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Programmed death ligand 1 (PD-L1) is widely known as an immune checkpoint, and immunotherapy through the inhibition of checkpoint molecules has become an important component in the successful treatment of tumours via programmed death 1 (PD-1)/PD-L1 signalling pathways. However, its biological functions and expression profile in colorectal cancer (CRC) are elusive. We previously found that PD-L1 can bind to PD-L1 and cause cell detachment. However, the detailed molecular mechanisms of how PD-L1 binds to PD-L1 and how it transmits signals to the cell remain unclear. In this study, we disclosed that PD-L1 expression was dramatically upregulated in CRC compared to normal tissues. Ectopic expression of PD-L1 inhibits cell adhesive capacity and promotes cell migration in CRC cell lines, while silencing PD-L1 had the opposite effects and suppressed invasion and proliferation. Mechanistically, PD-L1 was found to promote epithelial-mesenchymal transition (EMT) through the ERK signalling molecule pathway and interacted with the 1-86 aa fragment of KRAS to transduce signals. Collectively, our study demonstrated the role of PD-L1 after binding to PD-L1 in CRC, thereby providing a new theoretical basis for further improving immunotherapy with anti-PD-L1 antibodies.
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Antígeno B7-H1 , Neoplasias Colorrectales , Humanos , Antígeno B7-H1/metabolismo , Movimiento Celular , Neoplasias Colorrectales/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/farmacología , Transducción de Señal , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas ras/metabolismoRESUMEN
Spermatogenesis is a cyclical process in which different generations of spermatids undergo a series of developmental steps at a fixed time and finally produce spermatids. Here, we report that overexpression of PD-L1 (B7 homolog1) in the testis causes sperm developmental disorders and infertility in male mice, with severe malformation and sloughing during spermatid development, characterized by disorganized and collapsed seminiferous epithelium structure. PD-L1 needs to be simultaneously expressed on Sertoli cells and spermatogonia to cause spermatogenesis failure. After that, we excluded the influence of factors such as the PD-L1 receptor and humoral regulation, confirming that PD-L1 has an intrinsic function to interact with PD-L1. Studies have shown that PD-L1 not only serves as a ligand but also plays a receptor-like role in signal transduction. PD-L1 interacts with PD-L1 to affect the adhesive function of germ cells, causing malformation and spermatid sloughing. Taken together, these results indicate that PD-L1 can interact with PD-L1 to cause germ cell detachment and male infertility.
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Antígeno B7-H1 , Túbulos Seminíferos , Animales , Antígeno B7-H1/genética , Masculino , Ratones , Células de Sertoli , Espermatogénesis/genética , Espermatogonias , TestículoRESUMEN
Numerous clinical studies investigated how low expression of CD9 predicts poor prognosis of solid tumor. However, the results were inconclusive. This present meta-analysis was therefore performed to determine the prognostic value of CD9 expression in solid tumors. In this meta-analysis, 25 studies involving 5,555 participants were included; the result showed strong significant associations between declined expression of CD9 and all endpoints: overall survival (OS) (hazard ratio (HR) = 1.88, 95% CI = 1.45-2.43, p < 0.000) and time to progression (TTP) (HR = 2.0, 95% CI = 1.38-2.88, p < 0.000). The subgroup analysis was also performed, which revealed that the associations between CD9 downregulated expression related to poor OS in lung cancer and head and neck cancer. Also, low expression of CD9 was significantly connected with poor TTP in patients with head and neck cancer. The adverse prognostic impact of decreased expression of CD9 was observed in patients of different ethnicities. In conclusion, these results showed that declined expression of CD9 was associated with poor survival in human solid tumors. CD9 may be a valuable prognostic predictive biomarker and a potential therapeutic target in human solid tumors.
