RESUMO
Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) the etiology of which has not yet been fully clarified. Cytokine interleukin-10 (IL-10) plays a central role in downregulating inflammatory cascade in UC and is likely a candidate for therapeutic intervention. However, its intravenous administration is costly and inconvenient. Therefore, we established a novel IL-10 delivery system by transforming a hIL-10-containing plasmid into B. longum (BL-hIL-10) and investigated its effects on 5% dextran sulfate sodium (DSS)-induced ulcerative colitis in mice and the possible underlying mechanism. Our results show that (1) hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after L-arabinose induction in vitro as examined by Western blot, enzyme-linked immunosorbent assay (ELISA) and RT-PCR; (2) addition of BL-hIL-10 culture supernatant had no cytotoxic effect and morphological alteration, but significantly inhibited the enhancement of proinflammatory cytokines by lipopolysaccharide (LPS) in THP-1 cells; (3) oral administration of BL-hIL-10 alleviated colitis syndrome of the model mice, attenuated colitis-activated NF-κB pathway measured by DNA-binding assay and colitis-elevated expression of proinflammatory cytokines examined with CCK cytotoxic kits, and upregulated CD4+CD25+Foxp3+ Treg in blood and mesenteric lymph nodes measured by flow cytometry. In conclusion, BL-hIL-10 as a novel oral hIL-10 delivery system has been successfully established and oral administration of BL-hIL-10 alleviated inflammatory damage of colonic tissue in the model mice by blocking the colitis-activated NF-κB pathway and upregulating CD4+CD25+Foxp3+ Treg in blood and mesenteric lymph nodes in mice.
Assuntos
Bifidobacterium/metabolismo , Colite/tratamento farmacológico , Sulfato de Dextrana/toxicidade , Portadores de Fármacos , Interleucina-10/administração & dosagem , Interleucina-10/metabolismo , Administração Oral , Animais , Bifidobacterium/genética , Western Blotting , Colite/induzido quimicamente , Colite/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Peroxidase/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
Nuclear factor- κB (NF- κB) plays a pivotal role in the regulation of immune and inflammatory responses. The real-time expression level of NF- κB reflects the development of ulcerative colitis (UC). Polydatin has vast pharmacological activities, including inhibiting the production of inflammatory mediators, inducing the production of antioxidants, regulating immune function, etc. The purpose of this study was to investigate the potential inhibitory effects of polydatin on NF- κB pathway activation in a mouse UC model. The results showed that polydatin treatment downregulated NF- κB p65 activity and expression, blocked the expression of TNF- α, IL-6 and IL-1 ß at both mRNA and protein levels, decreased myeloperoxidase (MPO) activity, and alleviated inflammatory damage of colitis in mice with UC (p < 0.05), suggesting that the anti-inflammation effects of polydatin can be attributed, at least partially, to the blocking of the NF- κB pathway.
Assuntos
Anti-Inflamatórios/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Glucosídeos/uso terapêutico , NF-kappa B/antagonistas & inibidores , Estilbenos/uso terapêutico , Animais , Anti-Inflamatórios/química , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Glucosídeos/química , Mediadores da Inflamação/metabolismo , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/genética , NF-kappa B/metabolismo , Peroxidase/antagonistas & inibidores , Peroxidase/metabolismo , Projetos Piloto , RNA Mensageiro/metabolismo , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Estilbenos/química , Estilbenos/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE: Plenty of studies showed that the immune system was associated with cancer initiation and progression. This study aimed to explore the prognostic biomarkers from immune-related genes (IRGs) in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) and IRGs and transcription factors (TFs) were extracted. Then, the co-expression network between IRGs and TFs was constructed using the "WGCNA" package in R software. Furthermore, a gene expression signature according to IRGs was constructed to predict OSCC prognosis and its accuracy was validated by survival analysis. Subsequently, correlation analyses between risk-score and immune cells level and clinical parameters were performed. Finally, immune-related biomarkers were selected and further investigated using gain-of-function assays in vitro. RESULTS: A total of 32 normal cases and 317 OSCC cases were selected in our study. Differentially-expressed analysis indicated that there were 381 differentially-expressed IRGs and 62 TFs in OSCC. Among them, 25 TFs and 21 IRGs were enrolled in the co-expression network. Furthermore, we found that gene expression signature on the basis of 10 IRGs could predict the prognosis accurately and a high-risk score based on gene expression signature meant a high T classification, terminal clinical stage, and low immune cells level in OSCC. Finally, cathepsin G (CTSG) was identified as a potential immune-related biomarker and therapeutic target in OSCC. CONCLUSION: In conclusion, IRGs were directly involved in the development and progression of OSCC. Furthermore, CTSG was identified as a potential independent biomarker and might be an immunotherapeutic target in OSCC treatment.
