by exerting an influence on transcriptional and post-transcriptional processes. Here, we report that the lncRNA UCA1 increases the metastatic ability of gastric cancer (GC) cells by regulating GRK2 protein stability by promoting Cbl-c-mediated GRK2 ubiquitination and degradation. This process then activates the ERK-MMP9 signalling pathway. Furthermore, we demonstrate that GRK2 is downregulated in GC cells and that silencing of GRK2 might cause similar phenotypic changes and signalling pathway activation as those induced by elevated UCA1 in GC cells. Our results suggest that UCA1 might function as a mediator of protein ubiquitination and may be a promising molecular target for GC therapy.
Assuntos
Movimento Celular , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/secundário , RNA Longo não Codificante/genética , Neoplasias Gástricas/patologia , Animais , Apoptose , Proliferação de Células , Quinase 2 de Receptor Acoplado a Proteína G/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteólise , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ten-Eleven Translocation 1 (TET1) is a member of ten eleven translocation enzymes, which convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). TET1 can promote CpG islands demethylation in specific genes and often absent in various cancers. Herein, we found that TET1 expression and 5-hmC content were low in gastric tumors compared to its adjacent non-tumor tissues. Cell proliferation, migration and invasion were enhanced upon TET1 knockdown in gastric cancer cells in vitro. This phenomenon was confirmed by an animal xeongraft model. We also found that TET1 directly binds to the promoter region of PTEN and activates its transcription through demethylation of CpG islands. TET1 knockdown activated AKT and FAK pathways, which were suppressed by PTEN. The activation of AKT and FAK facilitated tumor migration, invasion and accelerated cell growth. In conclusion, we found a novel mechanism that TET1 suppresses tumor cell growth, migration and invasion through demethylation of CpG island in PTEN promoter by increasing 5-hmC content. The re-expressed PTEN subsequently down regulates AKT and FAK activity.
Assuntos
Regulação Neoplásica da Expressão Gênica , Oxigenases de Função Mista/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias Gástricas/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Ilhas de CpG/genética , Desmetilação , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Oxigenases de Função Mista/metabolismo , Metástase Neoplásica , PTEN Fosfo-Hidrolase/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transplante HeterólogoRESUMO
AIMS AND BACKGROUND: Keratinocyte growth factor (KGF) is reported to be implicated in the growth of some cancer cells. Matrix metalloproteinase 9 (MMP-9) is thought to enhance the tumor invasion and metastasis ability. This study was aimed at analyzing the relationship between KGF and MMP-9 expression and patients' clinicopathological characteristics to clarify the clinical significance of the expression of KGF and MMP-9 in gastric cancer. METHODS: Tissue samples from 161 patients with primary gastric cancer were investigated using immunohistochemistry. The relationship between KGF and/or MMP-9 expression and clinicopathological characteristics was analyzed. RESULTS: KGF expression and MMP-9 expression in gastric cancer tissue were observed in 62 cases (38.5%) and 97 cases (60.2%), respectively. MMP-9 was significantly associated with depth of invasion, lymph node metastasis and TNM stage. The prognosis of MMP-9-positive patients was significantly poorer than that of MMP-9-negative patients (p = 0.009). KGF expression was positively correlated with MMP-9 expression in gastric cancer, and the prognosis of patients with both KGF- and MMP-9-positive tumors was significantly worse than that of patients with negative tumors for either factor (p = 0.045). Expression of MMP-9 was revealed to be an independent prognostic factor (p = 0.026). CONCLUSIONS: Coexpression of KGF and MMP-9 in gastric cancer could be a useful prognostic factor, and MMP-9 might also serve as a novel target for both prognostic prediction and therapeutics.