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BACKGROUND: Hairy and enhancer of split 1 (Hes-1) protein is a downstream target of Notch signaling and is a basic helix-loop-helix transcriptional repressor. However, definitive evidence for a role in hepatocellular carcinoma (HCC) cells has not been reported. Here, Hes-1 was revealed to an important component of the Notch signaling cascade in HCC cell lines possessing different potential for lung metastasis. MATERIALS AND METHODS: RNAi mediated by plasmid constructs was used to analyze the role of Hes-1 in MHCC-97L HCC cells by assessing proliferation, apoptosis, cell migration and matrigel invasion following transfection. Hes-1 protein expression analysis in HCC tissue was also conducted by immunohistochemistry. RESULTS: Our studies revealed that Hes-1 was decreased in HCC cell lines with higher lung metastasis potential at both the mRNA and protein levels. Down-regulation of the Hes-1 gene in MHCC-97L cells resulted in increased cell proliferation, reduced apoptosis and increased migration and invasion. CONCLUSIONS: Hes-1 has potential prognostic value in post-surgical HCC patients and may be an independent prognostic indicator for overall survival and tumor recurrence. These findings have important implications for understanding the mechanisms by which Hes-1 participates in tumor proliferation and invasion.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma Hepatocelular/metabolismo , Movimiento Celular , Proliferación Celular , Proteínas de Homeodominio/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptor Notch1/metabolismo , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Western Blotting , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/secundario , Regulación hacia Abajo , Femenino , Citometría de Flujo , Estudios de Seguimiento , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/secundario , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Notch1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Tasa de Supervivencia , Factor de Transcripción HES-1 , Células Tumorales CultivadasRESUMEN
Previous studies have showed that patients with gout showed lower serum 25(OH)D levels. As the specific receptor of vitamin D, VDR plays an important role in regulating immune system by combining with vitamin D. In this study, we investigated whether the functional VDR polymorphisms were associated with susceptibility to gout in Chinese Han male population. A total of 504 patients with gout and 523 gout-free controls were recruited from the Affiliated Hospital of the Medical College, Qingdao University. Genotyping of VDR rs11568820, rs2228570 and rs1544410 was performed by TaqMan allele discrimination assays. An association analysis was carried out using the χ(2) test. A genotype-phenotype analysis was also conducted. Our results showed that polymorphisms of rs11568820 and rs1544410 in VDR were associated with gout in Chinese Han male population. The A allele of both rs11568820 and rs1544410 was associated with the risk of gout [P = 0.012 OR 1.251, 95% CI (1.051-1.490); P = 0.006, OR 1.574, 95% CI (1.139-2.175)]. However, there was no statistic significance between rs2228570 and gout (P = 0.186). Our study suggested that the polymorphisms of VDR may be relevant host susceptibility factors for the development of gout in Chinese Han male population. However, further study should be done in a larger size sample and other ethic to test and verify our result.
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Pueblo Asiatico/genética , Gota/genética , Polimorfismo Genético , Receptores de Calcitriol/genética , Adulto , Anciano , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , China , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Gota/diagnóstico , Gota/etnología , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Fenotipo , Factores de Riesgo , Factores SexualesRESUMEN
BACKGROUND: Metastasis is a major reason for poor prognosis in patients with cancer, including hepatocellular carcinoma (HCC). A salient feature is the ability of cancer cells to colonize different organs. Long non-coding RNAs (lncRNAs) play important roles in numerous cellular processes, including metastasis. MATERIALS AND METHODS: In this study, the lncRNA expression profiles of two HCC cell lines, one with high potential for metastasis to the lung (HCCLM3) and the other to lymph nodes (HCCLYM-H2) were assessed using the Arraystar Human LncRNA Array v2.0, which contains 33,045 lncRNAs and 30,215 mRNAs. Coding-non-coding gene co-expression (CNC) networks were constructed and gene set enrichment analysis (GSEA) was performed to identify lncRNAs with potential functions in organ-specific metastasis. Levels of two representative lncRNAs and one representative mRNA, RP5-1014O16.1, lincRNA-TSPAN8 and TSPAN8, were further detected in HCC cell lines with differing metastasis potential by qRT-PCR. RESULTS: Using microarray data, we identified 1,482 lncRNAs and 1,629 mRNAs that were differentially expressed (≥1.5 fold-change) between the two HCC cell lines. The most upregulated lncRNAs in H2 were RP11-672F9.1, RP5-1014O16.1, and RP11-501G6.1, while the most downregulated ones were lincRNA-TSPAN8, lincRNA-CALCA, C14orf132, NCRNA00173, and CR613944. The most upregulated mRNAs in H2 were C15orf48, PSG2, and PSG8, while the most downregulated ones were CALCB, CD81, CD24, TSPAN8, and SOST. Among them, lincRNA-TSPAN8 and TSPAN8 were found highly expressed in high lung metastatic potential HCC cells, while lowly expressed in no or low lung metastatic potential HCC cells. RP5- 1014O16.1 was highly expressed in high lymphatic metastatic potential HCC cell lines, while lowly expressed in no lymphatic metastatic potential HCC cell lines. CONCLUSIONS: We provide the first detailed description of lncRNA expression profiles related to organ-specific metastasis in HCC. We demonstrated that a large number of lncRNAs may play important roles in driving HCC cells to metastasize to different sites; these lncRNAs may provide novel molecular biomarkers and offer a new basis for combating metastasis in HCC cases.