RESUMO
Increasing evidence suggests that N6-methyladenosine(m6A) has a vital role in cancer progression. Therefore, we aimed to explore the prognostic relevance of m6A-related genes in oral squamous cell carcinoma (OSCC). First, Expression profiles were downloaded from The Cancer Genome Atlas (TCGA) and m6A-related genes were extracted afterwards. Then, cluster analysis and principal component analysis (PCA) were used to analyze m6A-related genes. And differentially-expressed analysis was performed in R software. Furthermore, a risk model was constructed, and crucial m6A genes were selected to explore its biological effects in OSCC cells. Total of 13 m6A-related genes were extracted and 8 differentially-expressed genes were identified. Subsequently, m6A-based clustering showed 2 subtypes with different clinical outcome. In addition, a risk model was successfully established. Of 13 m6A-related genes, only heterogeneous nuclear ribonucleoprotein C (HNRNPC) might be an independent biomarker and mean unfavorable overall survival in OSCC by univariate and multivariate cox regression analysis. Functional studies revealed that overexpression of HNRNPC promoted carcinogenesis of OSCC via epithelial- mesenchymal transition (EMT). In total, a risk model of m6A-related genes in OSCC was established. Subsequently, HNRNPC was proved to promote OSCC carcinogenesis and be an independent biomarker prognostic biomarker of OSCC, suggesting that it might be a new biomarker and therapeutic target of OSCC.
Assuntos
Adenosina/análogos & derivados , Biomarcadores Tumorais/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Neoplasias Bucais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Análise por Conglomerados , Biologia Computacional , Conjuntos de Dados como Assunto , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Masculino , Metilação , Pessoa de Meia-Idade , Neoplasias Bucais/mortalidade , Neoplasias Bucais/patologia , Análise de Componente Principal , Prognóstico , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Medição de Risco , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Oral squamous cell carcinoma (OSCC) is the most common oral cancer. The molecular mechanisms of this disease are not fully understood. Our previous studies confirmed that dysregulated function of long non-coding RNA (lncRNA) AC007271.3 was associated with a poor prognosis and overexpression of AC007271.3 promoted cell proliferation, migration, invasion, and inhibited cell apoptosis in vitro, and promoted tumor growth in vivo. However, the underlying mechanisms of AC007271.3 dysregulation remained obscure. In this study, our investigation showed that AC007271.3 functioned as competing endogenous RNA by binding to miR-125b-2-3p and by destabilizing primary miR-125b-2, resulted in the upregulating expression of Slug, which is a direct target of miR-125b-2-3p. Slug also inhibited the expression of E-cadherin but N-cadherin, vimentin, and ß-catenin had no obvious change. The expression of AC007271.3 was promoted by the canonical nuclear factor-κB (NF-κB) pathway. Taken together, these results suggested that the classical NF-κB pathway-activated AC007271.3 regulates EMT by miR-125b-2-3p/Slug/E-cadherin axis to promote the development of OSCC, implicating it as a novel potential target for therapeutic intervention in this disease.
