RESUMO
The aim of this study was to evaluate the effect of chronological age on the in vitro fertilization (IVF) outcome in patients with diminished ovarian reserve (DOR). Four hundred and forty-nine women with DOR who underwent IVF cycles were enrolled in the study. There were only 296 patients who obtained available embryos. The patients with no available embryos had a significantly lower antral follicle count (AFC) and higher basal follicle-stimulating hormone (FSH) concentrations than those of women with available embryos, but chronological age in the two groups was comparable. However, patients aged >40 obtained a significantly lower ongoing pregnancy rate (OPR) than patients aged 35 - 40 or <35 (6.38% versus 26.15% versus 28.17%, respectively). Multivariate analysis also showed that chronological age was the only parameter associated with clinical results. It implied that patients with DOR still have reasonable chances of achieving a pregnancy, but their prognosis is significantly affected by chronological age.Impact statementWhat is already known on this subject? Diminished ovarian reserve DOR is a disappointing issue in reproductive medicine. Ovarian reserve, which represents ovarian biological age, is closely related to chronological age. However, ovarian biological age does not always match chronological age. Some studies suggest that biological age is more important than chronological age in predicting the outcome of in vitro fertilization (IVF). However, these conclusions are controversial.What do the results of this study add? We found DOR patients with no available embryos had significantly lower antral follicle count (AFC) and higher follicle-stimulating hormone (FSH) concentrations than that of patients with available embryos, but chronological age in the two groups was comparable. However, for patients with available embryos, chronological age is the only parameter associated with clinical results. For women aged >40 with DOR, chronological age was significantly negatively associated with clinical results.What are the implications of these findings for clinical practice and/or further research? Patients with DOR can obtain available embryos and still have a reasonable chance of becoming pregnant, but their prognosis is greatly affected by chronological age. Therefore, patients with DOR should seek medical help for pregnancy as soon as possible. When DOR patients over the age of 40 plan IVF treatment, the cost-effectiveness of healthcare should be considered.
Assuntos
Fatores Etários , Infertilidade Feminina , Doenças Ovarianas , Reserva Ovariana , Hormônio Antimülleriano , Feminino , Fertilização in vitro/métodos , Hormônio Foliculoestimulante , Humanos , Infertilidade Feminina/etiologia , Infertilidade Feminina/terapia , Doenças Ovarianas/etiologia , Indução da Ovulação/métodos , Gravidez , Taxa de GravidezRESUMO
BACKGROUND: Ovarian reserve reflects the quality and quantity of available oocytes and has become an indispensable measure for the better understanding of reproductive potential. Proteomic approaches are especially helpful in discerning differential protein expression patterns associated with normal and diseased states and, thus, proteomic analyses are increasingly used to identify clinically useful biomarkers. The aim of this study was to investigate proteins secreted in the urine of patients with different ovarian reserve by proteomic techniques to identify potential markers for assessing ovarian reserve. METHODS: Urine samples were obtained from patients with polycystic ovary syndrome (PCOS) and diminished ovarian reserve (DOR), and from normal control (NC)participants. We used isobaric tags for relative and absolute quantification (iTRAQ) technology combined with mass spectrometry analysis to identify candidate urinary proteins in the three groups. The selected proteins were confirmed using western blot analysis and enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of the selected proteins was assessed using receiver operating characteristic analysis. RESULTS: When Compared with NC samples, 285 differentially expressed proteins (DEPs) were identified in the DOR samples and 372 in the PCOS samples. By analyzing the intersection of the two groups of DEPs, we found 26 proteins with different expression trends in the DOR and PCOS groups. Vitamin D-binding protein (VDBP) was the key protein for the protein-protein interaction network. ELISA quantification of urinary VDBP revealed the highest levels in the PCOS group, followed by the NC group and the lowest levels in the DOR group (115.90 ± 26.02, 81.86 ± 23.92 and 52.84 ± 21.37 ng/ml, respectively; P < 0.05). As a diagnostic marker, VDBP had a sensitivity of 67.4% and a specificity of 91.8% for DOR, and a sensitivity of 93.8% and a specificity of 77.6% for PCOS. CONCLUSIONS: Urinary VDBP is closely associated with ovarian reserve and can be considered as a novel noninvasive biomarker of ovarian reserve. However, studies including large sample sizes are needed to validate these results.