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Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Células Hep G2 , Humanos , Metástasis de la Neoplasia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Aims. To examine the potential differences between multiple daily injection (MDI) regimens based on new long-acting insulin analogues (glargine or detemir) plus prandial insulin aspart and continuous subcutaneous insulin aspart infusion (CSII) in patients with poorly controlled type 2 diabetes. Methods. Patients (n = 119) with poorly controlled type 2 diabetes of a duration exceeding five years were randomly assigned into three groups: Group A treated with CSII using insulin aspart; Group B treated with glargine-based MDI and Group C treated with detemir-based MDI. Results. Good glycemic control was achieved by patients in Group A in a significantly shorter duration than patients in Groups B and C. Total daily insulin, basal insulin dose and dose per kg body weight in Group A were significantly less than those in Groups B and C. Daily blood glucose fluctuation in Group A was significantly less than that in Groups B and C. There were no differences between Groups B and C. Conclusions. Aspart-based CSII may achieve good blood glucose control with less insulin doses over a shorter period compared with glargine or detemir-based MDI. No differences between glargine- and detemir-based MDI were detected in poorly controlled subjects with type 2 diabetes.
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Profound T cell inhibitory activity of hepatic stellate cells (HSCs) in vitro has recently been described in hepatocellular carcinoma (HCC). In the present study, we investigated the immune inhibitory activity of HSCs in vivo in an orthotopic rat HCC model with lung metastasis. Rats (n=24) were randomly sacrificed on days 7, 14, 21 and 28 (n=4 each). Lung tissues were stained with hematoxylin and eosin. Liver sections were stained for immunofluorescence analysis. T-cell apoptosis was detected using double staining for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Staining revealed marked and continuous accumulation of α-smooth muscle actin with tumor progression after orthotopic tumor implantation in rat liver. T lymphocyte numbers gradually increased following tumor progression, and subset analysis revealed an increase in the distribution of liver CD8+ and CD4+ T cells. Double staining for CD3 and TUNEL demonstrated T-cell apoptosis. Apoptotic T cells were more frequent in the HCC livers compared to the normal livers, and were spatially associated with intratumoral activated HSCs (tHSCs), suggesting a direct interaction. T-cell apoptosis was more frequently induced in the co-cultures of activated splenic T cells(aT)/tHSCs compared to aT/quiescent (q) HSCs or qT/tHSCs. tHSCs were positively correlated with T-cell apoptosis, and the percentage of T-cells undergoing apoptosis was positively correlated with the number of lung metastasis nodules. T-cell apoptosis may be promoted via an interaction with tHSCs, suggesting that tHSCs regulate T cells and contribute to lung metastasis in HCC.
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Apoptosis , Carcinoma Hepatocelular/patología , Células Estrelladas Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/secundario , Linfocitos T/inmunología , Animales , Western Blotting , Carcinoma Hepatocelular/inmunología , Proliferación Celular , Células Dendríticas/inmunología , Células Dendríticas/patología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células Estrelladas Hepáticas/patología , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/inmunología , Neoplasias Pulmonares/inmunología , Activación de Linfocitos , Ratas , Ratas Endogámicas BUF , Ratas Endogámicas F344 , Linfocitos T/patología , Células Tumorales CultivadasRESUMEN
AIM: To evaluate the correlation between nonalcoholic fatty liver disease (NAFLD) and microvascular complications in type 2 diabetes mellitus (T2DM). METHODS: Data were obtained from 1217 inpatients with T2DM (757 females, 460 males; aged 63.39 ± 12.28 years). NAFLD was diagnosed by hepatic ultrasonography. Diabetic nephropathy (DN), diabetic peripheral neuropathy (DPN), and diabetic retinopathy (DR) were diagnosed according to their respective criteria. The prevalence of NAFLD and the independent correlations of clinical characteristics with NAFLD were determined by cross-tabulation and logistic regression, respectively. RESULTS: Approximately 61% of inpatients with T2DM in Qingdao, China had NAFLD, which decreased significantly with increase in age and prolonged course of diabetes. The prevalence of NAFLD in patients presenting with DN, DPN and DR was 49.4%, 57.2% and 54.9%, respectively. These rates were significantly lower than those of patients without DN, DPN and DR (65.9%, 65.6% and 66.1%, respectively, P < 0.05). Participants with NAFLD had greater body weight, waist circumference (WC), body mass index (BMI), fasting blood glucose (FBG), hemoglobin A1c, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltransferase, blood pressure, as well as triglyceride (TG) levels and lower high-density lipoprotein (HDL) concentration than those without NAFLD (P < 0.05). NAFLD was positively correlated with BMI, WC, TG, FBG, diastolic blood pressure, and systolic blood pressure but negatively correlated with the duration of diabetes, DR, DPN, DN, and HDL. CONCLUSION: Despite the benign nature of NAFLD, efforts should be directed toward early diagnosis, intensive blood glucose and blood pressure control, and effective dyslipidemia correction.