Assuntos
Carcinogênese/genética , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Bucais/genética , NF-kappa B/metabolismo , RNA Longo não Codificante/metabolismo , Fatores de Transcrição da Família Snail/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Inativação Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Estabilidade de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Regulação para Cima/genéticaRESUMO
PURPOSE: Fibroblast activation protein (FAP) acts as a tumor promoter via epithelial-mesenchymal transition (EMT) in human oral squamous cell carcinoma (OSCC). The present study was designed to investigate the FAP targeting proteins and explore the precise mechanism by which FAP promotes EMT in OSCC. PATIENTS AND METHODS: Proteins interacting with FAP were found and filtered by immunoprecipitation-mass spectrometry (IP-MS). Both DPP9 protein and mRNA were examined in 90 paired OSCC samples and matched normal tissue. DPP9 knockdown was conducted to determine its function in OSCC in vitro and in vivo. RESULTS: Dipeptidyl peptidase 9 (DPP9) was identified as interacting with FAP intracellularly by IP-MS. The levels of both DPP9 protein and mRNA were down-regulated in OSCC tissue. Lower DPP9 expression was correlated with unfavorable survival rates of OSCC patients. DPP9 knockdown accelerates the proliferation of OSCC cells in vitro and in vivo. Overexpression of FAP leads to a reduction in DPP9 expression. Likewise, DPP9 overexpression reverses the proliferation, migration, invasion and EMT induced by FAP during OSCC. CONCLUSION: Our study finds that FAP promotes EMT of OSCC by down-regulating DPP9 in a non-enzymatic manner. FAP-DPP9 pathway could be a potential therapeutic target of OSCC.
RESUMO
AIMS: Long noncoding RNA (lncRNA) AC007271.3 has been identified to be dysregulated in oral squamous cell carcinoma (OSCC) in our previous study. However, the precise role of AC007271.3 in OSCC remains unclear. In this study, we investigated the potential functions and the underlying mechanisms of AC007271.3 in OSCC. MATERIALS AND METHODS: The expression levels of AC007271.3 in OSCC tissues and cell lines were examined using RT-qPCR. The relationship between AC007271.3 level and clinicopathological characteristics was analyzed, and its association with patient prognosis was assessed by the Kaplan-Meier method. The biological function of AC007271.3 and its role in the development of OSCC through Wnt/ß-catenin signaling pathway were studied. KEY FINDINGS: We identified that AC007271.3 was up-regulated and positively correlated with advanced clinical stage, lymph node metastasis, poor histological differentiation and unfavorable prognosis. We explored the expression, function, and molecular mechanism of AC007271.3 in OSCC cells. Overexpression of AC007271.3 remarkably promoted cell proliferation in vitro and in vivo, induced cell migration, invasion and inhibited apoptosis in vitro, while knockdown of AC007271.3 attenuated cell proliferation, migration, invasion and induced apoptosis. Mechanistically, AC007271.3 overexpression substantially increased the expression of ß-catenin and the downstream target molecules CyclinD1, c-myc and Bcl-2, while silencing of AC007271.3 has the opposite effect. Rescued experiments showed that the ability to promote cell proliferation, migration, invasion and inhibiting apoptosis could be reversed when treated with the Wnt/ß-catenin pathway inhibitor. SIGNIFICANCE: Our data indicated that AC007271.3 could promote cell proliferation, invasion and inhibit cell apoptosis of OSCC via the Wnt/ß-catenin signaling pathway, which might provide a novel therapeutic approach for OSCC.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , RNA Longo não Codificante/genética , Idoso , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , China , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Invasividade Neoplásica/genética , Prognóstico , Transdução de Sinais/genética , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: In most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells. METHODS: Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed. RESULTS: Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined. Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells. CONCLUSIONS: PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.
Assuntos
Neoplasias Colorretais/terapia , Lentivirus/genética , Fosfolipase C gama/antagonistas & inibidores , RNA Interferente Pequeno/uso terapêutico , Apoptose/efeitos dos fármacos , Adesão Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Fluoruracila/farmacologia , Humanos , Laminina/antagonistas & inibidores , Laminina/genética , Fosfolipase C gama/genética , Fosfolipase C gama/fisiologiaRESUMO
OBJECTIVE: To investigate the effects of inhibiting phospholipase C gamma1 signaling pathway on the apoptosis of human colorectal carcinoma cells. METHODS: SW620 cells were treated with U73122 in vitro to inhibit the phospholipase C gamma1 signalling pathway and examined under light microscope and transmission electron microscope for analyzing changes in apoptotic behavior of the cells. MTT assay was used to evaluate the cell killing effects, and the percentage of apoptotic cells analyzed using flow cytometry. RESULTS: After inhibition of the phospholipase C gamma1 signaling pathway by U73122, SW620 cells exhibited obvious apoptotic morphology, the viable cells decreased dramatically, and the percentage of apoptotic cells rose to above 50%. CONCLUSION: Inhibition of phospholipase C gamma1 signaling pathway can induce apoptosis of human colorectal carcinoma cells.
