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1.
Medicine (Baltimore) ; 100(11): e24818, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33725949

RESUMO

ABSTRACT: Osteosarcoma is a malignant tumor that develops from a mesenchymal cell line and is caused by gene-environment interactions. This study aimed to explore whether TIMP2/TIMP3 polymorphisms influenced the osteosarcoma risk.The expression of the TIMP2 and TIMP3 genes in osteosarcoma histiocytes was analyzed by immunohistochemistry. In this case-control study, which includes samples from 499 patients and 500 healthy controls, 10 single-nucleotide polymorphisms (SNPs) in TIMP2 and TIMP3 were selected. Furthermore, we used the Agena MassARRAY platform for genotyping. The statistical analysis was performed using χ2 test/Fisher exact test, and logistic regression analysis.The immunohistochemistry results showed that the expression of TIMP2 is obvious higher in osteosarcoma histiocytes than in the normal histiocytes. The association study indicated that the allele of rs2277698 and rs4789936 were protective SNPs reducing the risk of osteosarcoma (odds ratios  > 1, P < .05) by the χ2 test. In the genetic model, logistic regression analyses revealed that the rs2277698 and rs4789936 were associated with decreasing the risk of osteosarcoma under the codominant model, dominant model, and log-additive model. Stratification analysis revealed that 2 SNPs (rs2277698 and rs4789936) were significantly associated with a reduced risk of osteosarcoma in allele and genetic model after stratification by gender or age (P < .05). In addition, the haplotype "Trs2277698Crs2009169Crs7342880" of TIMP2 was associated with decreasing the osteosarcoma risk. The "Ars9609634Trs11547635" of TIMP3 was associated with reducing the osteosarcoma risk.This finding shed new light on the high expression of TIMP2 polymorphisms may contribute to decreasing the osteosarcoma risk in Zhejiang populations.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Neoplasias Ósseas/genética , Osteossarcoma/genética , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Adolescente , Idoso , Alelos , Neoplasias Ósseas/etnologia , Estudos de Casos e Controles , China/etnologia , Feminino , Interação Gene-Ambiente , Predisposição Genética para Doença/etnologia , Predisposição Genética para Doença/genética , Genótipo , Haplótipos , Humanos , Imuno-Histoquímica , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Osteossarcoma/etnologia , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Adulto Jovem
2.
Molecules ; 26(3)2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33504102

RESUMO

Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.


Assuntos
Antineoplásicos/farmacologia , Peptídeos/metabolismo , Pró-Fármacos/farmacologia , Inibidor Tecidual de Metaloproteinase-3/metabolismo , gama-Glutamil Hidrolase/metabolismo , Anticorpos Monoclonais/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Compostos de Mostarda Nitrogenada/farmacologia , Proteínas Recombinantes/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
3.
Molecules ; 26(1)2020 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-33379361

RESUMO

Doxorubicin is a cytotoxic anthracycline derivative that has been used as a chemotherapeutic in many different forms of human cancer with some success. However, doxorubicin treatment has several side-effects, the most serious of which is cardiomyopathy, that can be fatal. Doxorubicin encapsulation in PEGylated liposomes (Doxil®) has been shown to increase tumour localisation and decrease cardiotoxicity. Conversely, the stability of such liposomes also leads to increased circulation times and accumulation in the skin, resulting in palmar planter erythrodysesthesia, while also limiting release of the drug at the tumour site. Specific targeting of such liposomes to tumour cells has been attempted using various receptor-specific peptides and antibodies. However, targeting a single epitope limits the likely number of tumour targets and increases the risk of tumour resistance through mutation. In this report, Doxil® was coupled to peptide sequence p700 derived from tissue inhibitor of metalloproteinase 3. This Doxil® -P700 complex results in an approximately 100-fold increase in drug uptake, relative to Doxil® alone, by both mouse and human breast cancer cells and immortalised vascular cells resulting in an increase in cytotoxicity. Using p700 to target liposomes in this way may enable specific delivery of doxorubicin or other drugs to a broad range of cancers.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/análogos & derivados , Lipossomos/química , Peptídeos/metabolismo , Polietilenoglicóis/química , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis/farmacologia
4.
Prostate ; 80(12): 977-985, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32542727