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Diabetes Mellitus Tipo 2/epidemiología , Angiopatías Diabéticas/epidemiología , Hígado Graso/epidemiología , Microcirculación , Anciano , Distribución de Chi-Cuadrado , China/epidemiología , Estudios Transversales , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/fisiopatología , Angiopatías Diabéticas/diagnóstico , Angiopatías Diabéticas/fisiopatología , Nefropatías Diabéticas/epidemiología , Retinopatía Diabética/epidemiología , Dislipidemias/epidemiología , Hígado Graso/diagnóstico por imagen , Femenino , Humanos , Hipertensión/epidemiología , Modelos Logísticos , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico , Obesidad/epidemiología , Oportunidad Relativa , Valor Predictivo de las Pruebas , Prevalencia , Factores de Riesgo , UltrasonografíaRESUMEN
CXCR7, a recently identified chemokine receptor, has been implicated in directing cancer metastasis. In the present study, the potential roles of CXCR7 in the growth and metastasis of hepatocellular carcinoma (HCC) were evaluated. A chemokine receptor gene chip was used to compare the expression of CXCR7 in HCC cell lines with different metastatic potential. Effects of targeting CXCR7 by RNA interference (RNAi) on the proliferation and metastasis of HCCLM3 cells were observed in vitro and in vivo. CXCR7 expression in 116 specimens from patients with or without metastatic HCC was assessed by tissue microarray. As a result, the gene chip showed that expression of CXCR7 was significantly higher in the highly metastatic HCCLM3 cells, which was confirmed by real-time RT-PCR and Western blotting. Chemotaxis assays showed that HCCLM3 cells responded to SDF-1α from 1 to 100 µg/l and lung extractions (1 g/l). Furthermore, down-regulation of CXCR7 in HCCLM3 cells by RNAi inhibited the proliferation and invasion of tumor cells in vitro. Moreover, CXCR7 knockdown significantly reduced the activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. RNAi of CXCR7 in the HCCLM3 cells also decreased the growth of tumors and the number of lung metastases in nude mice. The tissue microarray showed that HCCs with high expression of CXCR7 were prone to metastasize to the lung. These findings suggest that CXCR7 plays critical roles in the growth and metastasis of HCC. RNAi of CXCR7 inhibits the growth and invasion of tumor cells, which indicates that CXCR7 may be a potential molecular target for use in HCC therapy.
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BACKGROUND: Hepatocellular carcinoma (HCC) is a common and aggressive form of cancer. Due to a high rate of postoperative recurrence, the prognosis for HCC is poor. Subclinical metastasis is the major cause of tumor recurrence and patient mortality. Currently, there is no reliable prognostic method of invasion. AIM: To investigate the feasibility of fingerprints of volatile organic compounds (VOCs) for the in-vitro prediction of metastasis. METHODS: Headspace gases were collected from 36 cell cultures (HCC with high and low metastatic potential and normal cells) and analyzed using nanomaterial-based sensors. Predictive models were built by employing discriminant factor analysis pattern recognition, and the classification success was determined using leave-one-out cross-validation. The chemical composition of each headspace sample was studied using gas chromatography coupled with mass spectrometry (GC-MS). RESULTS: Excellent discrimination was achieved using the nanomaterial-based sensors between (i) all HCC and normal controls; (ii) low metastatic HCC and normal controls; (iii) high metastatic HCC and normal controls; and (iv) high and low HCC. Several HCC-related VOCs that could be associated with biochemical cellular processes were identified through GC-MS analysis. CONCLUSION: The presented results constitute a proof-of-concept for the in-vitro prediction of the metastatic potential of HCC from VOC fingerprints using nanotechnology. Further studies on a larger number of more diverse cell cultures are needed to evaluate the robustness of the VOC patterns. These findings could benefit the development of a fast and potentially inexpensive laboratory test for subclinical HCC metastasis.
