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BACKGROUND: The new TomoDirect (TD) modality offers a nonrotational option with discrete beam angles. We aim to compare dosimetric parameters of TD, helical tomotherapy (HT), volumetric-modulated arc therapy (VMAT), and fixed-field intensity-modulated radiotherapy (ff-IMRT) for upper thoracic esophageal carcinoma (EC). METHODS: Twenty patients with cT2-4N0-1M0 upper thoracic esophageal squamous cell carcinoma (ESCC) were enrolled. Four plans were generated using the same dose objectives for each patient: TD, HT, VMAT with a single arc, and ff-IMRT with 5 fields (5F). The prescribed doses were used to deliver 50.4 Gy/28F to the planning target volume (PTV50.4) and then provided a 9 Gy/5F boost to PTV59.4. Dose-volume histogram (DVH) statistics, dose uniformity, and dose homogeneity were analyzed to compare treatment plans. RESULTS: For PTV59.4, the D2, D98, Dmean, and V100% values in HT were significantly lower than other plans (all p < 0.05), and those in TD were significantly lower than VMAT and ff-IMRT (all p < 0.05). However, there was no significant difference in the D2 and Dmean values between VMAT and ff-IMRT techniques (p > 0.05). The homogeneity index (HI) differed significantly for the 4 techniques of TD, HT, VMAT, and ff-IMRT (0.03 ± 0.01, 0.02 ± 0.01, 0.06 ± 0.02, and 0.05 ± 0.01, respectively; p â < 0.001). The HI for TD was similar to HT (pâ¯=â¯0.166), and had statistically significant improvement compared to VMAT (p < 0.001) and ff-IMRT (pâ¯=â¯0.003). In comparison with the 4 conformity indices (CIs), there was no significant difference (p > 0.05). For PTV50.4, the D2 and Dmean values in HT were significantly lower than other plans (all p < 0.05), and those in TD were significantly lower than VMAT and ff-IMRT (all p < 0.05). However, there was no significant difference in the D2 and Dmean values between VMAT and ff-IMRT techniques (p > 0.05). No D98 and V100% parameters differed significantly among the 4 treatment types (p > 0.05). HT plans were provided for statistically significant improvement in HI (0.03 ± 0.01) compared to TD plans (0.05 ± 0.01, pâ¯=â¯0.003), VMAT (0.08 ± 0.03, p < 0.001), ff-IMRT (0.08 ± 0.01, p < 0.001). The HI revealed that TD was superior to VMAT and ff-IMRT (p < 0.05). The CI differed significantly for the 4 techniques of TD, HT, VMAT, and ff-IMRT (0.59 ± 0.10, 0.69 ± 0.11, 0.64 ± 0.09, and 0.64 ± 0.11, respectively; pâ = 0.035). The best CI was yielded by HT. We found no significant difference for the V5, V10, V15, V30, and the mean lung dose (MLD) among the 4 techniques (all p > 0.05). However, the V20 differed significantly among TD, HT, VMAT, and ff-IMRT (21.50 ± 7.20%, 19.50 ± 5.55%, 17.65 ± 5.45%, and 16.35 ± 5.70%, respectively; pâ = 0.047). Average V20 for the lungs was significantly improved by the TD plans compared to VMAT (pâ¯=â¯0.047), and ff-IMRT (pâ¯=â¯0.008). The V5 value of the lung in TD was 49.30 ± 13.01%, lower than other plans, but there was no significant difference (p > 0.05). The D1 of the spinal cord showed no significant difference among the 4 techniques (pâ¯=â¯0.056). CONCLUSIONS: All techniques are able to provide a homogeneous and highly conformal dose distribution. The TD technique is a good option for treating upper thoracic EC involvement. It could achieve optimal low dose to the lungs and spinal cord with acceptable PTV coverage. HT is a good option as it could achieve quality dose conformality and uniformity, while TD generated superior conformality.