RESUMO

BACKGROUND: Recently, resveratrol (Res) has been suggested to suppress the migration and invasion of prostate cancer (PCa). In the present study, we aimed to investigate the effects of Res on genomic DNA methylation, as well as the migration and invasion of PCa cells. METHODS: The suppression by Res of the growth of PCa cells was verified through a cytotoxicity assay. In addition, the effects of Res on 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and ten-eleven translocation 1 (TET1) levels were assessed, and the cell migration and invasion were also determined. The expressions of TET1, tissue inhibitor of metalloproteinases (TIMP) 2, TIMP3, MMP2, and MMP9 were detected through Western blot analysis. Afterward, TET1 was silenced using lentiviral short hairpin RNA to examine the effect of TET1 on the Res-triggered inhibition of migration and invasion of PCa cells. RESULTS: Our results showed that Res upregulated the 5hmC and TET1 levels and downregulated the 5mC level. Moreover, Res also inhibited the migration and invasion of PCa cells, promoted the demethylation of TIMP2 and TIMP3 to upregulate their expressions, and suppressed the expressions of MMP2 and MMP9. The silencing of TET1 in the presence of Res showed that Res could exert its effect through TET1. CONCLUSIONS: Our findings indicated that Res inhibited the migration and invasion of PCa cells via the TET1/TIMP2/TIMP3 pathway, which might potentially serve as a target for the treatment of PCa.


Assuntos
Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Resveratrol/farmacologia , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Invasividade Neoplásica , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Resveratrol/farmacocinética , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Inibidor Tecidual de Metaloproteinase-3/genética , Regulação para Cima
5.
Medicine (Baltimore) ; 99(23): e20552, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32502021

RESUMO

BACKGROUND: To assess the association of the interaction between the rs9619311 and rs402007 polymorphisms and smoking with essential hypertension (EH) in a Chinese Han population. METHOD: Peripheral blood samples were extracted from 422 EH patients and 280 normotensive (NT) patients in a Chinese Han population. A whole blood genomic DNA extraction kit was used to extract genomic DNA from the blood samples. Polymerase chain reaction restriction fragment length polymorphism was used to detect the rs402007 polymorphism of a disintegrin and metalloproteinase with thrombospondin type motifs 1 gene and the rs9619311 polymorphism of the tissue inhibitor of metalloproteinase-3 gene. The distributions of the genotypes and alleles between the 2 study groups (EH and NT) were compared. The main risk factors for EH were determined by using logistic regression analysis. The effects of gene-gene and gene-smoking interactions on EH were analyzed using multifactor dimensional reduction. RESULTS: The frequencies of the rs402007 GC + CC genotype and the C allele were significantly different between the EH and NT groups (0.68 vs 0.57, χ = 8.99, P = .003, odds ratio [OR] = 1.19; 0.45 vs 0.32, χ = 22.16, P < .001, OR = 1.38). The frequencies of the rs9619311 TC + CC genotype and the C allele were also significantly different between the 2 groups (0.33 vs 0.25, χ = 4.51, P = .04, OR = 1.44; 0.18 vs 0.13, χ = 7.03, P = .01, OR = 1.50). Logistic regression analysis suggests that the rs402007 and rs9619311 polymorphisms are independent risk factors for EH (OR = 2.37, 1.86; P < .001, respectively). The multifactor dimensionality redundant analysis results showed that the interaction among rs402007, rs9619311, and smoking was statistically significant (P = .001). CONCLUSIONS: A disintegrin and metalloproteinase with thrombospondin type motifs 1 rs402007 and tissue inhibitor of metalloproteinase-3 rs9619311 polymorphisms are associated with EH in a Chinese Han population, and there was a positive interaction among rs402007, rs9619311, and smoking.


Assuntos
Proteína ADAMTS1/genética , Hipertensão Essencial/genética , Grupos Étnicos/genética , Predisposição Genética para Doença , Polimorfismo Genético , Fumar/epidemiologia , Inibidor Tecidual de Metaloproteinase-3/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , China/epidemiologia , Hipertensão Essencial/epidemiologia , Feminino , Genótipo , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase
6.
J Surg Res ; 254: 102-109, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32422429

