RESUMO
Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (Rh) measured by online DLS with the Rh values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The Rg/Rh ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.
Assuntos
Circovirus/química , Fracionamento por Campo e Fluxo/instrumentação , Theilovirus/química , Proteínas Virais/isolamento & purificação , Animais , Linhagem Celular , Fracionamento por Campo e Fluxo/métodos , Camundongos , Multimerização Proteica , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Virais/análiseRESUMO
The natural adjuvant properties of bacterial ghosts (BGs) lie within the presence of intact pathogen-associated molecular patterns on their surface. BGs can improve the direct delivery, natural processing and presentation of target antigens within dendritic cells (DCs). Moreover, sensitization of human DCs by cancer cell lysate (oncolysate)-loaded BGs in the presence of IFN-α and GM-CSF enhanced DC maturation as indicated by an increased expression of maturation markers and co-stimulatory molecules, higher production of IL-12p70 and stimulation of significantly increased proliferation of both autologous CD4+ and CD8+ T cells compared to DCs matured in the presence of purified lipopolysaccharide. The induced T cells efficiently recognized oncolysate-derived tumor-associated antigens expressed by cancer cells used for the production of oncolysate. Our optimized one-step simultaneous antigen delivery and DC maturation-inducing method emerges as a promising tool for the development and implementation of next-generation cellular cancer immunotherapies.
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Células Dendríticas/imunologia , Escherichia coli/imunologia , Imunoterapia Adotiva/métodos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Células Dendríticas/microbiologia , Células Dendríticas/transplante , Glioblastoma/imunologia , Glioblastoma/terapia , Humanos , Interleucina-12/biossíntese , Interleucina-12/imunologia , Lipopolissacarídeos/farmacologia , FenótipoRESUMO
Esophageal cancer is a malignant disease with poor prognosis, increasing incidence, and ineffective treatment options. MicroRNAs are post-transcriptional regulators of gene expression involved in many biological processes including carcinogenesis. We determined miR-205 expression levels in tumor/non-tumor tissues of 45 esophageal cancer patients using qPCR and found that decreased level of miR-205 in tumor tissue correlates with poor overall survival in esophageal adenocarcinoma patients. Further, we observed significantly higher levels of miR-205 in tumor tissue of esophageal squamous cell carcinoma. Ectopic overexpression of miR-205 in adenocarcinoma cell line SK-GT-4 led to decreased cell proliferation, cell cycle arrest in G1, and decreased migration ability. Conversely, in squamous cell line KYSE-150, same effects like inhibition of proliferation, migration, and colony-forming potential and cell cycle arrest in G2 were observed after silencing of miR-205. We performed global gene expression profiling and revealed that suppressive functioning of miR-205 in adenocarcinoma could be realized through regulation of epithelial-mesenchymal transition (EMT), whereas oncogenic in squamous cell carcinoma by regulation of metalloproteinase 10. Our results suggest that miR-205 could serve as biomarker in esophageal cancer and acts as a tumor suppressor in esophageal adenocarcinoma and oncogene in esophageal squamous cell carcinoma.
Assuntos
Adenocarcinoma/patologia , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/patologia , MicroRNAs/fisiologia , Oncogenes/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adulto , Idoso , Apoptose/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes Supressores de Tumor , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Taxa de SobrevidaRESUMO
In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain α and ß, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca(2+) imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca(2+) from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the ß-adrenergic pathway.