Assuntos
Apoptose/fisiologia , Neoplasias Colorretais/patologia , Estrenos/farmacologia , Fosfolipase C gama/antagonistas & inibidores , Pirrolidinonas/farmacologia , Transdução de Sinais , Humanos , Inibidores de Fosfodiesterase/farmacologia , Células Tumorais CultivadasRESUMO
OBJECTIVE: To detect the expression of human bone morphogenetic protein 7 (hBMP-7) mRNA in rabbit bone marrow-derived mesenchymal stem cells with hBMP-7 gene transfection mediated by retroviral vector. METHODS: Retroviral vector for hBMP-7 gene was constructed that was transferred into the packaging cell PT67 mediated by liposome. hBMP-7 gene-positive cell clones were selected with G418 (600 ng/ml) and amplified to obtain the retroviral supernatant containing the target genes that were subsequently used to transfect rabbit bone marrow-derived mesenchymal stem cells (MSCs). HBMP-7 mRNA expression in the MSCs was examined by way of in situ hybridization and reverse transcriptase-polymerase chain reaction. RESULTS: hBMP-7 retroviral vector was successfully constructed and transferred into PT67 cells with resistance to G418, and after transfection with the recombinant retrovirus, transcription and expression of hBMP-7 mRNA were detected in MSCs. CONCLUSION: Rabbit bone marrow stem cells transfected with hBMP gene via retroviral vector can secrete the correspondent protein, indicating the possibility of enhancing the osteogenic capacity of MSCs in the study of bone tissue engineering.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , RNA Mensageiro/metabolismo , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta , Células 3T3 , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 7 , Expressão Gênica , Vetores Genéticos/genética , Humanos , Hibridização In Situ , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , RNA Mensageiro/genética , Coelhos , Retroviridae/genética , Células-Tronco/citologia , TransfecçãoRESUMO
OBJECTIVE: To construct the eukaryotic expression vector of human full-length PLCgamma1 gene for further study of the role of PLCgamma1 in cancer invasion. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) technique was used to amplify human full-length PLCgamma1 gene from MG63 cells with a pair of specific primers containing the restriction sites for HindIII and NotI. After purification, the product of RT-PCR was digested with HindIII and NotI before insertion into the corresponding sites of eukaryotic expression vector pLNCX2, yielding the recombinant plasmid pLNCX2/PLCgamma1. PCR, restriction endonuclease analysis and DNA sequencing were performed to identify the recombinant eukaryotic expression vector pLNCX2/PLCgamma1. RT-PCR and Western blotting were used to detect the expression of the PLCgamma1 gene in LoVo cells after transient transfection via Lipofectamine TM 2000. RESULTS: A 3 878-bp full-length PLCgamma1 gene fragment was successfully amplified by RT-PCR and inserted into eukaryotic expression vector pLNCX2. After digestion by HindIII and NotI, the recombinant eukaryotic expression vector pLNCX2/PLCgamma1 yielded a 3 878-bp fragment (PLCgamma1 gene) and a 6 100 bp fragment (vector). HindIII-BglII digestion was also done to verify the correctness of the recombinant plasmid, resulting in the identification of the fragments as expected. Sequencing analysis further confirmed the results. In addition, RT-PCR and Western blotting verified that the PLCgamma1 could overexpress in LoVo cells after transfection with recombinant eukaryotic expression vector pLNCX2/PLCgamma1. CONCLUSION: The recombinant eukaryotic expression vector pLNCX2/PLCgamma1 has been constructed successfully.