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Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/radioterapia , Radioterapia de Intensidad Modulada/métodos , Neoplasias Torácicas/radioterapia , Anciano , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas de Esófago/secundario , Estudios de Factibilidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Radiometría , Dosificación Radioterapéutica , Neoplasias Torácicas/secundarioRESUMEN
BACKGROUND: Non-small-cell lung cancer (NSCLC) constitutes the leading cause of cancer death in humans. Previous studies revealed the essential role of the protein arginine methyltransferase 7 (PRMT7) in promoting metastasis in breast cancer. However, its function and potential mechanism in NSCLC remain unclear. MATERIALS AND METHODS: The gene expression of PRMT7 between lung cancer tissues and normal tissues was studied with online database (http://medicalgenome.kribb.re.kr/GENT/). NSCLC cell lines with specific gene overexpression were constructed with lentivirus transduction. Matrigel invasion and colony formation assays were performed to evaluate the invasion and colony formation abilities. Co-immunoprecipitation coupled with mass spectrometry analysis was performed to explore the potential interaction proteins of PRMT7. Bioinformatic analysis was performed with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. RESULTS: Online analysis of gene expression patterns revealed the relatively high expression of PRMT7 in lung cancer tissues. PRMT7 overexpression was able to promote the invasion and colony formation of A549 and SPC-A1 cells. A total of 19 in-common proteins shared by both NSCLC cell lines were identified to be interacting with PRMT7 and found to participate in a wide variety of pathways and protein-protein interactions according to bioinformatic analysis. Among them, HSPA5 and EEF2 were further investigated for their essential roles in PRMT7-promoted NSCLC cell invasion. CONCLUSION: Our results suggested PRMT7 overexpression was able to promote metastasis in NSCLC possibly through the interaction with HSPA5 and EEF2, which provides the potential mechanism of oncogenesis in lung cancer.
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BACKGROUND: This study aimed to select piperacillin/tazobactam (TZP) infusion mode guided by Sequential Organ Failure Assessment (SOFA) score in cancer patients with hospital-acquired pneumonia (HAP) postoperation. PATIENTS AND METHODS: A total of 120 cancer patients with postoperative HAP were divided into two groups: improved administration group (L group) and conventional treatment group (Con group). The Con group received traditional infusion of TZP and the L group received it as prolonged infusion. Blood drug concentration was detected at different time points. Based on the SOFA cut-off value of 9, the patients were regrouped into M (mild) and S (severe) groups. RESULTS: Percent time that the free drug concentrations remain above the minimum inhibitory concentration (%fT>MIC) was longer than 5 h in L group, but <4 h in Con group. Administration method (p=0.033, OX value 2.796, B value 1.028, 95% CI: 0.855-8.934) and SOFA score (p=0.038, OX value 0.080, B value -2.522, 95% CI: 0.007-0.874) were independent predictors of patient survival. In the S group, compared to conventional treatment, prolonged infusion mode resulted in shorter days of antibiotic use and shorter ventilator time, and achieved longer survival, better clinical efficacy, and lower 28-day mortality rate. CONCLUSION: For cancer patients with SOFA score ≥9, prolonged infusion of TZP could benefit the patients and obtain better clinical efficacy.
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In humans, males compared to females have increased visceral adipose tissue which contributes to their increased risk of early death. Mice display analogous sexual diamorphism whereby females are protected from weight gain when fed a high fat diet compared to males. A role has recently been reported for ß2-glycoprotein I, an abundant plasma protein, in healthy leanness in humans. In this study we investigated the role of ß2-glycoprotein I in fat metabolism in male and female mice fed a normal chow or high fat diet. We have made a number of novel insights into factors contributing to sexual diamorphism in obesity. Female wild type mice are protected from obesity when fed a high fat diet due to down regulation of lipogenesis in the visceral adipose tissues. This down regulation is due to ß2-glycoprotein I as female mice deficient in this protein have increased levels of lipogenesis enzymes in their visceral adipose tissues with an accompanying increase in weight compared to female wild type controls. Understanding female specific regulators of obesity may lead to sex specific anti-obesity therapies to address this major health problem.