Assuntos
Corpos Embrioides/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos , Retículo Sarcoplasmático/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Linhagem Celular , Forma Celular , Tamanho Celular , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Reprodutibilidade dos Testes , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Troponina T/metabolismoRESUMO
Dendritic cells (DCs) belong to the immune system and are particularly studied for their potential to direct either an activated or tolerogenic immune response. The roles of microRNAs (miRNAs) in posttranscriptional gene expression regulation are being increasingly investigated. This study's aim is to evaluate the miRNAs' expression changes in prepared human immature (iDCs), activated (aDCs), and tolerogenic dendritic cells (tDCs). The dendritic cells were prepared using GM-CSF and IL-4 (iDC) and subsequently maturated by adding LPS and IFN-γ (aDC) or IL-10 and TGF-ß (tDC). Surface markers, cytokine profiles, and miRNA profiles were evaluated in iDC, tDC, and aDC at 6 h and 24 h of maturation. We identified 4 miRNAs (miR-7, miR-9, miR-155 and miR-182), which were consistently overexpressed in aDC after 6 h and 24 h of maturation and 3 miRNAs (miR-17, miR-133b, and miR-203) and miR-23b cluster solely expressed in tDC. We found 5 miRNAs (miR-10a, miR-203, miR-210, miR-30a, and miR-449b) upregulated and 3 miRNAs downregulated (miR-134, miR-145, and miR-149) in both tDC and aDC. These results indicate that miRNAs are specifically modulated in human DC types. This work may contribute to identifying specific modulating miRNAs for aDC and tDC, which could in the future serve as therapeutic targets in the treatment of cancer and autoimmune diseases.
Assuntos
Células Dendríticas/metabolismo , MicroRNAs/genética , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-10/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Clinical behavior of neuroblastoma (NBL) is remarkably heterogeneous, as it ranges from spontaneous regression to aggressive clinical phenotype and death. There is increasing body of evidence demonstrating that microRNAs could be considered the potential biomarkers for clinical applications in NBL. In this report, we focus on molecular characterization of high-risk as well as low-risk and intermediate-risk NBL cases in the context of the microRNA expression profile that is specific for the given risk category of the disease. We investigated a total of 30 NBL patients, out of whom there were 19 patients with low- to intermediate-risk and 11 with high-risk NBLs as defined by the Clinical Oncology Group. We determined the expression profiles of 754 microRNAs (miRNAs), whereas the miRNA expression levels were normalized to RNU44, mean expression levels were calculated, and data were analyzed by use of the microarray biostatistical approaches. We identified the signature of 38 miRNAs differentially expressed between these groups of NBL patients (P < 0.05): 17 miRNAs were upregulated and 21 miRNAs were downregulated in the tumors of high-risk NBL patients. We confirm some of the previous observations and we report several new microRNAs associated with aggressive NBL, both being relevant subjects for further translational validation and functional studies.
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Neuroblastoma/genética , Biomarcadores Tumorais/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Masculino , PrognósticoRESUMO
Sporadic colorectal cancer (CRC) is a typical multifactorial disease. Isothiocyanates (ITC) have been recently shown to inhibit development of CRC in many experimental models. MicroRNAs (miRNAs) are short noncoding RNAs that posttranscriptionally regulate gene expression through binding to 3' untranslated regions (3'UTR) of target mRNAs. MiRNAs are regulated by natural agents, ITCs included. In our study, using global expression profiling based on TaqMan Low-Density Arrays, we identified 3 common miRNAs (miR-155, miR-23b, miR-27b) regulated by ITCs (sulforaphane, iberin) in colonic epithelial cell lines NCM460 and NCM356. In silico predictions allowed us to find 9 relevant single nucleotide polymorphisms (SNPs) localized within the 3'UTRs of genes (AGTR1, TNFAIP2, PRKCB, HSPA9, RABGAP1, DICER1, ADAM19, VWA5A, and SIRT5) targeted by these ITC-related miRNAs. Finally, we observed that homozygous CC genotype of DICER1, rs1057035, was significantly associated with decreased risk of CRC (odds ratio = 0.49; 95% confidence interval: 0.25-0.95, P = 0.036) when compared to TT homozygote genotype; also, the C allele tended to have a protective effect (P = 0.072). This study showed that miRNAs could be involved in chemoprotective effects of natural agents; their function alteration through SNPs in their binding sites and flanking regions presents a new class of CRC risk factors.