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Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/patología , Cromatografía de Gases y Espectrometría de Masas/métodos , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patología , Compuestos Orgánicos Volátiles/análisis , Animales , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Estudios de Casos y Controles , Línea Celular Tumoral , Análisis Discriminante , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Modelos Teóricos , Nanotecnología/instrumentación , Metástasis de la Neoplasia , Reconocimiento de Normas Patrones Automatizadas , Reproducibilidad de los Resultados , Estadísticas no Paramétricas , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
Metformin appears to be involved in altering energy expenditure and thermogenesis, and could affect hypothalamic feeding circuits. However, it is not clear whether metformin is able to cross the blood-brain barrier (BBB) to reach the hypothalamus and exert a direct effect on the central nervous system. Here we show the presence of metformin in cerebrospinal fluid (CSF) of diabetic rats administered orally with metformin which was confirmed by detecting the concentration of metformin with liquid chromatography-tandem mass spectrometry. Food intake of diabetic rats treated with metformin was reduced, and glucose homeostasis was gained. Expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related protein (AgRP) decreased in the hypothalamus of metformin-treated diabetic rats, though anorexigenic peptides pro-opiomelanocortin (POMC) did not change significantly. The phosphorylation of signal transducer and activator of transcription 3 (STAT3) was increased but phosphorylated AMP-activated kinase (AMPK) was similar in the hypothalamus of metformin-treated diabetic rats. Our findings suggest that metformin may cross BBB and play a central mechanism on regulation of food intake in the hypothalamus. The anorexic effect of metformin may be mediated by inhibition of NPY and AgRP gene expression through the STAT3 signaling pathway.
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Diabetes Mellitus Experimental , Ingestión de Alimentos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hipoglucemiantes/administración & dosificación , Hipotálamo/efectos de los fármacos , Metformina/administración & dosificación , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Administración Oral , Proteína Relacionada con Agouti/genética , Proteína Relacionada con Agouti/metabolismo , Animales , Glucemia/efectos de los fármacos , Cromatografía Liquida , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/fisiopatología , Modelos Animales de Enfermedad , Hipoglucemiantes/sangre , Hipoglucemiantes/líquido cefalorraquídeo , Hipotálamo/metabolismo , Masculino , Metformina/sangre , Metformina/líquido cefalorraquídeo , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Fosforilación/efectos de los fármacos , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Espectrometría de Masas en TándemRESUMEN
Alterations in the DNA methylation status particularly in CpG islands are involved in the initiation and progression of many types of human cancer. A number of DNA methylation alterations have been reported in hepatocellular carcinoma (HCC). However, a systematic analysis is required to elucidate the relationship between differential DNA methylation status and the characteristics and progression of HCC. In the present study, a global analysis of DNA methylation using a human CpG-island 12K array was performed on a number of HCC cell lines of different origin and metastatic potential. Based on a standard methylation alteration ratio of ≥2 or ≤0.5, 58 CpG island sites and 66 tumor-related genes upstream, downstream or within were identified. This study showed a series of CpG island methylation alterations in the HCC cell lines. The expression of various oncogenes, tumor suppressor genes and other key genes were up- or downregulated, respectively, resulting in CpG island hypomethylation or hypermethylation accordingly. To conclude, a foundation has been provided for screening CpG island methylation profiles as HCC biological markers.