Assuntos
Doenças Ovarianas/urina , Reserva Ovariana , Síndrome do Ovário Policístico/urina , Proteína de Ligação a Vitamina D/urina , Adulto , Hormônio Antimülleriano/sangue , Western Blotting , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Folículo Ovariano/diagnóstico por imagem , ProteômicaRESUMO
OBJECTIVE: To evaluate the efficacy and safety of intracytoplasmic morphologically selected sperm injection (IMSI) versus intracytoplasmic sperm injection (ICSI) in in vitro fertilization (IVF) for couples with male factor infertility. METHODS: Using the Cochrane system evaluation method, we searched MEDLINE, EMBASE, CENTRAL, ClinicalTrials.gov, and SinoMed and manually searched the reference lists of the included studies and relevant reviews for randomized controlled trials (RCT) comparing ICSI and IMSI published from 1992 to July 2017. We performed a meta-analysis on the included literature with the RevMan 5.3 software and subgroup analyses due to the prominent clinical heterogeneity of the patients. RESULTS: Of the 280 articles retrieved, 8 RCTs were included, involving 1 741 IVF cycles (842 cycles of IMSI versus 899 cycles of ICSI). There was no evidence for any significant difference between IMSI and ICSI in the live birth rate in the subgroup of infertility induced by pure male factors (RR = 1.31, 95% CI: 0.68ï¼2.51; very low quality evidence from 1 RCT with 77 cycles) but an association of IMSI with an increased clinical pregnancy rate (RR = 1.46, 95% CI: 1.02ï¼2.07; low quality evidence from 4 RCTs with 813 cycles), nor was there any evidence for that in the live birth rate (RR = 0.88, 95% CI: 0.60ï¼1.31; low quality evidence from 1 RCT with 255 cycles) or clinical pregnancy rate (RR = 1.03, 95% CI: 0.86ï¼1.23; moderate quality evidence from 3 RCTs with 851 cycles) in the subgroup of infertility caused by accompanying male factors. CONCLUSIONS: The evidence is of low quality for the association of IMSI with an increased rate of clinical pregnancy and is not sufficient to support the routine use of IMSI in IVF for male factor infertility.
Assuntos
Fertilização in vitro/métodos , Infertilidade Masculina/terapia , Injeções de Esperma Intracitoplásmicas , Feminino , Humanos , Masculino , Gravidez , Taxa de Gravidez , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
This systematic review aimed to evaluate the efficacy and safety of assisted hatching (AH) performed in couples with advanced maternal age. We searched for randomized controlled trials (RCTs) in electronic databases, including MEDLINE, EMBASE and CENTRAL (from inception to January 2018); in addition, we hand-searched the reference lists of included studies and similar reviews. We included RCTs comparing AH versus no treatment (control). The meta-analysis was performed by RevMan 5.3 software. The search retrieved 943 records and 8 RCTs were included, comprising 870 cycles (n=440 for AH, and n=430 for control). There was no significant difference in the rates of live birth (RR 0.88, 95% CI 0.65 to 1.18, 3 RCTs, n=427, I2=0%), clinical pregnancy (RR 1.00, 95% CI 0.83 to 1.19, 8 RCTs, n=870, I2=22%), implantation (RR 1.07, 95% CI 0.83 to 1.39, 4 RCTs, n=1359, I2=0%), miscarriage (RR 1.13, 95% CI 0.66 to 1.94, 2 RCTs, n=116, I2=0%) and multiple pregnancy (RR 0.89, 95% CI 0.31 to 2.52, 1 RCT, n=97, I2=not applicable) between the treatment group and control group. No reasonable conclusions could be drawn regarding reproductive outcomes after AH in patients with advanced maternal age due to the small sample pooled in meta-analyses. Studies of high methodological quality and with adequate power are necessary to further investigate the value of AH in assisted conception of those patients.