Assuntos
Biomarcadores Tumorais/análise , Fator 7 de Crescimento de Fibroblastos/análise , Metaloproteinase 9 da Matriz/análise , Neoplasias Gástricas/química , Neoplasias Gástricas/patologia , Adulto , Idoso , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Razão de Chances , Valor Preditivo dos Testes , Prognóstico , Fatores de RiscoRESUMO
Androgen receptor (AR) plays an important role in many kinds of cancers. However, the molecular mechanisms of AR in gastric cancer (GC) are poorly characterized. Here, we investigated the role of AR in GC cell migration, invasion and metastatic potential. Our data showed that AR expression was positively correlated with lymph node metastasis and late TNM stages. These findings were accompanied by activation of AKT and upregulation of matrix metalloproteinase 9 (MMP9). AR overexpression induced increases in GC cell migration, invasion and proliferation in vitro and in vivo. These effects were attenuated by inhibition of AKT, AR and MMP9. AR overexpression upregulated MMP9 protein levels, whereas this effect was counteracted by AR siRNA. Inhibition of AKT by siRNA or an inhibitor (MK-2206 2HC) decreased AR protein expression in both stably transfected and parental SGC-7901 cells. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that AR bound to the AR-binding sites of the MMP9 promoter. In summary, AR overexpression induced by AKT phosphorylation upregulated MMP9 by binding to its promoter region to promote gastric carcinogenesis. The AKT/AR/MMP9 pathway plays an important role in GC metastasis and may be a novel therapeutic target for GC treatment.
Assuntos
Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/metabolismo , Neoplasias Gástricas/patologia , Animais , Apoptose , Sequência de Bases , Western Blotting , Adesão Celular , Feminino , Humanos , Técnicas Imunoenzimáticas , Metástase Linfática , Masculino , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Dados de Sequência Molecular , Invasividade Neoplásica , Estadiamento de Neoplasias , Fosforilação , Prognóstico , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Receptores Androgênicos/química , Receptores Androgênicos/genética , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MPS-1 (metallopanstimulin-1), also known as ribosomal protein S27, was overexpressed in gastric cancer cells. However, how MPS-1 contributes to gastric carcinogenesis has not been well characterized. Here, we show that high expression of MPS-1 was observed in gastric cancer tissues and associated with gastric cancer cell metastasis. Alteration of MPS-1 expression regulates invasion and migration of gastric cancer cells both in vitro and in vivo. Furthermore, by using Signal-Net and cluster analyses of microarray data we identified integrin ß4 (ITGB4) as a downstream target of MPS-1 that mediates its effects on cell metastasis. Knockdown of MPS-1 expression in gastric cancer cells led to significant reduction of ITGB4 expression at both the RNA and protein levels. Mechanically, we found that overexpression of ITGB4 in MPS-1 knockdown cells largely recovers the ability of invasion and migration. Conversely, knockdown of ITGB4 partially reduced cell invading/migrating ability induced by MPS-1 overexpression. Moreover, MPS-1 and ITGB4 expressions are positively correlated in gastric cancer cell lines and tissues. Finally, the survival analyses show that the expression of MPS-1 and ITGB4 is associated with poor outcomes in gastric cancer patients. Collectively, our findings suggest that MPS-1 regulates cell invasiveness and migration partially through ITGB4 and that MPS-1/ITGB4 signaling axis may serve as therapeutic targets in the treatment of gastric cancer.
Assuntos
Movimento Celular/genética , Integrina beta4/genética , Integrina beta4/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Invasividade Neoplásica/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Neoplasias Gástricas/patologiaRESUMO
Lysyl oxidase (LOX) initiates the enzymatic stage of collagen and elastin cross-linking. It also has intracellular functions involved in the regulation of cell differentiation, motility/migration and gene transcription. Aberrant expression of the LOX gene has been reported in multiple tumors. However, the correlation of its expression with clinicopathological parameters and its prognostic significance in gastric cancer remains largely unknown. In order to address this problem, total RNA of paired tissue samples (n=10) and a tissue microarray containing 161 paired tissues from patients with gastric cancers at different stages were collected. Quantitative real-time PCR and immunochemistry assay were conducted to investigate the expression of LOX. Based on the results, LOX mRNA was increased in gastric cancer tissues compared with the adjacent normal mucosa. Immunohistochemical detection revealed that expression of LOX was associated with depth of tumor invasion (P<0.05), lymph node status (P<0.05), TNM stage (P<0.05) and survival (P<0.05). Cox regression analysis revealed that positive expression of LOX (P=0.026) was an independent prognostic marker for survival in patients with gastric cancer.