Assuntos
Células Eucarióticas/metabolismo , Vetores Genéticos , Fosfolipase C gama/biossíntese , Transfecção , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Humanos , Dados de Sequência Molecular , Osteoblastos/citologia , Fosfolipase C gama/genética , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To study the relationship between gene p53 codon 72 polymorphism and pathological scar formation occurrence after caesarean section. METHODS: The method of molecular beacon with real-time PCR was applied to detect gene polymorphism of p53 codon 72 in blood samples taken from 303 pregnant women (within a week after caesarea section). The clinical visits were taken 3 times for 12th to 18th months to ascertain clinical formation of pathological scar and its relationship to genotype of p53. The chi-square method was used to analyze the relationship of p53 gene polymorphism and abnormal scar formation occurrence by statistical software SPSS 13.0. RESULTS: Total of 303 pregnant women were assayed. 30 patients were found with pathological scar by clinical visit in the total 303 pregnant women. The genotype frequencies of total three types (C/C, C/G and G/G) of p53 gene codon 72 in patients with pathological scar are significantly different from that of normal pregnant woman. The frequency of C/C genotype in patients are higher than that of normal pregnant women (P < 0.01). The frequency of C/C genotype in these patients with pathological scar is higher (46.7%, 14/30) than C/G (33.0%, 10/30, P < 0.01) or G/G (20%, 6/30) genotype (P < 0.01). The C allele frequency in the patients is 63.7%. It is also higher than G allele (36.7%, P < 0.01). The OR value is 2.30. Therefore the C allele of p53 gene codon 72 is a risk factor for pathological scar. CONCLUSIONS: There was a certain relationship between p53 gene codon 72 C allele and pathological scar formation after caesarean section.
Assuntos
Cesárea , Cicatriz/genética , Códon , Genes p53 , Polimorfismo Genético , Alelos , Feminino , Frequência do Gene , Genótipo , Humanos , Gravidez , Fatores de RiscoRESUMO
OBJECTIVE: To establish a method based on molecular beacon real-time PCR for detecting single nucleotide polymorphisms (SNP) in codon 72 of scar-related p53 gene. METHODS: Two fluorescence-labeled molecular beacon probes were synthesized targeting CCC/CGC SNP of p53 codon 72. The genomic DNA was extracted from the peripheral blood of 28 patients with keloid, and the CCC/CGC SNP of P53 gene codon 72 were assayed with molecular beacon real-time PCR. The results of SNP typing were compared with the results of reverse dot hybridization and confirmed by direct DNA sequencing. RESULTS: The goodness of fit of this method was 100% in comparison with direct DNA sequencing, higher than that of reverse dot hybridization. CONCLUSION: Molecular beacon real-time PCR is suitable for rapid clinical detection of SNPs in p53 gene.
Assuntos
Queloide/genética , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteína Supressora de Tumor p53/genética , Sequência de Bases , Códon/genética , Humanos , Dados de Sequência MolecularRESUMO
OBJECTIVE: To investigate the relationship between cell apoptosis and the quality of early mouse embryos, understand the significance of apoptosis-regulatory genes in early embryonic development, and explore a new approach to improving the embryo quality. METHODS: The levels of cell apoptosis and proliferation in early mouse embryos in different developmental status (morphologically normal embryos, arrested embryos and fragmented embryos) were analyzed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), caspase in situ fluorescence and Bcl-2 immunofluorescence, and immunofluorescent detection of proliferating cell nuclear antigen (PCNA). RESULTS: The cells in arrested embryos and embryonic fragments showed positive results in TUNEL assay with enhanced caspase activity and lowered expressions of Bcl-2 and PCNA. CONCLUSION: Cell apoptosis in early mouse embryos may be closely related to embryonic arrest and fragmentation.
Assuntos
Apoptose , Embrião de Mamíferos/citologia , Animais , Caspases/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos , Gravidez , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
BACKGROUND AND AIMS: Oxidant/antioxidant balance is suggested to be an important factor for the recurrence and progression of ulcerative colitis (UC). The aim of the study is to investigate the potential protective role of resveratrol (Res) against dextran sodium sulfate (DSS)-induced oxidative damage in colon of mice with UC. METHODS: UC was induced in mice by oral administration of synthetic DSS (molecular weight 5000) for 7 days. Mice were divided into normal group, colitis control group, low-dose Res-treated group (RLD-treated group), and high-dose Res-treated group (RHD-treated group). Inhibitory effects of concomitant treatment with Res were assessed daily using a Disease Activity Index (DAI) and severity of histological changes. MDA, MPO, SOD and GSH-PX activity of colonic tissue were determined in colon samples by chemical colorimetry. TNF-alpha, IL-8, IFN-gamma, p22(phox) and gp91(phox) expression levels were detected using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), ELISA, and Western blot analysis. RESULT: Administration of Res significantly inhibited the severity of UC compared to the colitis control group. Colonic tissue MDA and MPO activities decreased significantly in Res-treated groups compared to colitis control groups. Furthermore, colonic tissue SOD and GSH-Px activities increased significantly in Res-treated groups compared to colitis control groups. The expression levels of TNF-alpha, IL-8, IFN-gamma, p22(phox), and gp91(phox) also decreased significantly in the Res-treated group compared to the colitis control group. CONCLUSIONS: Oral administration of Res exerts marked inhibitory effects on UC in mice. Resveratrol may play an important role in preventing DSS-induced oxidative damage.