Assuntos
Características da Família , Idade Materna , Técnicas de Reprodução Assistida , Aborto Espontâneo/epidemiologia , Estudos de Casos e Controles , Anormalidades Congênitas/etiologia , Implantação do Embrião , Feminino , Humanos , Gravidez , Resultado da Gravidez , Taxa de Gravidez , Gravidez Múltipla/estatística & dados numéricos , Viés de Publicação , Técnicas de Reprodução Assistida/efeitos adversos , Fatores de RiscoRESUMO
PURPOSE: This study aimed to determine the effect of paternal hepatitis B virus (HBV) infection on reproductive outcomes of couples undergoing frozen-thawed embryo transfer (FET). METHODS: This retrospective cohort study included FET cycles performed between January 2014 and March 2017 in couples with a hepatitis B surface antigen (HBsAg)-positive male partner and an HBsAg-negative female partner, which was categorized as HBsAg group. The FET cycles underwent by couples with both HBsAg-negative partners were randomly selected as controls. The primary outcome was clinical pregnancy. RESULTS: A total of 117 FET cycles, comprising 39 in the HBsAg group and 78 in the control group, were included. Couples with HBsAg-positive male partners had significantly lower clinical pregnancy rate (17.9 vs 41.0%, P = 0.013), lower implantation rate (11.1 vs 24.5%, P = 0.014), and lower live birth rate (12.8 vs 30.8%, P = 0.034) compared with the control group. Moreover, the multivariate logistic regression analysis showed that paternal HBV infection was negatively associated with clinical pregnancy (odds ratio = 0.297, 95% confidence interval 0.108-0.817, P = 0.019). The miscarriage rate was not significantly different between the two groups (28.6 vs 25.0%, P = 1.000). CONCLUSIONS: Paternal HBV infection resulted in a lower frequency of clinical pregnancy after FET, a difference that was probably attributed to a detrimental effect of HBV on the ability of embryos to survive freezing and thawing.
Assuntos
Transferência Embrionária , Hepatite B/complicações , Taxa de Gravidez , Aborto Espontâneo/epidemiologia , Adulto , Transferência Embrionária/métodos , Pai , Feminino , Antígenos de Superfície da Hepatite B/sangue , Humanos , Modelos Logísticos , Masculino , Gravidez , Estudos RetrospectivosRESUMO
Despite the use of adjuvant therapies, the cumulative proportion of live births remains at ~40%. Accumulating data show that low pregnancy rates, even in the presence of high fertility rates, are due to implantation failure. The present study aimed to identify and construct a profile of proteins that react with preimplantation factor (PIF) and to provide an understanding into the molecular mechanisms by which PIF promotes trophoblast invasion. Cytoplasmic proteins were immunoprecipitated with biotinlabeled synthetic PIF or intralipid and scrambled PIF (PIFscr). The protein profiles were analyzed using isobaric tags for relative and absolute quantification coupled with mass spectrometry. Immunoprecipitation and western blot analyses were used to assess the interactions between PIF and myosin heavy chain 10 (MYH10) and heat shock protein family D1. Small interfering RNAbased silencing was performed to examine the function of MYH10. In the results of the present study, 21 proteins were identified with interactions with PIF. The immunoprecipitation and western blot analyses revealed an interaction between PIF and MYH10. Silencing of the expression of MYH10 in HEC1B cells significantly attenuated cell migration and invasion capacities. These data support the conclusion that MYH10mediated cell migration and invasion act in conjunction with PIF to promote the trophoblast invasion procedure.