RESUMO
Resistance to 5-fluorouracil (5-FU) in patients with gastric cancer is a serious therapeutic problem and major efforts are underway to understand the underlying mechanisms. We have previously identified RhoGDI2 as a contributor to 5-FU resistance in colon cancer cells using 2D electrophoresis and mass spectrometry and the current study aimed to further investigate this role. The expression of RhoGDI2 in seven gastric cancer cell lines was positively correlated with resistance to 5-FU. Lower 5-FU sensitivity of isolated tumor cells from patients with gastric cancer was also associated with higher RhoGDI2 expression. Ectopic expression of RhoGDI2 in gastric cancer cells increased the resistance to 5-FU and reverted low dose 5-FU-induced G2/M phase arrest without affecting the population of sub-G1 cells. Overall, these findings suggest that RhoGDI2 is associated with 5-FU resistance and is a potential therapeutic target for enhancing chemotherapy efficacy in gastric cancer.
RESUMO
micrornas (miRNAs) play an important role in a wide range of physiological and developmental processes by negatively regulating the expression of target genes at the post-transcriptional level. In this study, we investigated the differential miRNA expression signature between gastric cancer cells and normal gastric mucosa to determine changes in miRNA expression during gastric cancer development. We analyzed the global miRNA expression profiles of 9 gastric cancer cell lines and 6 normal gastric mucosa lines using miRNA microarrays. In addition, we performed quantitative real-time PCR (Q-PCR) to validate the results. Correlations between the miRNA expression profile and tumor clinicopathological parameters were analyzed. We found that 17 miRNAs were upregulated in gastric cancer cell lines and 146 miRNAs were downregulated compared to normal gastric mucosa. Using microarray data and Q-PCR validation, 15 miRNAs were finally selected. These candidate miRNAs were associated with gastric cancer clinicopathology to various degrees. High expression levels of hsa-miR-93 were found to predict poor survival (median, 16 vs. 40 months; log-rank test p<0.05). These findings suggest that miRNAs play vital roles in human gastric cancer. The findings may also provide clues toward understanding the molecular functions of miRNAs in various biological processes.
Assuntos
Mucosa Gástrica/metabolismo , MicroRNAs/metabolismo , Linhagem Celular , Biologia Computacional , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologiaRESUMO
The ribosomal protein S27 (metallopanstimulin-1, MPS-1) has been reported to be a multifunctional protein, with increased expression in a number of cancers. We reported previously that MPS-1 was highly expressed in human gastric cancer. Knockdown of MPS-1 led to spontaneous apoptosis and repressed proliferation of human gastric cancer cells in vitro and in vivo. However, how does MPS-1 regulate these processes is unclear. Here we performed microarray and pathway analyses to investigate possible pathways involved in MPS-1 knockdown-induced apoptosis in gastric cancer cells. Our results showed that knockdown of MPS-1 inhibited NF-κB activity by reducing phosphorylation of p65 at Ser536 and IκBα at Ser32, inhibiting NF-κB nuclear translocation, and down-regulating its DNA binding activity. Furthermore, data-mining the Gene-Regulatory-Network revealed that growth arrest DNA damage inducible gene 45ß (Gadd45ß), a direct NF-κB target gene, played a critical role in MPS-1 knockdown-induced apoptosis. Over-expression of Gadd45ß inhibited MPS-1 knockdown-induced apoptosis via inhibition of JNK phosphorylation. Taken together, these data revealed a novel pathway, the MPS-1/NF-κB/Gadd45ß signal pathway, played an important role in MPS-1 knockdown-induced apoptosis of gastric cancer cells. This study sheds new light on the role of MPS-1/NF-κB in apoptosis and the possible use of MPS-1 targeting strategy in the treatment of gastric cancer.