Assuntos
Neoplasias Colorretais/genética , RNA Helicases DEAD-box/genética , Isotiocianatos/farmacologia , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Ribonuclease III/genética , Regiões 3' não Traduzidas , Idoso , Sítios de Ligação/genética , Estudos de Casos e Controles , Linhagem Celular/efeitos dos fármacos , Simulação por Computador , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Substâncias Protetoras/farmacologia , SulfóxidosRESUMO
Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV-C light necessary for baculovirus inactivation by measurement of fluorescent GFP protein produced by baculovirus expression system after the irradiation of baculovirus culture in doses ranging from 3.5 to 42 J/m2 was determined. At a dose of 36.8 J/m2, only 0.5% of GFP-expressing cells were detected by flow cytometry and confocal microscopy. The stability of purified VP1-PCV2bCap protein produced by baculovirus expression system was analyzed after the irradiation at doses ranging from 3.5 to 19.3 J/m2. Up to the dose of 11 J/m2, no significant effect of UV-C light on the stability of VP1-PCV2bCap was detected. We observed a dose-dependent increase in VP1-PCV2bCap-specific immune response in BALB/c mice immunized by recombinant protein sterilized by irradiation in dose 11 J/m2 with no significant difference between vaccines sterilized by UV-C light and filtration. A substantial difference in the production of VP1-PCV2bCap specific IgG was observed in piglets immunized with VP1-PCV2bCap sterilized by UV-C in comparison with protein sterilized by filtration in combination with the inactivation of baculovirus by binary ethylenimine. UV-C irradiation represents an effective method for vaccine sterilization, where commonly used methods of sterilization are not possible.
Assuntos
Vacinas Sintéticas , Vírus , Camundongos , Animais , Suínos , Esterilização , Proteínas Recombinantes/genética , Raios UltravioletaRESUMO
Bovine papillomavirus type 1 L1 protein was produced in a baculovirus expression system and purified as virus-like particles (VLPs) by affinity chromatography using lectins. The morphological integrity of VLPs was confirmed by electron microscopy. Differences between the two detected variants were deciphered by mass spectrometry of peptides (MALDI-TOF). Mice were immunized with purified VLPs in doses of 10, 25, or 50 µg in combination with 1% saponin and 15% alhydrogel per dose as adjuvants. Analysis of the humoral immune response revealed increased levels of specific antibodies detected 3 weeks after the first immunization in all groups of animals. This was further significantly increased by the booster applied 3 weeks after the first dose, with the best immune response in a group of mice immunized by the largest dose of antigen. BPV1 L1 VLPs purified by affinity chromatography using lectins could be used for prophylactic immunization in veterinary medicine.
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Angiotensinogen (AGT) represents a key component of the renin-angiotensin-aldosterone system (RAAS). Polymorphisms in the 3' untranslated region (3'UTR) of the AGT gene may alter miRNA binding and cause disbalance in the RAAS. Within this study, we evaluated the possible association of AGT +11525C/A (rs7079) with the clinical characteristics of patients with coronary artery diseases (CAD). Selective coronarography was performed in 652 consecutive CAD patients. Clinical characteristics of the patients, together with peripheral blood samples for DNA isolation, were collected. The genotyping of rs7079 polymorphism was performed with TaqMan® SNP Genotyping Assays. We observed that patients with the CC genotype were referred for coronarography at a younger age compared to those with the AA+CA genotypes (CC vs. AA+CA: 59.1 ± 9.64 vs. 60.91 ± 9.5 (years), p = 0.045). Moreover, according to the logistic regression model, patients with the CC genotype presented more often with restenosis than those with the CA genotype (p = 0.0081). In conclusion, CC homozygotes for rs7079 present with CAD symptoms at a younger age compared with those with the AA+CA genotype, and they are more prone to present with restenosis compared with heterozygotes.