RESUMO

BACKGROUND: Wound healing is a complex process aiming at repairing the damaged skin. MiR-23b has been reported to be upregulated during wound healing. In this study, we intended to explore the working mechanism of miR-23b during wound healing. METHODS: Quantitative real-time polymerase chain reaction was performed to detect the enrichment of miR-23b and tissue inhibitor of metalloproteinase-3 (TIMP3) in HaCaT cells. Scratch wound assay was carried out to measure the migration of HaCaT cells. The target of miR-23b was predicted by microT-CDS software, and the combination was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. The abundance of TIMP3 protein was detected by Western blot assay. RESULTS: The abundance of miR-23b was positively related to the concentration and time of transforming growth factor ß1 treatment in HaCaT cells. MiR-23b promoted the migration of keratinocytes. TIMP3 was a direct target of miR-23b and was negatively regulated by miR-23b. TIMP3 inhibited the migration of keratinocytes. MiR-23b accelerated the migration of keratinocytes by downregulating the abundance of TIMP3. CONCLUSIONS: MiR-23b promoted the migration of keratinocytes partly through reducing the enrichment of TIMP3. MiR-23b might be a promising target for the treatment of wound healing-associated diseases.


Assuntos
Movimento Celular/genética , Regulação para Baixo/genética , Queratinócitos/fisiologia , MicroRNAs/fisiologia , Inibidor Tecidual de Metaloproteinase-3/genética , Linhagem Celular , Movimento Celular/fisiologia , Queratinócitos/química , MicroRNAs/análise , Inibidor Tecidual de Metaloproteinase-3/análise , Transfecção , Fator de Crescimento Transformador beta1/farmacologia , Cicatrização/genética
7.
Invest Ophthalmol Vis Sci ; 61(5): 17, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32407518

RESUMO

Purpose: To investigate the relationship of growth in drusen size with genetic susceptibility and adherence to the alternate Mediterranean diet. Methods: Participants in this analysis had complete ocular, genetic, and dietary data with mean follow-up time of 10.2 years in the Age-Related Eye Disease database. Maximal drusen size was graded on an ordinal scale and two-step progression was determined. A genetic risk score using variants associated with advanced AMD and derived from a stepwise regression model yielded 11 variants in 8 genes. Adherence to the alternate Mediterranean diet was assessed using a nine-component score based on intake of vegetables, fruits, legumes, whole cereals, fish, meat, nuts, alcohol, and monounsaturated-to-saturated fatty acids ratio. Multivariate Cox proportional hazards models were used. Results: Among 3023 eligible eyes, 19% had drusen growth. In the stepwise selection, common and rare risk alleles for CFH Y402H, CFH rs1410996, CFH R1210C, C3 R102G, C3 K155Q, and ARMS2/HTRA1, as well as VEGF-A, TIMP3, NPLOC4, and HSPH1 variants were significantly associated with 2-step progression in drusen size, and the C2 E318D protective allele conferred decreased risk, adjusting for other covariates. A higher genetic risk score conferred a higher risk (hazard ratio per 1-unit increase, 2.68; 95% confidence interval, 2.23-3.23; P < 0.001), and a medium/high adherence to alternate Mediterranean diet score (4-9) tended to lower risk (hazard ratio, 0.83; 95% confidence interval, 0.68-0.99; P = 0.049), adjusting for all covariates. Conclusions: Genetic susceptibility was independently related to drusen growth. A Mediterranean-style diet with healthful nutrient-rich foods (fruits, vegetables, legumes and fish), may reduce enlargement of drusen, the hallmark of AMD.


Assuntos
Dieta Mediterrânea , Progressão da Doença , Predisposição Genética para Doença , Degeneração Macular/dietoterapia , Degeneração Macular/genética , Drusas Retinianas/patologia , Idoso , Idoso de 80 Anos ou mais , Alelos , Complemento C3/genética , Fator H do Complemento/genética , Feminino , Seguimentos , Proteínas de Choque Térmico HSP110/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Fotografação , Proteínas/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Fator A de Crescimento do Endotélio Vascular/genética
8.
Sci Rep ; 10(1): 5844, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32246022

RESUMO

As the primary indication for corneal transplantation, the pathogenesis of keratoconus remains elusive. Aiming to identify whether any mutation from extracellular-matrix (ECM)-related genes contributes to the patients with sporadic cases of keratoconus (KC) from Chinese Han population, one hundred and fifty-three participants in total were enrolled in our study, including fifty-three KC patients and one hundred healthy controls. Mutational analysis of three ECM-related genes (LOX, COL5A1 and TIMP3) with next-generation sequencing and Sanger sequencing was performed. To further confirm the function of three ECM-related genes in the pathogenesis of keratoconus, we performed Real-time Quantitative PCR in vitro. Results showed that three new sequence variants (c.95 G > A in LOX, c.1372 C > T in COL5A1 and c.476 C > T in TIMP3) were identified in aforementioned ECM-related genes in KC patients without being detected among the healthy controls. According to the results of QPCR, we found that the expression levels of LOX and TIMP3 were decreased in the KC patients, while COL5A1 showed no significant difference of expression. This is the first time to screen so many ECM-related genes in Chinese keratoconus patients using next-generation sequencing. We find numerous underlying causal variants, enlarging lots of mutation spectrums and thus providing new sites for other investigators to replicate and for further research.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Colágeno Tipo V/genética , Matriz Extracelular/genética , Predisposição Genética para Doença/genética , Ceratocone/genética , Proteína-Lisina 6-Oxidase/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Adulto , Estudos de Casos e Controles , China , Matriz Extracelular/metabolismo , Feminino , Genes/genética , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real
9.
Am J Surg Pathol ; 44(7): 970-981, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32271187