Assuntos
Implantação do Embrião/efeitos dos fármacos , Peptídeos/farmacologia , Proteoma/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Células HEK293 , Humanos , Espectrometria de Massas , Cadeias Pesadas de Miosina/antagonistas & inibidores , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/antagonistas & inibidores , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Proteoma/efeitos dos fármacos , Proteômica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: Liver fibrosis can lead to cirrhosis and hepatocellular carcinoma if not treated in the early stages. The molecular mechanisms of the pathogenesis of hepatic fibrosis remain unclear. AIM: To identify the molecules involved in the pathogenesis of liver fibrosis and to investigate the potential effect and mechanism of Annexin A2 up-regulation during liver fibrosis progression. METHODS: Twenty Sprague-Dawley rats were divided into two groups: the carbon tetrachloride (CCl4)-induced liver fibrosis group and the normal control group. Hematoxylin and eosin staining or Masson Trichrome staining and enzyme-linked immunosorbent assay were applied to assess the degree of liver damage and fibrosis in rats with CCl4-induced liver fibrosis. Liver tissue protein profiles were analyzed using iTRAQ and mass spectrometry. RT-PCR and western blotting analyses were employed to validate differentially expressed proteins. Small interfering RNA-based silencing was performed to study the function of Annexin A2. RESULTS: Twelve weeks after CCl4 injection, significant body weight changes and liver injury and liver fibrosis were observed in rats. In addition, 130 proteins were differentially expressed in the liver fibrosis group. Overexpression of Annexin A2 was confirmed by RT-PCR and Western blotting analysis. Silencing of Annexin A2 expression in HepG2 and LX-2 cells significantly reduced the secretion of von Willebrand factor (vWF). CONCLUSION: Annexin A2 promotes liver fibrosis by mediating vWF secretion, which can be used to mitigate the progression of liver fibrosis.
Assuntos
Anexina A2/metabolismo , Cirrose Hepática Experimental/metabolismo , Fator de von Willebrand/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Inativação Gênica , Cirrose Hepática Experimental/etiologia , Espectrometria de Massas , Ligação Proteica , RNA Interferente Pequeno , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
BACKGROUND AND AIMS: Hepatitis B virus (HBV) infection is a major risk factor for liver cirrhosis and hepatocellular carcinoma (HCC). To gain a better understanding of the pathogenesis of HBV infection, this study aimed to investigate the differentially expressed proteins (DEPs) in liver tissues from patients with chronic hepatitis B (CHB) infection. RESULTS: Seventy-one DEPs were identified. Overexpression of multi-drug resistance protein 1 (MDR1) was validated by RT-qPCR and western blot analyses. Moreover, its expression was increased at both the mRNA and protein levels in response to overexpression of HBV large surface protein (LHBs). Furthermore, screening of transcription factors suggested the possible involvement of hypoxia-inducible factor 1α (HIF-1α) in the interaction between LHBs and MDR1. The function of HIF-1α in the MDR1 activation was confirmed by EMSA and reporter gene analyses. MATERIALS AND METHODS: Liver samples from CHB patients and controls without HBV infection were collected and subjected to isobaric tags for relative and absolute quantitation (iTRAQ) and mass spectrometric analysis. CONCLUSIONS: These results imply that LHBs, in association with HIF-1α, induces MDR1 overexpression, which may contribute to the pathogenic changes in CHB infection.
Assuntos
Carcinoma Hepatocelular/virologia , Hepatite B Crônica/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Hepáticas/virologia , Proteínas do Envelope Viral/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Estudos de Casos e Controles , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/genética , Humanos , Fígado/metabolismo , Fígado/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica/métodos , Adulto JovemRESUMO
Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), the sixth most common cancer worldwide. To explore potential biomarkers for HCC, iTRAQ coupled with mass spectrometry was used to analyze proteins in plasma from individuals with HBV-associated HCC, nonmalignant cirrhosis, chronic hepatitis B, and healthy individuals. Twenty-one aberrantly expressed proteins were identified from HCC patients as compared with nontumor controls. Overexpression of von Willebrand factor (vWF) was confirmed by Western blotting, and immunohistochemical analysis from liver biopsies and ELISA from plasma samples revealed a correlation between vWF expression and HCC clinicopathologic staging. Furthermore, siRNA-induced vWF silencing reduced HBV replication by over two-fold via the interferon-signaling pathway and impaired the invasion and migration of HCC cells in vitro. These results indicate that vWF can serve as a biomarker, and perhaps an alternative target for therapeutic intervention of HCC progression and HBV viral infection. BIOLOGICAL SIGNIFICANCE: We report comparative plasma proteome profiles of HBV-associated HCC and nonmalignant chronic liver diseases, including chronic hepatitis B and cirrhosis. The quantification of these datasets showed altered abundance of 21 proteins in HBV-related HCC and provides a reference point for future applied and basic research. In addition, we have demonstrated that the candidate protein vWF is involved in the pathogenesis of HBV infection and replication, and also associated with clinicopathologic staging of HCC patients with HBV infection. Overall these findings provide information on the mechanism of HCC development, which may assist in the development of novel cancer and HBV therapeutic drugs.