Assuntos
Antígenos de Diferenciação/metabolismo , Apoptose/genética , Metaloproteínas/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Neoplasias Gástricas/metabolismo , Antígenos de Diferenciação/biossíntese , Linhagem Celular Tumoral , Proliferação de Células , Etoposídeo/farmacologia , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Potencial da Membrana Mitocondrial , Metaloproteínas/genética , NF-kappa B/biossíntese , NF-kappa B/genética , Proteínas Nucleares/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Neoplasias Gástricas/genética , Fator de Transcrição RelA/metabolismoRESUMO
Salinomycin is a novel identified cancer stem cells (CSCs) killer. Higher ALDH activity represents CSCs characterization. Here, we screened ALDH activities on several gastric cancer cell lines and divided them into ALDH(high) and ALDH(low) gastric cancer groups. ALDH(high) cancer cells (NCI-N87 and SNU-1) disclosed more CSCs characteristics, such as higher levels of Sox2, Nanog and Nestin, more floating spheroid bodies, more colony formation and more resistance to conventional chemotherapeutic drugs 5-Fu and CDDP, compared to these parameters observed in ALDH(low) cancer cells (P<0.01). Importantly, ALDH(high) cancer cells are relatively sensitive to salinomycin when compared to ALDH(low) cancer cells (P<0.01). Our results confirmed ALDH as functional marker of CSCs population on gastric cancer. Salinomycin might be selective therapy for CSCs fraction, which is resistant to conventional anticancer drugs 5-Fu and CDDP.
Assuntos
Aldeído Desidrogenase/análise , Antibióticos Antineoplásicos/farmacologia , Proteínas de Neoplasias/análise , Células-Tronco Neoplásicas/efeitos dos fármacos , Piranos/farmacologia , Neoplasias Gástricas/patologia , Aldeído Desidrogenase/biossíntese , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos Alquilantes/farmacologia , Cisplatino/farmacologia , Indução Enzimática/efeitos dos fármacos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Proteínas de Filamentos Intermediários/biossíntese , Proteínas de Filamentos Intermediários/genética , Proteína Homeobox Nanog , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/enzimologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Nestina , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Retinal Desidrogenase , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Esferoides Celulares/efeitos dos fármacos , Neoplasias Gástricas/enzimologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Ensaio Tumoral de Célula-TroncoRESUMO
BACKGROUND AND AIM: Gene silence of IRX1 tumor suppressor by promoter CpG methylation combined with loss of heterozygosity (LOH) has been identified in human gastric cancer. This study investigated the association between methylation of IRX1 and Helicobacter pylori infection in gastric mucosa tissues and cell line. METHODS: IRX1 methylation was studied by methylation specific polymerase chain reaction (MSP) and bisulfate sequencing polymerase chain reaction (BSP) methods in gastric mucosa tissues from H. pylori-positive chronic gastritis patients or H. pylori-negative chronic gastritis patients. Promoter activity, methylation status and gene expressing level of IRX1 were evaluated by persistent infecting H. pylori on human gastric cells GES-1 in vitro. Electron microscopy was used to observe the effect of H. pylori infection on GES-1 gastric mucosa cells. RESULTS: The methylation level of IRX1 promoter in H. pylori positive chronic gastritis and H. pylori negative chronic gastritis was 55.30%±13.17 versus 5.20%±6.31, respectively (P<0.01). H. pylori infection stimulated increased microvillus, and mucous secretion on GES-1 cells. Infection of H. pylori induced IRX1 promoter methylation and downregulation of the promoter activity as well as gene expression significantly. CONCLUSIONS: This study firstly demonstrated that H. pylori infection contributes to IRX1 promoter methylation on gastric mucosa.