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OBJECTIVE: Hepatic stellate cells (HSC) in hepatocellular carcinoma (HCC) transdifferentiate into extracellular matrix-producing myofibroblasts. Activated HSC can promote invasion and metastasis of HCC. To understand the differences of HSC in normal liver and HCC, we compared the gene expression patterns in HCC cell induction-activated and culture-activated rat HSC. METHODS: HSC were isolated by density centrifugation and exposed to conditioned medium from rat HCC cell line C5F. Expression of 22 012 genes in quiescent HSC, culture-activated HSC and HCC induction-activated HSC was analyzed by cDNA microarray and confirmed by real-time RT-PCR and Western blot. RESULTS: 1672 genes were differentially expressed in culture-activated HSC, including proinflammatory factors, cell adhesion molecules, cell surface receptors, signaling transduction molecules and immune factors. 711 genes were differentially expressed in HCC induction-activated HSC. Some of them were identical to those in culture-activated HSC. HCC Induction-activated HSC showed specific gene expression patterns, including Raf1, Rac2, Adam17, Wnt6, MMP-9 and TNF, suggesting that HCC cells can specifically induce HSC activation. CONCLUSION: The gene expression patterns in HCC induction-activated HSC are different from those in culture-activated HSC. HCC induction-activated HSC may play a major role in the invasion and metastasis of HCC. In vivo activation should be considered as the standard for the study of HSC biology. HCC induction-activated HSC should be considered as the standard for HSC biology studies.
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Carcinoma Hepatocelular/metabolismo , Perfilación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Neoplasias Hepáticas/metabolismo , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Células Cultivadas , Regulación Neoplásica de la Expresión Génica , Células Estrelladas Hepáticas/patología , Neoplasias Hepáticas/patología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas F344RESUMEN
OBJECTIVE: To analyze the expression of genes in the Slit/Robo signaling pathway, and the methylation status of their promoters in hepatocellular carcinoma (HCC) cell lines. METHODS: Genomic DNA and total RNA were isolated from 9 HCC cell lines of different metastatic ability (Hep3B, HepG2, PLC/PRF/5, SMMC-7721, BEL-7402, MHCC97-H, MHCC97-L, LM3, LM6) and a control cell line L-02. The expression profiles of Slit1, Slit2, Slit3, Robo1, and Robo3 were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The methylation status of the promoters was detected by methylation specific polymerase chain reaction (MSP). RESULTS: The promoters of Slit1, Slit2 and Slit3 genes were almost methylated in all the HCC cell lines. The Slit1 and Slit3 RNAs were not detected in most of the cell lines. Furthermore, the mRNA Slit2 was decreased gradually as the metastatic potential of the cell lines increased. As the candidate ligand of the Slit2 gene, Robo1 was frequently methylated in HCC cell lines whereas its mRNA was detected in all of these cells except SMMC-7721, BEL-7402 and L-02. Robo3 was unmethylated in HCC cell lines while its mRNA was not detected in these HCC cell lines. CONCLUSION: The hypermethylation status of Slit/Robo signaling pathway related genes is a universal event in the HCC. The hypermethylation status of Slit1, Slit2, Slit3 genes associated with the loss of expression or reduced expression. Those data suggest that Slit/Robo pathway may play a significant role in the progress or metastasis of HCC.
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Metilación de ADN , Péptidos y Proteínas de Señalización Intercelular/genética , Neoplasias Hepáticas/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Receptores Inmunológicos/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Islas de CpG/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Receptores de Superficie Celular , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
PURPOSE: To investigate the differential proteins and related molecular mechanism of CDA-II (cell differentiation agent-II) induced therapy on a human hepatocellular carcinoma model in nude mice with high metastatic potential (LCI-D20). METHODS: After tumors were transplanted 11 days, mice were intraperitoneally injected with CDA-II (1,800 mg/kg) for 20 days continuously. The tumor growth-inhibitory efficiency in CDA-II treated groups was calculated. Proteins extracted from tumor tissue were separated by two-dimensional gel electrophoresis (2DE) and the differential proteins were identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Western blotting (WB) was performed to verify the expression of certain candidate proteins. Reverse transcription-polymerase chain reaction (RT-PCR) was engaged to study the molecular mechanism of the therapy. RESULTS: CDA-II suppressed the growth and metastasis of tumor. The tumor growth-inhibitory efficiency was 41.8%. In total, 27 differentially expressed proteins were identified, including HSP27, UGDH, CK8, Hsp60, ENOA and AnxA5, with functions involved in oncogene expression and/or cell differentiation. In addition, apparent alternations of HSP60 and beta-actin expression levels and their different posttranslational modifications (PTMs) were investigated. RT-PCR analysis confirmed that the cancer related genes c-myc, N-ras and MMP-9 were significantly down-regulated. CONCLUSION: Our results demonstrate that CDA-II presence can change the proteome profiling and favors of the tumor suppression in LCI-D20 cell differentiation. Our results also suggest that the dynamic PTM of HSP60 expression levels could be used to predict HCC and might be a promising and useful biomarker to prognosticate CDA-II therapeutic efficacy.