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A subset of regulatory B cells in humans has been identified as B10 cell which has the function of secreting interleukin-10. We evaluated the significance of B10 cell in patients with thymoma complicated with myasthenia gravis. In this study, 156 patients diagnosed with thymoma were enrolled, FCM was used to detected the percentage of Breg/CD19+B cells and CD19+B cells/PBMC, ELISA to evaluate the serum concentration of the relevant immunological markers; purified CD19+B cells in tissues by MACS; gene and protein expressions of CD19 and IL-10 by Real-time PCR and Western-Blot; double immunofluorescence staining to detect the distribution of CD19 and IL-10 in thymus tissues. Thymoma patients without MG mainly display the types A and AB of thymoma, whereas the thymoma patients with MG mainly display type B (B1, B2 and B3) thymoma; AChR-Ab in Tm + MG group was the highest, with the progress of the disease, the percentage of Breg/CD19+B cells increased and B10/CD19+B cells decreased (p < 0.05); ROC curve showed that B10 had the greatest significance for the clinical directivity of Tm+MG and cut-off point = 0.55%; in accordance with the Con, Tm and Tm+MG group, the content of CD19+IL-10+B10 cells increased gradually (p < 0.05); meanwhile, the gene and protein expression levels of CD19 and IL-10 gradually increased in the same way. It is concluded that with the progress of thymoma, the infiltration of Breg in tumour tissue increases; however, as the severity of MG increases, the function of Breg (B10 cell) in peripheral blood decreases and the cut-off point is 0.55%.
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Malignant fibrous histiocytoma (MFH) is an aggressive soft tissue sarcoma known to occur in various organs. Primary MFH arising in the lung is quite rare. Herein we report a case of a 61-year-old male with primary pulmonary MFH and explore the underlying molecular mechanisms by next-generation sequencing (NGS). Five gene mutations in TSC2, ARID1B, CDK8, KDM5C and CASP8 were detected, and the mTOR inhibitor might be an effective treatment for this patient. In addition, we reviewed the scientific literature of approximately 23 primary pulmonary MFH case reports since 1990 and summarized the clinical features and prognosis of this rare pulmonary malignant tumor.
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The immune responses of males and females to bacterial infections display differences. The mechanisms that underlie this sexual dimorphism are multifactorial. Lipopolysaccharide (LPS) contributes to the pathogenesis of endotoxaemia. We have previously demonstrated that the plasma protein beta-2 glycoprotein-1 (ß2GPI) reduces LPS-induced inflammation in male mice. In the present study using a more robust infection model of septicaemia the role of ß2GPI is examined in both male and female wild type (WT) and ß2GPI deficient (ß2GPI-/-) mice challenged with Escherichia coli (E. coli) intravenously. ß2GPI deficiency led to an increase of E. coli colony forming units (CFU) in the circulation of both male and female mice. In male ß2GPI-/- mice this was associated with a worse clinical severity score. This difference was not observed between female ß2GPI-/- and female WT mice. Male WT mice had decreased levels of total and increased levels of free thiol ß2GPI following administration of LPS or E. coli. This pattern of sexual dimorphic response was also observed in our cohort of humans with sepsis. These findings support a role for ß2GPI in modulating the sex-specific susceptibility to gram-negative septicaemia.
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Endotoxemia/genética , Endotoxemia/inmunología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , beta 2 Glicoproteína I/genética , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endotoxemia/sangre , Endotoxemia/diagnóstico , Infecciones por Escherichia coli/sangre , Infecciones por Escherichia coli/diagnóstico , Femenino , Predisposición Genética a la Enfermedad , Humanos , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Noqueados , Especificidad de Órganos , Sepsis/genética , Sepsis/inmunología , Índice de Severidad de la Enfermedad , Factores Sexuales , beta 2 Glicoproteína I/sangreRESUMEN
Purpose Emerging evidence indicates that circulating microRNAs (miRs) might act as noninvasive biomarkers for cancer diagnosis and prognosis. We examined the expression pattern and clinical significance of plasma miR-9 in patients with esophageal squamous cell carcinoma (ESCC). Methods Venous blood samples (6 mL) were collected from 131 patients with ESCC and 131 healthy controls, and the plasma miR-9 concentration was detected by reverse transcription polymerase chain reaction. The association of plasma miR-9 expression with clinicopathologic factors and survival of patients with ESCC was evaluated. Receiver operating characteristic (ROC) curve analysis was applied to evaluate the clinical value of plasma miR-9 for ESCC diagnosis. Results The plasma miR-9 expression levels in patients with ESCC were significantly upregulated compared with normal controls. High plasma miR-9 concentrations were significantly correlated with poor tumor differentiation, large tumor size, deep local invasion, lymph node metastasis, advanced clinical stage, and poor survival. ROC curve analysis showed that the plasma miR-9 concentration could efficiently distinguish patients with ESCC from healthy controls. Multivariate survival analysis confirmed plasma miR-9 as an independent prognostic factor for ESCC. Conclusions Plasma miR-9 expression was upregulated in ESCC and might act as a novel diagnostic and prognostic biomarker.