Assuntos
Metilação de DNA , Mucosa Gástrica/microbiologia , Gastrite/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Estudos de Casos e Controles , Linhagem Celular , Doença Crônica , Regulação para Baixo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/ultraestrutura , Gastrite/genética , Gastrite/metabolismo , Genes Reporter , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/patologia , Proteínas de Homeodomínio/metabolismo , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
BACKGROUND/AIMS: To investigate the cell cycle dependent genes involved in gastric tumorigenesis, possibly determining the relationship between the cell cycle and tumorigenesis. METHODOLOGY: MKN45 cells were collected every hour from Oh to 12h after release from G2/M and G1/S blocks. Nine samples (a-i), chosen at key times of the cell cycle, were prepared for RNA isolation and cDNA microarray analysis. RESULTS: In 2001 viable clones, 959 genes showed periodic variations during the cell cycle. Among 2001 genes that were clustered, a series of up-regulated genes were assigned to different cell cycle phases. Many periodically dependent genes in the cell cycle were ubiquitously expressed and participated in various cell physiological functions, such as transcription, translation, ubiquitination and signal transduction. These cell cycle dependent genes could affect cancer cell proliferation, apoptosis, activation of oncogenes and inactivation of tumor suppressor genes. CONCLUSIONS: We provided a comprehensive understanding of the gene expression profile involved in gastric cancer cell cycles and laid a foundation for further research on mechanisms of gastric tumorigenesis.
Assuntos
Ciclo Celular/genética , Perfilação da Expressão Gênica , Neoplasias Gástricas/etiologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Genes Supressores de Tumor , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Oncogenes , Biossíntese de Proteínas , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Transcrição Gênica , UbiquitinaçãoRESUMO
BACKGROUND: Genetic modification of dendritic cells (DCs) has been used as an effective approach to enhance anti-tumor immunity. RNA interference (RNAi), which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA (siRNA) specific for the Ii gene was transfected into DCs, and the anti-tumor immunity of Ii-silenced DCs was assessed. METHODS: The silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96(®) non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used. RESULTS: The Ii expression of DCs was significantly reduced after Ii siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with Ii siRNA plus endogenous tumor antigen (P < 0.05). Furthermore, tumor growth was greatly inhibited when mice were immunized with DCs transfected with Ii siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4(+) and CD8(+) T cells were significantly activated in the Ii siRNA group (P < 0.05). CONCLUSION: Silencing of the Ii gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Inativação Gênica/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Neoplasias/imunologia , RNA Interferente Pequeno/fisiologia , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Western Blotting , Células Cultivadas , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/genética , Camundongos , Interferência de RNA/fisiologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIM: To prepare PHF10 antibody and check the expression of PHF10 protein in the tissues of gastric cancer and adjacent tissue. METHODS: His-tagged PHF10 was expressed in E.coli BL21. Rabbit PHF10 polyclonal antiserum was generated by injecting the purified recombinant His-tagged PHF10 inclusion body as the antigen, and further separated by affinity purification. To confirm the specificity of the PHF10 antibody, transiently expressed Flag-PHF10 fusion protein was analyzed by immunoblotting with anti-flag monoclonal antibody control. The produced antibody Was used to check the expression of PHF10 protein in gastric cancer and adjacent tissues by Western blot. RESULTS: Antibodies specifically binding to PHF10 could be obtained by immunization, and expression of PHF10 was significantly higher in gastric cancerous tissues comparing with adjacent normal tissues and GES-1 shows more PHF10 expression than gastric cancer cell lines with the generated antibody. CONCLUSION: The specific anti-PHF10 antibody is obtained and it could be used to detect the expression of PHF10 protein in gastric cancer cell lines and tissues, in which PHF10 is unregulated in gastric cancer.