Assuntos
Antioxidantes/farmacologia , Colite Ulcerativa/metabolismo , Sulfato de Dextrana/toxicidade , Estilbenos/farmacologia , Animais , Sequência de Bases , Western Blotting , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/enzimologia , Colo/enzimologia , Colo/metabolismo , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Glutationa Peroxidase/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To investigate the inhibitory effect of small interfering RNA (siRNA) targeting Bax-Bak on the apoptosis of human granulosa cells. METHODS: Human granulosa cells were transfected with Bax-siRNA and Bak-siRNA either alone or in comibnation, and the cell morphological changes were obsered and the cell apoptosis was detected with flow cytometry. Western blotting was performed to examine the changes in Bax and Bak expressions in the transfected cells. RESULTS: Western blotting demonstrated significantly weakened expressions of Bax and Bak in the transfected cells. The cell morphology of the cells tranfected with Bak siRNA and with both Bak and Bax siRNA remained normal; the cells with exclusive Bax siRNA transfection presented with basically normal cell morphology, but black spots were noted in the cytoplasm. In the positive and negative control groups, the cells became rounded and shrank with expanded intercellular spaces and numerous black spots in the cytoplasm. Flow cytometry showed apoptotic indexes of 3.44% and 3.97% in cells transfected with Bak siRNA and Bax-Bak siRNA, respectively, significantly lower than that in the negative group. Bax siRNA transfection resulted in an apoptotic index of 19.98%, similar to that in the negative group. CONCLUSION: Interference of the expression of Bak gene inhibits the apoptosis of human granulosa cells, and the inhibitory effect can be enhanced by simultaneous Bax interference, which, when used alone, does not obviosuly inhibit the apoptosis of human granulosa cells.
Assuntos
Apoptose/genética , Células da Granulosa/citologia , RNA Interferente Pequeno/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Células Cultivadas , Feminino , Humanos , Interferência de RNA , Transfecção , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Reactive oxygen species (ROSs) are involved in osteoporosis by inhibiting osteoblastic differentiation and stimulating osteoclastgenesis. Little is known about the role and how ROS controls proliferation of osteoblasts. Mammalian target of rapamycin, mTOR, is a central regulator of cell growth and proliferation. Here, we report for the first time that 5-200 microM hydrogen peroxide (H(2)O(2)) dose- and time-dependently suppressed cell proliferation without affecting cell viability in mouse osteoblast cell line, MC3T3-E1, and in human osteoblast-like cell line, MG63. Further study revealed that protein level of cyclin B1 decreased markedly and the percentage of the cells in G(2)/M phase increased about 2-4 fold by 200 microM H(2)O(2) treatment for 24-72 hr. A total of 0.5-5 mM of H(2)O(2) but not lower concentrations (5-200 microM) of H(2)O(2) inhibited mTOR signaling, as manifested by dephosphorylation of S6K (T389), 4E-BP1 (T37/46), and S6(S235/236) in MC3T3-E1 and MG63 cells. Rapamycin, which could inhibit mTOR signaling and cell proliferation, however, did not reduce the protein level of cyclin B1. In a summary, H(2)O(2) prevents cell proliferation of osteoblasts by down-regulating cyclin B1 and inducing G(2) cell cycle arrest. Inhibition of mTOR signaling by H(2)O(2) may not be involved in this process.