Assuntos
Carcinoma Hepatocelular/sangue , Regulação Neoplásica da Expressão Gênica , Hepatite B Crônica/sangue , Neoplasias Hepáticas/sangue , Fator de von Willebrand/metabolismo , Adulto , Apolipoproteínas E/sangue , Biomarcadores/sangue , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Feminino , Frutose-Bifosfato Aldolase/sangue , Inativação Gênica , Vírus da Hepatite B , Humanos , Neoplasias Hepáticas/virologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteoma , Proteômica , RNA Interferente Pequeno/metabolismo , Proteína Amiloide A Sérica/metabolismo , Transdução de SinaisRESUMO
Hepatitis B virus (HBV) is the most common of the hepatitis viruses that cause chronic liver infections in humans, and it is considered to be a major global health problem. To gain a better understanding of HBV pathogenesis, and identify novel putative targets for anti-HBV therapy, this study was designed to elucidate the differential expression of host proteins in liver tissue from HBV-transgenic mice. Liver samples from two groups, (1) HBV-transgenic (Tg) mice, (2) corresponding background normal mice, wild-type (WT) mice, were collected and subjected to iTRAQ and mass spectrometry analysis. In total, 1950 unique proteins were identified, and 68 proteins were found to be differentially expressed in HBV-Tg mice as compared with that in WT mice. Several differentially expressed proteins were further validated by real-time quantitative RT-PCR, Western blot and immunohistochemical analysis. Furthermore, the association of HBV replication with fatty acid synthase (FASN), one of the highly expressed proteins in HBV-Tg mice, was verified. Silencing of FASN expression in HepG2.2.15 cells suppressed viral replication through the IFN signaling pathway, and some downstream antiviral effectors. The implicated role of FASN in HBV replication provides an opportunity to test existing compounds against FASN for adjuvant therapy and/or treatment of HBV replication.
Assuntos
Ácido Graxo Sintase Tipo I/genética , Regulação da Expressão Gênica , Hepatite B/genética , Fígado/metabolismo , Proteoma/análise , Sequência de Aminoácidos , Animais , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/metabolismo , Feminino , Perfilação da Expressão Gênica , Inativação Gênica , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Fígado/química , Fígado/virologia , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Transdução de Sinais , Replicação Viral/fisiologiaRESUMO
Hepatitis B virus (HBV) is the most common of the hepatitis viruses that cause chronic liver infections in humans and it is considered a major global health problem. However, the mechanisms of HBV replication are complex and not yet fully understood. In this study, the HBV DNA-transfected HepG2.2.15 cell line and its parental HepG2 cell line were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass-spectrophotometry (2D LC-MS/MS), a successfully exploited high-throughput proteomic technology. In total, 2,028 unique proteins were identified and 170 proteins were differentially expressed in HepG2.2.15 cells as compared with that in HepG2. Several differentially expressed proteins were further validated by Western blot and real-time quantitative reverse transcription-PCR. Furthermore, the association of HBV replication with heat shock protein B1, one of the highly expressed proteins in HepG2.2.15 cells, was verified. HSPB1 functions as a anti-viral protein during HBV infection by specifically inducing type interferon and some downstream antiviral effectors. This study is the first to report the application of iTRAQ technology to analyze the underlying mechanisms of HBV replication. Many of the differentially expressed proteins identified have not been linked to HBV replication before, and may provide valuable novel insights into HBV replication.