RESUMO

As inflammatory myofibroblastic tumors (IMTs) have become more widely recognized in the female genital tract, an intriguing subset of uterine tumors associated with pregnancy has emerged. Whether uterine IMTs occurring in the setting of pregnancy are clinically or biologically distinct from other uterine IMTs is unknown. Furthermore, little is known about the perinatal factors that may influence the development of these tumors. Here, we report the largest case series of 8 pregnancy-associated IMTs. All pregnancy-associated IMTs in this series occurred in association with pregnancy complications, including abnormal implantation (n=1), gestational diabetes (n=2), preeclampsia and/or HELLP syndrome (n=2), antiphospholipid syndrome (n=1), premature rupture of membranes (n=1), and hepatitis B (n=1). Notably, all IMTs were expelled at the time of delivery or immediately postpartum and were either adherent to the placenta or presented as separate, detached tissue. Tumors ranged from 2.0 to 6.0 cm (median, 3.9 cm), were well-circumscribed and showed classic histologic features of IMTs, including myxoid stroma and a lymphoplasmacytic infiltrate. Seven of 8 cases were positive by ALK immunohistochemistry and confirmed to have an ALK gene rearrangement by fluorescent in situ hybridization and RNA sequencing. The ALK-rearranged IMTs were found to be particularly enriched for TIMP3-ALK (n=5) and THBS1-ALK (n=2) fusions. The single case that was negative for an ALK rearrangement exhibited the classic morphology of an IMT. None of the 4 cases with available clinical follow-up recurred. The clinicopathologic features of pregnancy-associated IMTs in this series in conjunction with those reported in the literature suggests that these may be transient tumors that develop during pregnancy and shed at parturition; they appear to have a relatively indolent clinical course and favorable outcome, although studies with a longer duration of follow-up are still required.


Assuntos
Quinase do Linfoma Anaplásico/genética , Neoplasias de Tecido Muscular/diagnóstico , Proteínas de Fusão Oncogênica/genética , Complicações Neoplásicas na Gravidez/diagnóstico , Trombospondina 1/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Neoplasias Uterinas/diagnóstico , Adulto , Quinase do Linfoma Anaplásico/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias de Tecido Muscular/genética , Neoplasias de Tecido Muscular/patologia , Fusão Oncogênica , Proteínas de Fusão Oncogênica/metabolismo , Gravidez , Complicações Neoplásicas na Gravidez/genética , Complicações Neoplásicas na Gravidez/patologia , Análise de Sequência de RNA , Trombospondina 1/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia
10.
Gene ; 747: 144700, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32330537

RESUMO

OBJECTIVE: Survival rate of laryngeal squamous cell carcinoma (LSCC) patients is not improving. To understand more complete biology of LSCC, studies focused on identification of new specific and prognostic markers are performed. The aim of current study was to evaluate the impact of five different single nucleotide polymorphisms (SNP) (IL6 rs1800795, BLK rs13277113, TIMP3 rs9621532, IL1RL1 rs1041973 and IL1RAP rs4624606) on LSCC development. MATERIAL AND METHODS: A total of 891 subjects (353 histologically verified LSCC patients and 538 healthy controls) were involved in this study. The genotyping was carried out using the real-time-PCR. RESULTS: Statistical analysis revealed statistically significant associations between TIMP3 rs96215332 variants and LSCC in the codominant (OR = 0.600; 95% CI: 0.390-0.922; p = 0.020), overdominant (OR = 0.599; 95% CI: 0.390-0.922; p = 0.020) and additive (OR = 0.675; 95% CI: 0.459-0.991; p = 0.045) models. Also, significant variants of IL1RAP rs4624606 were determined in the codominant (OR = 1.372; 95% CI: 1.031-1.827; p = 0.030), overdominant (OR = 1.353; 95% CI: 1.018-1.798; p = 0.037) and additive (OR = 1.337; 95% CI: 1.038-1.724; p = 0.025) models. CONCLUSION: Results of the current study indicate significant associations between TIMP3 rs9621532 and IL1RAP rs4624606 gene polymorphisms and LSCC development.