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Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Metástasis de la Neoplasia/patología , Péptidos/uso terapéutico , Fenilacetatos/uso terapéutico , Proteómica , Animales , División Celular , Línea Celular Tumoral , Cartilla de ADN , Genes myc , Genes ras , Humanos , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Desnudos , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trasplante HeterólogoRESUMEN
OBJECTIVE: To examine how the thymidine phosphorylase (TP) gene expression is upregulated by interferon-alpha (IFN-alpha) in human hepatocellular carcinoma SMMC-7721 cells. METHODS: TP mRNA levels were determined by RT-PCR. Whether the JAK-STAT cascade mediates IFN-alpha-induced TP mRNA expression was studied by pretreatment with Janus Kinase (JAK) inhibitor, AG-490. Effects of IFN-alpha on TP mRNA stability were detected with additional actinomycin D. RESULTS: The expression of TP mRNA was induced by IFN-alpha in a dose- and time-dependent manner in SMMC-7721 (human hepatocellular carcinoma) cells. TP mRNA levels rose at 8 h, reached the peak value at 12 h, and remained at a high level up to 72 h in SMMC-7721 cells treated with IFN-alpha 10000 U/ml. IFN-alpha at a dose of 5000 or 10000 U/ml up-regulated TP expression about 3 fold compared with that of non-treated cells (P < 0.05). Induction of TP mRNA expression by IFN-alpha was significantly inhibited in SMMC-7721 cells by pretreatment with AG-490, in comparison with that treated with IFN-alpha alone. Pretreatment of SMMC-7721 cells with IFN-alpha 10000 U/ml for 24 h caused a substantial stabilization of TP mRNA, with a half-live of 35.8 h, compared with 8.5 hr in the control SMMC-7721 cells. CONCLUSION: IFN-alpha at certain doses upregulates TP mRNA expression via both JAK-STAT transcriptional activation and post-transcriptional mRNA stabilization in human hepatocellular carcinoma SMMC-7721 cells.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Interferón-alfa/farmacología , Neoplasias Hepáticas/enzimología , Timidina Fosforilasa/biosíntesis , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Interferón-alfa/administración & dosificación , Quinasas Janus/metabolismo , Neoplasias Hepáticas/patología , ARN Mensajero/metabolismo , Factor de Transcripción STAT1/metabolismo , Timidina Fosforilasa/genética , Activación Transcripcional/efectos de los fármacos , Tirfostinos/farmacologíaRESUMEN
OBJECTIVES: To study the biological function and its possible underlying mechanism of peroxiredoxin II (PrxII) in liver cancer cell line Hep3B. METHODS: Two pairs of double-stranded small interfering RNA (siRNA) targeted on PrxII gene were transfected into Hep3B cells using LipofectamineTM 2000. After confirming the inhibited effects of these siRNAs through Quant SYBR Green polymerase chain reaction and Western blot, the biological characters of Hep3B cell were analyzed by flow cytometry analysis, MTT and colony formation assays. Furthermore, dichlorodihydrofluorescein diacetate (DCFH-DA) and thiobarbituric acid (TBA) assays, for measuring the products of oxidative reaction, such as the reactive oxygen species (ROS) and malondialdehyde (MDA), were applied to explore whether the antioxidant mechanism was involved in the effects of PrxII functioning on Hep3B cell. RESULTS: The two pairs of siRNA significantly inhibited PrxII mRNA and protein expression. Compared to the mock and blank control groups, the two PrxII-silent groups showed decreased rates of cell growth and clone formation and increased rates of cell apoptosis. The numbers of the formed colonies were 42.0+/-2.8 and 40.5+/-0.7 respectively in the two PrxII-silent groups, while they were 121.5+/-2.1 and 130.0+/-1.4 in the mock and blank control groups (P less than 0.05). The levels of endogenous ROS and MDA were significantly higher in the two PrxII-silent groups than those in the mock and blank control groups (P less than 0.05). CONCLUSION: PrxII might play an important role in the hepatocarcinogenesis, possibly through an antioxidant function which may provide a favorable microenvironment for cancer cell survival and progression.