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Carcinoma de Células Escamosas/sangre , Neoplasias Esofágicas/sangre , MicroARNs/sangre , Biomarcadores de Tumor/sangre , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/mortalidad , Supervivencia sin Enfermedad , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/mortalidad , Carcinoma de Células Escamosas de Esófago , Femenino , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Curva ROCRESUMEN
BACKGROUND: Esmolol is a highly selective beta 1 receptor blocker with various effects such as slowing heart rate, lowering blood pressure and reducing myocardial oxygen consumption. However, few studies have reported the use of beta blockers in sepsis with multiple organ dysfunctions. This study aimed to investigate the effects of esmolol on reducing apoptosis and inflammation in early sepsis rats with abdominal infection. METHODS: Rats were randomly divided into sham operation group, sepsis group, antibiotic group, Esmolol + antibiotic group with low, median and high dose Esmolol (L group, M group and H group). Values between two or more groups were compared by independent t-tests. RESULTS: In the liver and kidney, we found inflammatory infiltration in sepsis group while pathological aspects reduced in L, M and H groups. Bcl-2 mRNA and protein levels increased while Bax mRNA and protein levels decreased in the liver and kidney of L, M and H groups. Serum IL-6, HMGB-1 and TNF-α levels decreased but IL-10 level increased in L, M and H groups, compared to sepsis group. Compared to sepsis and antibiotic groups, the levels of myocardial enzymes were lower in L, M and H groups. CONCLUSION: The administration of esmolol in early sepsis may reduce inflammation, inhibit apoptosis and protect key organs.
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Antagonistas de Receptores Adrenérgicos beta 1/uso terapéutico , Propanolaminas/uso terapéutico , Sepsis/tratamiento farmacológico , Sepsis/patología , Infección de la Herida Quirúrgica/patología , Abdomen/cirugía , Animales , Antibacterianos/uso terapéutico , Apoptosis , Modelos Animales de Enfermedad , Proteína HMGB1/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Sepsis/metabolismo , Infección de la Herida Quirúrgica/complicaciones , Infección de la Herida Quirúrgica/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Targeting CD47 efficiently enhances macrophage phagocytosis in both physiological and pathological conditions. Anti-CD47 antibodies have been shown to inhibit the progression of several types of cancer. However, the mechanism of anti-CD47 monoclonal antibody (mAb) treatment remains controversial. In this study, we confirmed that CD47 protein is highly expressed in ovarian cancer, and is correlated with poor clinical characteristics and prognosis. CD47 knockdown in the ovarian cancer cell line, SK-OV-3, promoted phagocytosis by macrophages in vitro and inhibited tumor growth in vivo. These data combined suggest that CD47 inhibition is a potential strategy for cancer treatment. Using an anti-CD47 mAb, we found that CD47 inhibition in both SK-OV-3 cells and primary cancer cells was able to recapitulate our knockdown results and led to an increase in the number of infiltrating macrophages. In addition, the CD133+ tumor initiating cells expressed a high level of CD47, and anti-CD47 mAb treatment was able to trigger the phagocytosis of this cell population. In conclusion, our results indicate that CD47 inhibits macrophage phagocytosis of ovarian cancer cells, and down-regulation of CD47 or inhibiting CD47 by mAb was able to reverse the negative effect. Thus, CD47 antibody therapy may be a promising strategy to treat ovarian cancer.