Assuntos
Carcinoma de Células Escamosas/genética , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-6/genética , Neoplasias Laríngeas/genética , Polimorfismo de Nucleotídeo Único/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Quinases da Família src/genética , Estudos de Casos e Controles , Feminino , Frequência do Gene/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas/genética
12.
BMC Cancer ; 20(1): 217, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32171282

RESUMO

BACKGROUND: Colorectal cancer (CRC) is the most common cancer and a leading cause of death worldwide. Extracellular matrix (ECM) proteins regulate tumor growth and development in CRC. Elastin (ELN) is a component of ECM proteins involved in the tumor microenvironment. However, the role of ELN in CRC remains unclear. METHODS: In this study, we analyzed ELN gene expression in tumors from CRC patients and adjacent non-tumor colon tissues and healthy controls from two existing microarray datasets. ELN protein was measured in human normal colon cells and colon cancer epithelial cells and tumor development was assessed in colon epithelial cells cultured in medium with or without ELN peptide on plates coated with ELN recombinant protein. Control plates were coated with PBS only. RESULTS: We found ELN gene expression was increased in tumors from CRC patients compared to adjacent non-tumor tissues and healthy controls. ELN protein was increased in cancer cells compared to normal colon epithelial cells. Transforming growth factor beta (TGF-ß) was a key cytokine to induce production of ECM proteins, but it did not induce ELN expression in colon cancer cells. Matrix metalloproteinase 9 (MMP9) gene expression was increased, but that of MMP12 (elastase) did not change between CRC patients and control. Tissue inhibitor of metalloproteinases 3 (TIMP3) gene expression was decreased in colon tissues from CRC patients compared to healthy controls. However, MMP9, MMP12 and TIMP3 proteins were increased in colon cancer cells. ELN recombinant protein increased proliferation and wound healing in colon cancer epithelial cells. This had further increased in cancer cells incubated in plates coated with recombinant ELN coated plate and in culture media containing ELN peptide. A potential mechanism was that ELN induced epithelial mesenchymal transition with increased alpha-smooth muscle actin and vimentin proteins but decreased E-cadherin protein. Tumor necrosis factor alpha (TNF) mRNA was also increased in CRC patients compared to controls. ELN recombinant protein induced further increases in TNF protein in mouse bone marrow derived macrophages after lipopolysaccharide stimulation. CONCLUSIONS: These data suggest ELN regulates tumor development and the microenvironment in CRC.


Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Elastina/genética , Elastina/metabolismo , Animais , Células CACO-2 , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/farmacologia , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Microambiente Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
13.
Sci Rep ; 10(1): 2833, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071343

RESUMO

Ubiquinol-cytochrome c reductase (UQCRB), a subunit of the mitochondrial complex III, is highly expressed in tissues from colorectal cancer patients. Since UQCRB is highly expressed in colorectal cancer, we investigated miRNAs from mutant UQCRB-expressing cell lines to identify new miRNA biomarkers. After sequencing miRNAs in the mutant UQCRB-expressing cell lines, miR-4435 was selected as a potential biomarker candidate from the six up-regulated miRNAs. The expression level of miR-4435 in the mutant UQCRB-expressing cell lines and colon cancer was increased. Notably, the expression level of miR-4435 was increased in exosomes isolated from cell culture medium, suggesting that miR-4435 is closely related to colon cancer and that large amounts of miR-4435 may be secreted outside of the cells through exosomes. Additionally, exosomes extracted from the serum samples of colorectal cancer patients showed increased miR-4435 levels depending on the cancer progression stage. Moreover, analyses of a miRNA database and mRNA-sequencing data of the mutant UQCRB-expressing cell lines revealed that TIMP3, a tumor suppressor, could be a target of miR-4435. Additionally, the expression of miR-4435 was suppressed by UQCRB inhibitor treatment whereas TIMP3 was up-regulated. Upregulation of TIMP3 decreased proliferation of the mutant UQCRB-expressing cell lines and a colorectal cancer cell line. TIMP3 was also upregulated in response to miR-4435 inhibitor and UQCRB inhibitor treatments. Furthermore, these findings suggest that miR-4435 is related to an oncogenic function in UQCRB related disease, CRC, and that effects migration and invasion on mutant UQCRB-expressing cell lines and colorectal cancer cell. In conclusion, our results identified miR-4435 as a potential circulating miRNA biomarker of colorectal cancer associated with UQCRB.


Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais/genética , MicroRNAs/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Movimento Celular/genética , Proliferação de Células/genética , MicroRNA Circulante/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/patologia , Exossomos/metabolismo , Exossomos/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade
14.
Am J Physiol Cell Physiol ; 318(4): C751-C761, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32023075

RESUMO

In this study, we identified P14 alternate reading frame (P14ARF) as a novel regulator of inflammation and vascularization in intervertebral disk degeneration (IVDD). We collected IVD tissues from IVDD patients and normal individuals for analysis of P14ARF expression. We also induced experimental IVDD by needle puncture injuries in the caudal intervertebral disks of Sprague-Dawley (SD) rats and achieved recombinant adenovirus-mediated P14ARF overexpression in experimental IVDD rats. Regulation relationships between P14ARF and tissue inhibitors of metalloproteinases-3 (TIMP3) were confirmed in P14ARF-overexpressed and TIMP3-depleted nucleus pulposus (NP) cells. Tube formation in vitro was evaluated in coculture systems of human umbilical vein endothelial cells (HUVECs) and rat degenerated NP cells (DNPCs). Inflammatory response was assessed from levels of TNF-α, IL-1ß, and IL-6 and neovascularization from expression of endothelial growth factor (VEGF). The P14ARF and TIMP3 were downregulated in degenerated IVD tissue derived from patients and experimental IVDD rats. Overexpressed P14ARF suppressed inflammatory cytokine levels and vascularization. There was decreased in vitro tube formation in response to P14ARF overexpression and TIMP3 elevation. Finally, attenuated inflammatory responses and suppression of VEGF were achieved by P14ARF-mediated promotion of TIMP3 in rat DNPCs. Taken together, the present study reveals that P14ARF/TIMP3 modulation of inflammatory response and vascularization in the context of IVDD highlights a potential target for future therapeutic strategies.


Assuntos
Células Endoteliais/metabolismo , Inflamação/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Animais , Citocinas/metabolismo , Humanos , Disco Intervertebral/metabolismo , Neovascularização Patológica/metabolismo , Ratos , Ativação Transcricional/fisiologia , Regulação para Cima
15.
Biochem Biophys Res Commun ; 523(2): 522-526, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31902587

RESUMO

Isolation of trabecular meshwork stem cells in vitro provides the foundation of a novel treatment for glaucoma. Trabecular meshwork stem cells (TMSCs) of the fetal calve were extracted and cultured for this experiment. TMSCs were isolated through side population cell sorting. TMSCs were then identified using immunofluorescent staining. Extracellular matrix (ECM) expression in TM cells derived from TMSCs was evaluated with Western blot. Our results showed a positive expression of stem cell markers Notch1 and OCT-3/4 in TMSCs, but no TM cells markers TIMP3 or AQP1. In contrast, primary TM cells expressed these TM cell markers but no stem cell markers. Our result confirmed that there are expression of ECM components, such as fibronectin, laminin, collagen I and collagen IV in TM cells differentiated from TMSCs. CONCLUSION: TM cells derived from TMSCs can secrete ECM components which is important for sustain the physiological function.


Assuntos
Matriz Extracelular/metabolismo , Células-Tronco/citologia , Malha Trabecular/citologia , Animais , Biomarcadores/metabolismo , Bovinos , Separação Celular/métodos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Células-Tronco/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo
16.
Gene ; 731: 144348, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-31927006

RESUMO

Mounting evidence demonstrates that N6-methyladenosine (m6A) play critical roles of m6A in the epigenetic regulation, especially for human cancer. The m6A modification is installed by methyltransferase and erased demethylases, leading to the significant modification for gene expression and cell fate. Here, we investigated the biological roles and mechanism of demethylase alkylation repair homolog protein 5 (ALKBH5) in the non-small cell lung cancer (NSCLC). Results revealed that ALKBH5 was ectopically up-regulated in the NSCLC tissue and cells, and closely correlated with the poor prognosis. Functionally, ALKBH5 promoted the proliferation and reduced apoptosis of NSCLC cells in vitro, and knockdown of ALKBH5 repressed the tumor growth in vivo. Mechanistically, RNA immunoprecipitation sequencing (RIP-Seq) revealed that ALKBH5 targeted the TIMP3. Moreover, ALKBH5 repressed TIMP3 mRNA stability and protein production. In conclusion, the present research confirmed the ALKBH5/TIMP3 pathway in the NSCLC oncogenesis progress, providing a novel insight for the epitranscriptome and potential therapeutic target for NSCLC.