Assuntos
Anticorpos , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Anticorpos/imunologia , Western Blotting , Linhagem Celular Tumoral/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Soros Imunes/análise , Soros Imunes/imunologia , Imunização , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/imunologia , Neoplasias Gástricas/patologiaRESUMO
AIM: To isolate and identify differentially expressed proteins between cancer and normal tissues of gastric cancer by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). METHODS: Soluble fraction proteins of gastric cancer tissues and paired normal tissues were separated by 2-DE. The differentially expressed proteins were selected and identified by MALDI-TOF-MS and database search. RESULTS: 2-DE profiles with high resolution and reproducibility were obtained. Twenty-three protein spots were excised from sliver staining gel and digested in gel by trypsin, in which fifteen protein spots were identified successfully. Among the identified proteins, there were ten over-expressed and five under-expressed proteins in stomach cancer tissues compared with normal tissues. CONCLUSION: In this study, the well-resolved, reproducible 2-DE patterns of human gastric cancer tissue and paired normal tissue were established and optimized and certain differentially-expressed proteins were identified. The combined use of 2-DE and MS provides an effective approach to screen for potential tumor markers.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Neoplasias/análise , Proteômica , Neoplasias Gástricas/química , Adulto , Idoso , Sequência de Aminoácidos , Diferenciação Celular , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estadiamento de Neoplasias , Proteômica/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Neoplasias Gástricas/patologia , Espectrometria de Massas em TandemRESUMO
PURPOSE: Frizzled motif associated with bone development (FRZB) was a member of secreted frizzled related proteins (sFRPs) family. Previous evidences showed that FRZB played role in embryogenesis and diseases such as osteoarthritis and prostate cancer. The purpose of our study is to clarify the role of FRZB in gastric cancer cell proliferation and differentiation. METHODS: The expression of FRZB in gastric cancer tissues were detected by immunohistochemistry. The expression of FRZB in eight gastric cancer cell lines and one immortal gastric epithelial cell GES-1 were detected by western blotting and real-time quantitative PCR. To investigate the role of over-expressed FRZB in gastric cancer cells, FRZB/pcDNA3.1 plasmid was constructed and transfected into gastric cancer cell line SGC7901. The changes of biological features in these stable transfectants were examined. RESULTS: FRZB was highly expressed in gastric cancer (90%), intestinal metaplasia (100%) and gastric dysplasia (90%), but no or just weakly (3/40) expressed in normal gastric mucosa. FRZB staining was stronger in intestinal-type gastric cancer tissues than that in diffuse-type ones and was positive correlated with differentiation grade. The expression of FRZB in eight gastric cancer cell lines was higher than in GES-1. Over-expressed FRZB inhibited cell proliferation in vitro and in vivo which was first caused by prolonged cell division progression in G2/M phase, and second by higher sensitivity to apoptotic inducing factors and spontaneous apoptosis. Our findings gave evidences that FRZB suppressed gastric cancer cell proliferation and modulated the balance between proliferation and differentiation in gastric cancer.
Assuntos
Glicoproteínas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Apoptose , Diferenciação Celular , Divisão Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Fase G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , TransfecçãoRESUMO
AIM: To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein (HSP) as a strategy to enhance DNA vaccine potency. METHODS: A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was performed in BALB/c mice to monitor anti-tumor immune responses. RESULTS: In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg (44% +/- 5% vs 30% +/- 6% in E:T > 50:1, P < 0.05). ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe-HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC class I or class II epitopes derived from HBeAg (74% +/- 9% vs 31% +/- 6%, P < 0.01). ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV-HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV-HBe-HSP when challenged with CT26-HBeAg. CONCLUSION: The results of this study demonstrate a broad enhancement of antigen-specific CD4+ helper, CD8+ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.