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Antígeno CD47/metabolismo , Macrófagos/metabolismo , Neoplasias Ováricas/patología , Escape del Tumor/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Biomarcadores de Tumor/análisis , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Humanos , Inmunoterapia/métodos , Estimación de Kaplan-Meier , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/mortalidad , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Identification of biomarkers for predicting radiosensitivity would be useful for administering individualized radiotherapy (RT) to patients with esophageal cancer. The aim of the present study was to evaluate the association between cyclooxygenase-2 (COX-2), X-ray repair cross complementing group 1 (XRCC1), ras association domain family 1 (RASSF1) protein expression, clinicopathological characteristics, radiosensitivity and survival rate in 76 patients with esophageal squamous cell carcinoma (ESCC) who were treated with RT. Positive expression of COX-2, XRCC1 and RASSF1 was identified by immunohistochemistry in 81.6, 52.6 and 59.2% of ESCC cases, respectively. Negative COX-2 expression was associated with tumor (T) stage, node (N) stage, clinical stage and complete response (P<0.05), but not with gender, age, tumor location, differentiation degree, lesion length, progression-free survival (PFS) or overall survival (OS; P>0.05). XRCC1 expression was not associated with the clinicopathological features of ESCC, response to RT, PFS or OS. Positive RASSF1 expression was associated with the clinical stage, response to RT, PFS and OS (P<0.05), but not with gender, age, tumor location, T stage, N stage, differentiation degree or the lesion length (P>0.05). In the subgroup analysis, RASSF1 positive/XRCC1 negative expression was correlated with a longer median OS and PFS (P<0.05). Multivariate analyses revealed that the tumor response and RASSF1 expression were significant prognostic factors. Therefore, positive RASSF1 expression is associated with ESCC RT sensitivity, and may be a useful independent prognostic factor for ESCC.
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Lipopolysaccharide (LPS) is a major component of the outer wall of gram negative bacteria. In high doses LPS contributes to the inflammation in gram negative sepsis, and in low doses contributes to the low grade inflammation characteristic of the metabolic syndrome. We wanted to assess the role of beta2-glycoprotein I (ß2GPI) a highly conserved plasma protein and its different biochemical forms in a mouse model of LPS systemic inflammation. Normal and ß2GPI deficient mice were administered LPS through their veins and assessed for a range of inflammation markers in their blood and liver. Different biochemical forms of ß2GPI were measured in normal mice given either saline or LPS. We show that ß2GPI has a significant role in inhibiting LPS induced inflammation. In this study we provide some evidence that ß2GPI serves a protective role in a mouse model of LPS inflammation. This resolves the controversy of previous studies which used LPS and ß2GPI in test tube based models of LPS induced activation of white cells. We also highlight the potential relevance of a newly discovered biochemical form of ß2GPI in LPS mediated inflammation and we speculate that this form has a protective role against LPS induced pathology.
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The present study examined the role of the PRDI-BF1-RIZ (PR) domain of tumor suppressor retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) as an anticancer domain and its ability to induce apoptosis in esophageal squamous cell carcinoma (ESCC) cells. The TE13 ESCC cell line was transfected with pcDNA3.1(+) eukaryotic expression vectors bearing the open reading frames of either the human RIZ1 gene or the PR domain, and the mRNA and protein expression levels were then detected using quantitative reverse transcription polymerase chain reaction and western blotting, respectively. The rate of apoptosis was determined by flow cytometry and the cell invasion ability was determined by an invasion assay. RIZ1 and the PR domain induced apoptosis and reduced the cell invasion ability (P<0.01). These findings indicate that the RIZ1 gene possesses anticancer activity in the PR domain, which may be important in inhibiting the development of ESCC.