Assuntos
Adenosina/análogos & derivados , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/fisiologia , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Estabilidade de RNA/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Células A549 , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Animais , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Mensageiro/metabolismo
17.
Ecotoxicol Environ Saf ; 190: 110126, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31918251

RESUMO

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate pesticide used for crop protection and storage. But, it's widespread utilization is associated with deleterious growth plate cartilage disorder in broilers termed as avian tibial dyschondroplasia (TD). TD results in non-mineralized and less vascularized proximal tibial growth plate cartilage causing lameness and poor growth performance. This study investigated the therapeutic potential of puerarin against thiram toxicity in TD affected chickens. One-day-old broiler chickens (n = 240) were alienated into three equal groups i.e. control, TD and puerarin (n = 80) and were offered standard feed. Additionally, TD and puerarin groups were offered thiram at 50 mg/kg of feed from 4 to 7 days for TD induction followed by puerarin therapy at 120 mg/kg to puerarin group only from 8 to 18 days for TD treatment. Thiram feeding to TD and puerarin group chickens caused lameness, mortality, and increased the aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) levels and growth plate (GP) size and upregulated HIF-1α expression. Besides, the production parameters, alkaline phosphatase (ALP), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels and the expressions of TIMP-3 and BCL-2 were decreased (p < 0.05). Puerarin alleviated lameness, enhanced angiogenesis and growth performance and serum and antioxidant enzymes, decreased apoptosis and recuperated GP width by significantly downregulating HIF-1α and upregulating the TIMP-3 and BCL-2 mRNA and protein expressions in puerarin group chickens (p < 0.05). In conclusion, the toxic effects associated with thiram can be mitigated using puerarin.


Assuntos
Fungicidas Industriais/toxicidade , Isoflavonas/farmacologia , Osteocondrodisplasias/veterinária , Tiram/toxicidade , Vasodilatadores/farmacologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Galinhas/metabolismo , Glutationa Peroxidase/metabolismo , Lâmina de Crescimento/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Malondialdeído/metabolismo , Neovascularização Patológica/induzido quimicamente , Doenças das Aves Domésticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tíbia/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-3/metabolismo
18.
Sci Rep ; 10(1): 572, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953445

RESUMO

Recent evidences indicate that mitochondrial genes and function are decreased in active ulcerative colitis (UC) patients, in particular, the activity of Complex I of the electron transport chain is heavily compromised. MCJ is a mitochondrial inner membrane protein identified as a natural inhibitor of respiratory chain Complex I. The induction of experimental colitis in MCJ-deficient mice leads to the upregulation of Timp3 expression resulting in the inhibition of TACE activity that likely inhibits Tnf and Tnfr1 shedding from the cell membrane in the colon. MCJ-deficient mice also show higher expression of Myd88 and Tlr9, proinflammatory genes and disease severity. Interestingly, the absence of MCJ resulted in distinct microbiota metabolism and composition, including a member of the gut community in UC patients, Ruminococcus gnavus. These changes provoked an effect on IgA levels. Gene expression analyses in UC patients showed decreased levels of MCJ and higher expression of TIMP3, suggesting a relevant role of mitochondrial genes and function among active UC. The MCJ deficiency disturbs the regulatory relationship between the host mitochondria and microbiota affecting disease severity. Our results indicate that mitochondria function may be an important factor in the pathogenesis. All together support the importance of MCJ regulation during UC.