Assuntos
Proteínas de Choque Térmico HSP27/genética , Células Hep G2/metabolismo , Células Hep G2/virologia , Vírus da Hepatite B/fisiologia , Transcriptoma , Sequência de Aminoácidos , Cromatografia Líquida/métodos , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Células Hep G2/imunologia , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Chaperonas Moleculares , Dados de Sequência Molecular , Proteômica , Espectrometria de Massas em Tandem/métodos , Transfecção , Replicação Viral/fisiologiaRESUMO
Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Here, we comparatively analyzed the proteome profiles of ovarian cancer tissues and normal ovarian epithelial tissues. Using the high-throughput proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ)-coupled with two-dimensional-liquid chromatography-tandem mass spectrometry, 1,259 unique proteins were identified. Of those, 205 were potentially differentially expressed between ovarian cancer and normal ovarian tissues. Several of the potentially differentially expressed proteins were validated by Western blotting and real-time quantitative RT-PCR analyses. Furthermore, up-regulation of KRT8, PPA1, IDH2, and S100A11 were validated in ovarian tissue microarrays by immunohistochemistry. Silencing of S100A11 expression suppressed the migration and invasion properties of ovarian cancer cells in vitro. Our study represents the successful application of iTRAQ technology to an investigation of ovarian cancer. Many of the potentially differentially expressed proteins identified had not been linked to ovarian cancer before, and provide valuable novel insights into the underlying mechanisms of carcinogenesis in human ovarian cancer.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Ovarianas/metabolismo , Proteoma/análise , Proteômica/métodos , Biomarcadores Tumorais/metabolismo , Western Blotting , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular , Cromatografia Líquida , Epitélio/metabolismo , Epitélio/patologia , Feminino , Inativação Gênica , Humanos , Imuno-Histoquímica , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteoma/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas S100/genética , Proteínas S100/metabolismo , Coloração e Rotulagem , Espectrometria de Massas em Tandem , Análise Serial de Tecidos , TransfecçãoRESUMO
Legumain is a member of the asparaginyl endopeptidase family that is over-expressed in response to hypoxic stress on mammary adenocarcinoma, colorectal cancer, proliferating endothelial cells, and tumor-associated macrophages (TAMs). Here, we demonstrate that elevated expression of legumain in ovarian cancer by a proteomic approach using isobaric tags for relative and absolute quantification (iTRAQ) followed by liquid chromatography-mass spectrometry (LC-MS/MS). To investigate the relationship between legumain expression and ovarian cancer development, we tested legumain expression in malignant human ovarian tumors (n = 60), borderline ovarian tumors (n = 20), benign ovarian tumors (n = 20), and normal ovary samples (n = 20) using immunohistochemical assay (IHC). A correlation between legumain expression, and clinocopathologic and biological variables was also established. Importantly, increased legumain expression was validated by real-time PCR and Western blots, correlated positively with an increased malignancy of ovarian tumors (P < 0.01). In fact, patients with strong legumain expression had a worse prognosis (P = 0.03). In addition, results of in vitro experiments revealed that over-expression of legumain correlates with increased cell migration and invasion of ovarian cancer cells. Although legumain's functional role and clinical utility remain to be established, our results indicated that a sensitive assay for early expression of legumain may serve as both a potential biomarker and a molecular target for treatment of ovarian cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Cisteína Endopeptidases/metabolismo , Neoplasias Ovarianas/metabolismo , Biomarcadores Tumorais/genética , Western Blotting , Movimento Celular/genética , Movimento Celular/fisiologia , Cisteína Endopeptidases/genética , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Multi-drug resistance (MDR) is a major obstacle towards a successful treatment of hepatocellular carcinoma (HCC). The mechanisms of MDR are intricate and have not been fully understood. Therefore, we employed a cell-line model consisting of the 5-fluorouracil (5-FU) resistant BEL7402/5-FU cell line and its parental BEL7402 cell line. Using relative and absolute quantification (iTRAQ)-coupled 2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, in total, 660 unique proteins were identified and 52 proteins showed to be differentially expressed in BEL7402/5-FU compared with BEL7402. Several differentially expressed proteins were further validated by Western blot and real-time quantitative RT-PCR analysis. Furthermore, the association of MDR with ANXA3, one of the highly expressed proteins in BEL7402/5-FU, was verified. Our study represents the first successful application of iTRAQ technology for MDR mechanisms analysis in HCC. Many of the differentially expressed proteins identified had not been linked to MDR in HCC before, which provide valuable information for further understanding of MDR.