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The present study aimed to investigate the expression and role of SET and MYND domain-containing protein 3 (SMYD3) in esophageal squamous cell carcinoma; to observe the proliferation of esophageal squamous cell carcinoma after suppression of SMYD3 expression; and to explore the effect of SMYD3 downregulation on the expression of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1). Tissues from 11 patients, including cancer and normal esophageal tissues, were obtained by surgery to observe the SMYD3 protein expression immunohistochemistry. Esophageal squamous cell carcinoma TE13 cells were transfected with four different SMYD3-shRNA plasmids, and SMYD3 mRNA expression levels were assessed to select the most efficient interfering plasmid. After SMYD3 downregulation in TE13 cells, mRNA and protein expression levels of SMYD3 and RIZ1 were determined using RT-PCR and western blotting, and cell proliferation was evaluated by the MTT method. In all 11 tissue paired samples, SMYD3 protein expression was higher in the cancer tissues (72.7%; 8/11), than that in the normal tissues (18.2%; 2/11) (Fisher's exact test, P=0.03). The mRNA expression levels of SMYD3 were significantly decreased by RNA interference (P<0.05), and plasmid SMYD3-shRNA-1242 was determined to be the most effective. Compared with the controls, transfection with the SMYD3-shRNA interfering plasmid significantly reduced the SMYD3 mRNA and protein expression levels in TE13 cells (P<0.05), whereas the expression levels of the anti-oncogene RIZ1 were increased (P<0.05). The MTT assay showed that ablation of SMYD3 expression significantly inhibited proliferation of TE13 cells (P<0.05). SMYD3 may participate in the biological activity of esophageal squamous cell carcinoma, as overexpression of SMYD3 correlates with its occurrence and its downregulation inhibits cancer cell proliferation. The shRNA efficiently downregulated SMYD3 in TE13 cells, which represents an SMYD3-interfered cell-test-model for future experiments. RNAi suppression of SMYD3 promoted the expression of RIZ1 in TE13 cells, suggesting a signal transduction pathway between SMYD3 and RIZ1. The SMYD3-RIZ1 pathway may represent a therapeutic target for esophageal squamous cell carcinoma.
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Carcinoma de Células Escamosas/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Neoplasias Esofágicas/patología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Interferencia de ARN , Transducción de SeñalRESUMEN
OBJECTIVE: PR domain is responsible for the tumor suppressing activity of RIZ1. The study aimed to construct human PR domain eukaryotic expression vectors, transfect human esophageal cancer cells (TE13), and evaluate the anticancer activity of PR domain on human esophageal cancer TE13 cells. METHODS: First, mRNA was extracted from human esophageal cancer tissue by RT-PCR, then reverse-transcribed to cDNA. After amplifying from the DNA template, PR domain was linked to T vector. Second, after extraction, PR domain was cut using enzyme and linked to pcDNA3.1(+). Then, the plasmid was transfered to Trans1-T1 phage resistant competent cells, following by extracting the ultrapure plasmid, and transfecting into TE13 cells. In the end, the protein expression of pcDNA3.1(+)/PR domain in TE13 was detected by Western blot, and the apoptosis of TE13 by technique of flow cytometry. RESULTS: More than 5,000 bp purposed band of pcDNA3.1(+)/PR domain plasmid was found by agarose gel electrophoresis. After transfection, the PR domain (molecular weight of about 28 Da) was found only in 3, 4 and 5 groups by Western blot. Flow cytometry assay showed apoptosis in experimental group was significantly more than that in the control group (P<0.05). CONCLUSIONS: The PR domain eukaryotic expression vector was constructed successfully. The protein of the PR domain could be expressed in esophageal cancer TE13 cells firmly after transfection, and a single PR domain could promote apoptosis of TE13 cells.