Assuntos
Bactérias/classificação , Colite Ulcerativa/genética , Disbiose/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Proteína ADAM17/genética , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Colite Ulcerativa/microbiologia , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Microbiota , Filogenia , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Índice de Gravidade de Doença , Inibidor Tecidual de Metaloproteinase-3/genética , Fator de Necrose Tumoral alfa/genética
19.
Clin Exp Metastasis ; 37(1): 69-76, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31894441

RESUMO

TIMP-3 is one of four tissue inhibitors of matrix metalloproteinases, the endogenous inhibitors of the matrix metalloproteinase enzymes. These enzymes have an important role in metastasis, in the invasion of cancer cells through the basement membrane and extracellular matrix. TIMP-1, -2 and -4 both promote and inhibit tumour development, in a context-dependent manner, however TIMP-3 is consistently anti-tumourigenic. TIMP-3 is also the only insoluble member of the family, being either bound to the extracellular matrix or the low density lipoprotein-related protein-1, through which it can be endocytosed. Levels of TIMP-3 have also been shown to be regulated by micro RNAs and promoter hypermethylation, resulting in frequent silencing in many tumour types, to the extent that its expression has been suggested as a prognostic marker in some tumours, being associated with lower levels of metastasis, or better response to treatment. TIMP-3 has been shown to have anti-metastatic effects, both through inhibition of matrix metalloproteinases and ADAM family members and downregulation of angiogenesis. This occurs via interactions with receptors including VEGF, via modulation of signaling pathways and due to protease inhibition. TIMP-3 has also been shown to reduce tumour growth rate, most often by inducing apoptosis by stabilisation of death receptors. A number of successful mechanisms of delivery of TIMP-3 to tumour or inflammatory sites have been investigated in vitro or in animal studies. It may therefore be worthwhile further exploring the use of TIMP-3 as a potential anti-metastatic or anti-tumorigenic therapy for many tumour types.


Assuntos
Carcinogênese/patologia , Metástase Neoplásica/patologia , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/genética , Membrana Basal/metabolismo , Carcinogênese/genética , Metilação de DNA , Modelos Animais de Doenças , Regulação para Baixo , Epigênese Genética , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/genética , Prognóstico , Regiões Promotoras Genéticas , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/uso terapêutico , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/uso terapêutico
20.
Hum Pathol ; 97: 29-39, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31917155

RESUMO

Uterine inflammatory myofibroblastic tumors (IMTs) have been reported in association with pregnancy and, in some instances, secondarily involve the placenta. The clinicopathological spectrum of these tumors in the setting of pregnancy is not well defined. We investigated the clinical, morphologic, immunohistochemical, molecular cytogenetic, and genetic features of 6 uterine IMTs occurring in pregnant women. Each tumor was discovered at parturition, and none was identified by prenatal ultrasound. Patient age ranged from 25 to 41 years (mean 31.5). Tumor size ranged from 1.5 to 9 cm (mean 4.7). Four of 6 had usual IMT features, with at least focal deciduoid change in 3. Necrosis was identified in 3 tumors; and multinucleated cells, in 3 tumors. Sex hormone receptor expression was consistent with estrogen receptor negative or focally weakly positive and progesterone receptor diffusely moderately or moderately to strongly positive in all 6 tumors. ALK immunohistochemistry was strongly positive in 5 tumors, and all of these had an ALK rearrangement detected by break-apart fluorescence in situ hybridization. Subsequent RNA sequencing of these 5 tumors identified a TIMP3-ALK fusion in 4 and a THBS1-ALK in 1. In the ALK-negative tumor, RNA sequencing detected a novel TIMP3-RET fusion that was confirmed by RET break-apart fluorescence in situ hybridization. Follow-up was available for 2 of 6 patients 5 and 19 months after diagnosis. Neither patient developed recurrence. ALK immunohistochemistry will distinguish most uterine IMTs, but if ALK expression and gene studies are negative, in the appropriate morphologic context, evaluation of other tyrosine kinase genes known to be more commonly altered in extrauterine IMTs such as ROS1, NTRK3, PDGFRß, and RET may be necessary for diagnostic confirmation.


Assuntos
Biomarcadores Tumorais/genética , Fusão Gênica , Miofibroblastos/patologia , Neoplasias de Tecido Fibroso/genética , Placenta/patologia , Complicações Neoplásicas na Gravidez/genética , Proteínas Proto-Oncogênicas c-ret/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Neoplasias Uterinas/genética , Adulto , Quinase do Linfoma Anaplásico/genética , Feminino , Rearranjo Gênico , Predisposição Genética para Doença , Humanos , Necrose , Neoplasias de Tecido Fibroso/patologia , Neoplasias de Tecido Fibroso/terapia , Fenótipo , Gravidez , Complicações Neoplásicas na Gravidez/patologia , Complicações Neoplásicas na Gravidez/terapia , Resultado do Tratamento , Carga Tumoral , Neoplasias Uterinas/patologia , Neoplasias Uterinas/terapia
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