Assuntos
Anexina A3/metabolismo , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Fluoruracila/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Adulto , Idoso , Anexina A3/antagonistas & inibidores , Anexina A3/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos/genética , Resistência a Múltiplos Medicamentos/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Interferente Pequeno/genética , Espectrometria de Massas em TandemRESUMO
Ski is an avian sarcoma virus oncogene homolog best known for inhibiting TGF beta signaling through its association with the SMAD proteins. Anti-Ski antibodies (MAbs) of high titer were prepared by immunizing BALB/c mice with multifocal intradermal injections and fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Three MAbs were selected for further characterization as classes and subclasses. Antibodies were produced by these three clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin IgG1 and IgG2b subclass with kappa light chain. They could recognize Ski as determined by Western blot analysis. The produced MAbs will be a useful tool for further investigation of Ski functions in organisms.
Assuntos
Anticorpos Monoclonais/química , Proteínas de Ligação a DNA/imunologia , Imunoglobulina G/química , Fragmentos de Peptídeos/imunologia , Proteínas Proto-Oncogênicas/imunologia , Animais , Afinidade de Anticorpos , Clonagem Molecular , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Multidrug resistance (MDR) is a major obstacle towards a successful treatment of gastric cancer. However, the mechanisms of MDR are intricate and have not been fully understood. To elucidate the molecular mechanisms of MDR in gastric cancer, we employed the proteomic approach of isobaric tags for relative and absolute quantification (iTRAQ), followed by LC-MS/MS, using the vincristine-resistant SGC7901/VCR cell line and its parental SGC7901 cell line as a model. In total, 820 unique proteins were identified and 91 proteins showed to be differentially expressed in SGC7901/VCR compared with SGC7901. Several differentially expressed proteins were further validated by western blot analysis. Furthermore, the association of MVP, one of the highly expressed proteins in SGC7901/VCR, with MDR was verified. Our study is the first application of iTRAQ technology for MDR mechanisms analysis in gastric cancer, and many of the differentially expressed proteins identified have not been linked to MDR in gastric cancer before, which showed the value of this technology in identifying differentially expressed proteins in cancer.
Assuntos
Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Marcação por Isótopo/métodos , Proteômica/métodos , Neoplasias Gástricas/metabolismo , Sequência de Aminoácidos , Western Blotting , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
In order to understand the molecular mechanisms of multidrug resistance (MDR) in ovarian cancer, we employed the proteomic approach of isobaric tags for relative and absolute quantification (iTRAQ), followed by LC-MS/MS, using the cisplatin-resistant COC1/DDP cell line and its parental COC1 cell line as a model. A total number of 28 proteins differentially expressed were identified, and then the differential expression levels of partially identified proteins were confirmed by Western blot analysis and/or real-time RT-PCR. Furthermore, the association of PKM2 and HSPD1, two differentially expressed proteins, with MDR were analyzed, and the results showed that they could contribute considerably to the cisplatin resistance in ovarian cancer cell. The differential expression proteins could be classified into eight categories based on their functions, that is, calcium binding proteins, chaperones, extracellular matrix, proteins involved in drug detoxification or repair of DNA damage, metabolic enzymes, transcription factor, proteins related to cellular structure and proteins relative to signal transduction. These data will be valuable for further study of the mechanisms of MDR in the ovarian cancer.