Asunto(s)
Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Peroxirredoxinas/genética , ARN Interferente Pequeño , Línea Celular Tumoral , Humanos , Neoplasias Hepáticas/patología , Estrés Oxidativo , Especies Reactivas de Oxígeno , Transducción de Señal , TransfecciónRESUMEN
OBJECTIVE: To study survivin expression in human hepatoma cells and the effects of survivin siRNA on the malignant phenotypes of human hepatocellular cell line HCCLM6. METHODS: Four hepatocellular carcinoma (HCC) cell lines were used. Semi-quantitative RT-PCR and Western blot were used to measure and compare their survivin expressions. The siRNA expression vector pshRNA-survivin targeting the mRNA of survivin and vector pGPU6/GFP/Neo-NC (as a control) were constructed, and then transfected into HCCLM6 cells. FQ-PCR was used to quantify the mRNA levels of survivin. The malignant phenotypes of transfected HCCLM6 cells, including invasive activities and adhesive capabilities, were analyzed. RESULTS: Survivin expression gradually increased with the increase of the invasion and metastasis behaviors of the four HCC cell lines (P<0.05). The expression of survivin was highest in cell line HCCLM6. Survivin mRNA level was decreased by 93.500%+/-3.117% after the pshRNA-survivin transfection. The cell adhesion rates significantly decreased in the cells transfected with pshRNA-survivin (cell adhesion rates were 11.403%+/-1.256% vs 32.545%+/-1.367%, t=20.732, P<0.01). The migrating number of HCCLM6 cells (13.5+/-0.9) transfected with pshRNA-survivin was also significantly decreased (t=14.5, P<0.01) as compared with the control group (32.6+/-1.4). CONCLUSION: The expression of survivin in HCC might have a close relationship to their invasion and metastasis properties. Sequence-specific shRNA can significantly reduce the survivin expression in the HCCLM6 cell line. Suppression of survivin expression in HCCLM6 cells transfected with pshRNA-survivin can reduce their invasive and adhesive capabilities.
Asunto(s)
Neoplasias Hepáticas/patología , Proteínas Asociadas a Microtúbulos/genética , ARN Interferente Pequeño , Línea Celular Tumoral , Humanos , Proteínas Inhibidoras de la Apoptosis , Neoplasias Hepáticas/genética , SurvivinRESUMEN
OBJECTIVE: We previously showed that introduction of a normal, neomycin-tagged human chromosome 8 reduced the metastatic capacity of C5F rat liver cancer cell line, which had high metastatic potential without affecting tumorigenicity, suggesting the presence of one or more metastasis suppressor genes encoded on human chromosome 8. We proceeded to define further the region harboring the metastasis suppressor gene(s) and to determine the random loss of human chromosome 8 by PCR amplification of sequence tag site (STS) markers. METHODS: The national Center for Biotechnology Information (NCBI) databases were used as references of the relative genetic distances of the STS markers. C5F genomic DNA and A9/neo8 genomic DNA were used as negative and positive controls for chromosome 8 amplification, respectively. Genomic DNA was isolated and quantified from cultured hybrid clones (A9/C5F-1 and A9/C5F-2 microcell hybrid clones served as metastasis-unsuppressed groups; A9/C5F-4, A9/C5F-8 and A9/C5F-10 microcell hybrid clones served as metastasis suppressed groups). STS-PCR products were separated by electrophoresis through 2% agarose gel. RESULTS: Metastasis-suppressed microcell hybrid clones (A9/C5F-4, A9/C5F-8 and A9/C5F-10) conserved STS markers between D8S542 --> D8S1973 (8p21.1-23.1). In contrast, metastasis-unsuppressed clones (A9/C5F-1 and A9/C5F-2) lacked several markers in this region. In attempts to refine the region retained in the microcell suppressed clones, more densely spaced STS markers in the human chromosome 8p21.1-23.1 were used. We found that the metastasis-suppressed clones retained 18cM region between D8S542 and D8S1973 (8P21.1-23.1), where as the metastasis-unsuppressed clones lacked the region. CONCLUSION: Our results suggest that a metastasis suppressor gene is located within the interval between D8S542 and D8S1973 on human chromosome 8p21.1-23.1.