Assuntos
Células Dendríticas/imunologia , Proteínas de Choque Térmico/uso terapêutico , Antígenos E da Hepatite B/uso terapêutico , Vírus da Hepatite B/imunologia , Hepatite B/prevenção & controle , Vacinas de DNA/uso terapêutico , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico/genética , Hepatite B/complicações , Hepatite B/imunologia , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinas de DNA/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA interference (RNAi), which causes the degradation of any RNA in a sequence specific manner, is a posttranscriptional gene silencing mechanism. Targeting the invariant chain (Ii) in DCs has been used as an approach to enhance antitumor immunity. It is demonstrated in this article that transfection of H-2(K) DCs with siRNA specific for Ii gene can significantly knock down Ii. When exposed to TNF-alpha, immature DCs transfected with Ii siRNA can differentiate into mature DCs without reducing viability or IL-12p70 production. Ii siRNA-treated H-2(K) DCs exhibited an increased allostimulatory capacity in a lymphocyte proliferation assay. Furthermore, Ii siRNA-transfected H-2(K) DCs enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production, and much stronger cytotoxic activity was observed when DCs were co-transfected with Ii siRNA and an endogenous tumor antigen in vitro. Our findings indicate that silencing the Ii gene in DCs with siRNA may offer a potential approach to enhancing antitumor immunotherapy.
Assuntos
Antígenos de Diferenciação de Linfócitos B/genética , Células Dendríticas/imunologia , Inativação Gênica , Antígenos de Histocompatibilidade Classe II/genética , RNA Interferente Pequeno/farmacologia , Células Th1/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Sobrevivência Celular , Feminino , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/metabolismo , Interleucina-12/metabolismo , Ativação Linfocitária , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos , Transplante de Neoplasias , Linfócitos T Citotóxicos/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND: Bcl-2, the anti-apoptotic protein is overexpressed in the majority of gastric cancers and associated with its pathogenesis. To better understanding of the role of Bcl-2, RNA interference (RNAi) was used to inhibit Bcl-2 expression in the human gastric cancer cells in vitro and in vivo. METHODS: Bcl-2 small interfering RNA (siRNA) was transfected into human gastric cancer cells SGC-7901, and Bcl-2 expression was monitored by real-time polymerase chain reaction (PCR) and Western blot. Cell proliferation, apoptosis, and telomerase activity were examined by MTT, flow cytometry, and TRAP assay, respectively. Gastric cancer cells treated with 100 nmol/L Bcl-2 siRNA were subcutaneously transplanted into nude mice and tumor growth was assessed. RESULTS: Bcl-2 siRNA significantly inhibited the expression of Bcl-2 in human gastric cancer cells at both mRNA and protein levels in a time- and dose-dependent manner. Bcl-2 siRNA also decreased telomerase activity (by 78.76%) and increased the rate of apoptosis (by 37.47%). SGC-7901 cell growth was also significantly suppressed in vivo and in vitro. CONCLUSIONS: Bcl-2 expression knockdown suppressed the growth of gastric cancer cells. Thus, Bcl-2 may play a very important role in carcinogenesis of gastric cancer and its knockdown may offer a new potential gene therapy approach for human gastric cancer in future.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , RNA Interferente Pequeno/genética , Neoplasias Gástricas/terapia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , Neoplasias Gástricas/patologia , TransfecçãoRESUMO
OBJECTIVE: To investigate the expression of Ezrin in gastric cancer, its role in tumor metastasis. METHODS: Ezrin expression in tumor tissues from 90 gastric cancer cases and in normal gastric mucosa from 12 cases with benign disease was examined by immunohistochemical staining. Ezrin expression in gastric cancer cell lines was also detected by Western blot, and in vitro invasion assay was used to examine the invasive ability of the cell lines. RESULTS: The expression rate of Ezrin was significantly higher in gastric cancer tissues than that in normal tissues (P< 0.05), and significantly correlated with lymph node metastasis (P< 0.05). Western blot showed that MKN-45 cell line had the highest expression of Ezrin among 5 gastric cancer cells. MKN-45 possessed highest invasion ability. CONCLUSION: Ezrin expression is up-regulated, and may be associated with lymph node metastasis in gastric cancer.