Assuntos
Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Osteoblastos/efeitos dos fármacos , Animais , Linhagem Celular , Ciclina B/metabolismo , Ciclina B1 , Regulação para Baixo , Humanos , Camundongos , Osteoblastos/citologia , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Fatores de TempoRESUMO
OBJECTIVE: To observe the effect of pBBADs-OXM-transformed bifidobacteria on the body weight of obese mice. METHODS: B. longum was transformed with pBBADs-OXM by electroporation, and arabopyranose-induced oxyntomodulin expression by the bacterium was detected by ELISA. pBBADs-OXM-transformed bifidobacteria was administered orally obese mice on a daily basis with pBBADs-GFP-transformed bifidobacteria as the negative control, and the body weight changes of the mice were observed. RESULTS: OXM was detected by ELISA not only in the supernatant but also the precipitant of the transformed bacterial culture. The body weight of the obese mice fed with pBBADs-OXM-transformed bifidobacteria decreased significantly compared with that of the mice in the obese model group (P<0.05). CONCLUSION: Administration of pBBADs-OXM-transformed B.longum can reduce the body weight of obese mice.
Assuntos
Bifidobacterium , Obesidade/tratamento farmacológico , Oxintomodulina/administração & dosagem , Oxintomodulina/biossíntese , Administração Oral , Animais , Depressores do Apetite/administração & dosagem , Depressores do Apetite/metabolismo , Bifidobacterium/genética , Bifidobacterium/metabolismo , Peso Corporal/efeitos dos fármacos , Eletroporação , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Oxintomodulina/genética , Distribuição Aleatória , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
OBJECTIVE: To study the regulatory role of BRCA1 in the expression of progesterone receptors A and B (PRA and PRB) in breast cancer cells. METHODS: Breast cancer MCF-7 cells were transfected with pFlag-CMV2-BRCA1 wt plasmid containing a full-length BRCA1 cDNA or with BRCA1-specific siRNA via lipofectamine 2000 to induce overexpression or suppressed expression of BRCA1, respectively. Twenty-four hours after the transfection, the cells were incubated in fresh culture medium containing 100 nmol/L progesterone for 24 h. The total RNA extract or whole cell lysate was prepared for detecting BRCA1, PRA and PRB expressions using RT-PCR and Western blotting. RESULTS: The protein expressions of PRA and PRB were significantly decreased whereas their mRNA expressions remained unchanged in MCF-7 cells overexpressing BRCA1. In MCF-7 cells with BRCA1 knock-down, in contrast, the PRA and PRB protein expressions were markedly increased. CONCLUSION: In breast cancer cells, exogenous and endogenous BRCA1 can both down-regulate the expressions of PRA and PRB at the protein level.
Assuntos
Proteína BRCA1/genética , RNA Interferente Pequeno/genética , Receptores de Progesterona/genética , Proteína BRCA1/biossíntese , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Progesterona/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
BACKGROUND & OBJECTIVE: Phospholipase C-gamma 1 (PLC-gamma1) is a vital signal transducer in transmembrane signaling, which regulates cell proliferation and apoptosis. It is overexpressed in many cancers, such as colorectal cancer, which indicates that it is closely related to the genesis and development of tumors. This study was to explore the effects of blocking PLC-gamma1 signaling pathway on the proliferation and apoptosis of human colorectal cancer cell line LoVo, and investigate the signaling mechanisms. METHODS: LoVo cells were treated with PLC-gamma1-specific chemical blocking agent U73122. Cell proliferation was examined by cell counting, MTT assay, and flow cytometry (FCM). Cell apoptosis was observed under a microscope, and measured by agarose gel electrophoresis and FCM with PI simple staining. The expression of hot shock protein 70(HSP70) and Caspase-3 in LoVo cells were detected by Western blot. RESULTS: The proliferation of LoVo cells was inhibited after blocking PLC-gamma1 signaling pathway and the effect was enhanced along with the increasing concentration of U73122. The inhibition rate reached 35% and 45% when treated with 10 micromol/L U73122 for 24 h and 48 h respectively. After blocking PLC-gamma1 signaling pathway, the G1 phase proportion of LoVo cells was increased while the S phase proportion was decreasedû no apoptosis-specific cell shrinkage was found under a light microscope, and no apoptosis-specific DNA ladder was found by agarose gel electrophoresisû no activated Caspase-3 was detected by Western blot, while increased expression of HSP70 was detected. CONCLUSIONS: Blocking PLC-gamma1 signaling pathway can inhibit the proliferation and cell cycle progress of LoVo cells, which may be due to the up-regulated expression of HSP70. PLC-gamma1 is not a vital signal molecule regulating the apoptosis of LoVo cells.