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The tumor suppressor protein retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) is downregulated in several types of cancer, including esophageal squamous cell carcinoma (ESCC). The present study used two in vitro methods to re-express RIZ1 in the human ESCC TE13 cell line in order to induce apoptosis. RIZ1 was re-expressed in the TE13 cells by reintroducing the gene through transfection or by removal of transcriptional repression through treatment with a DNA methyltransferase (DNMT) inhibitor. To reintroduce the gene, the open reading frame of the RIZ1 gene was inserted into the eukaryotic expression pcDNA3.1(+) vector and pcDNA3.1(+)/RIZ1 was purified and transfected into the TE13 ESCC cells. Removing transcriptional repression involved treating the TE13 cells with 5-aza-2'-deoxycytidine (5-aza-CdR), a DNMT inhibitor. RIZ1 mRNA and protein expression were determined by quantitative polymerase chain reaction (qPCR) and western blotting. The rate of apoptosis of the cells was determined by flow cytometry. A recombinant eukaryotic human RIZ1 expression plasmid, pcDNA3.1(+)/RIZ1, was constructed and confirmed by sequencing. RIZ1 mRNA and protein expression increased in pcDNA3.1(+)/RIZ1 stably transfected cells. Treatment with 5-aza-CdR for 48 and 72 h resulted in increased RIZ1 protein expression and increased the rate of apoptosis in the TE13 cells (P<0.01). In conclusion, transfection of the TE13 cells with the eukaryotic pcDNA3.1(+)/RIZ1 expression vector and reversal of transcriptional repression of RIZ1 using 5-aza-CdR demonstrate that it is possible to re-express RIZ1 in TE13 cells. Furthermore, the re-expression of RIZ1 led to an increased rate of apoptosis and this method may provide new therapeutic possibilities.
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AIM: To investigate the effect of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) upregulation in gene expression profile and oncogenicity of human esophageal squamous cell carcinoma (ESCC) cell line TE13. METHODS: TE13 cells were transfected with pcDNA3.1(+)/RIZ1 and pcDNA3.1(+). Changes in gene expression profile were screened and the microarray results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Nude mice were inoculated with TE13 cells to establish ESCC xenografts. After two weeks, the inoculated mice were randomly divided into three groups. Tumors were injected with normal saline, transfection reagent pcDNA3.1(+) and transfection reagent pcDNA3.1(+)/RIZ1, respectively. Tumor development was quantified, and changes in gene expression of RIZ1 transfected tumors were detected by RT-PCR and Western blotting. RESULTS: DNA microarray data showed that RIZ1 transfection induced widespread changes in gene expression profile of cell line TE13, with 960 genes upregulated and 1163 downregulated. Treatment of tumor xenografts with RIZ1 recombinant plasmid significantly inhibited tumor growth, decreased tumor size, and increased expression of RIZ1 mRNA compared to control groups. The changes in gene expression profile were also observed in vivo after RIZ1 transfection. Most of the differentially expressed genes were associated with cell development, supervision of viral replication, lymphocyte costimulatory and immune system development in esophageal cells. RIZ1 gene may be involved in multiple cancer pathways, such as cytokine receptor interaction and transforming growth factor beta signaling. CONCLUSION: The development and progression of esophageal cancer are related to the inactivation of RIZ1. Virus infection may also be an important factor.
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Carcinoma de Células Escamosas/genética , Proteínas de Unión al ADN/genética , Neoplasias Esofágicas/genética , Perfilación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Proteínas Nucleares/genética , Oncogenes , Factores de Transcripción/genética , Animales , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección , Carga Tumoral , Regulación hacia ArribaRESUMEN
BACKGROUND: To study the expression of Syk (spleen tyrosine kinase) gene in human ESCC (esophageal squamous cell carcinoma). METHODS: We used immunohistochemical staining to detect Syk protein expression in esophageal carcinoma tissues and RT-PCR (semi-quantitative reverse transcription PCR), Real-time PCR (Real-time quantitative PCR) and western blotting technique to detect the expression of Syk mRNA and protein in specimens from 4 esophageal cell lines. RESULTS: Immunohistochemistry analyses showed that in the esophageal cancer tissues Syk protein was expressed inferior to the adjacent normal one, p < 0.05. Real-time PCR and Western blotting showed that low expression of the Syk gene was detected in the 4 esophageal cancer cell lines compared with positive and negative cell lines, p < 0.05. CONCLUSIONS: The low expression of the Syk gene may play an important role in tumorigenesis in esophageal cancer.