Assuntos
Doença da Artéria Coronariana , MicroRNAs , Humanos , Regiões 3' não Traduzidas , Angiotensinogênio/genética , Sítios de Ligação , Doença da Artéria Coronariana/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Polimorfismo GenéticoRESUMO
Regulatory T cells (Tregs) are critical regulators of autoimmune diseases, including type 1 diabetes mellitus. It is hypothesised that Tregs' function can be influenced by changes in the expression of specific microRNAs (miRNAs). Thus, we performed miRNAs profiling in a population of Tregs separated from peripheral blood of five type 1 diabetic patients and six healthy donors. For more detailed molecular characterisation of Tregs, we additionally compared miRNAs expression profiles of Tregs and conventional T cells. Tregs were isolated according to CD3+, CD4+, CD25(hi)+ and CD127- by flow cytometry, and miRNA expression profiling was performed using TaqMan Array Human MicroRNA Panel-1 (384-well low density array). In Tregs of diabetic patients we found significantly increased expression of miRNA-510 (p=0.05) and decreased expression of both miRNA-342 (p<0.0001) and miRNA-191 (p=0.0079). When comparing Tregs and T cells, we revealed that Tregs had significant higher expression of miRNA-146a and lower expression of eight specific miRNAs (20b, 31, 99a, 100, 125b, 151, 335, and 365). To our knowledge, this is the first study demonstrating changes in miRNA expression profiles occurring in Tregs of T1D patients and a miRNAs signature of adult Tregs.
Assuntos
Diabetes Mellitus Tipo 1/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , Linfócitos T Reguladores/metabolismo , Adulto , Análise por Conglomerados , Diabetes Mellitus Tipo 1/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linfócitos T/metabolismo , Adulto JovemRESUMO
MicroRNAs are short non-coding RNAs involved in post-trascriptional regulation of gene expression. They can be found in various organisms including animals, plants and viruses and they play a key role in diverse biological processes, such as embryogenesis, differentiation and proliferation of cells, production of cytokines or apoptosis. Since the initial observation, more than 700 microRNAs have been identified in mammalian cells and up to one third of all protein-encoding genes is estimated to be regulated by these small molecules. In order to biological functions of these mRNA targets, it is apparent today, that altered microRNA expression can contribute not only to carcinogenesis but also to serious failure of the immune system. The aim of this review is to summarize the latest findings about the biogenesis and function of microRNAs and to refer on their role in the regulation of ontogenesis of immune system, haematopoiesis, immune responses and viral infections. Possible contribution of microRNAs to the therapy of viral infections is discussed.
Assuntos
Imunidade/genética , Imunidade/fisiologia , MicroRNAs/fisiologia , Animais , Hematopoese/genética , HumanosRESUMO
Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bß chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.
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New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblr™. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.
Assuntos
Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Lipossomos/imunologia , Mananas/imunologia , Lectinas de Ligação a Manose/imunologia , Nanopartículas/química , Receptores de Superfície Celular/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Superfície/metabolismo , Candida glabrata/química , Química Click , Humanos , Hidroxilaminas/síntese química , Hidroxilaminas/química , Lipídeos/síntese química , Lipídeos/química , Lipossomos/química , Lipossomos/farmacologia , Mananas/química , Mananas/farmacologia , Receptor de Manose , Camundongos Endogâmicos BALB C , Microfluídica/métodos , Tamanho da PartículaRESUMO
BACKGROUND: WWOX (WW domain-containing oxidoreductase) gene, located on chromosome 16q 23.3-24.1 in the region recognized as the common fragile site FRA16D is considered to be a tumor suppressor gene involved in various cancers: breast, ovarian, prostate, esophageal, lung, pancreatic, gastric and hepatic. The aim of this study was to describe (i) putative protein interactions of WWOX (ii) the molecular mechanisms of tumor suppressor activity (iii) present an overview of WWOX in relation to nervous system and breast, prostate and ovarian cancers. METHODS AND RESULTS: WWOX expression is up-regulated in endocrine organs indicating its importance in these tissues. In many cancers WWOX expression is down-regulated and low WWOX expression is related to poor prognosis. CONCLUSION: All the evidence suggest that WWOX can be considered as a new tumor suppressor gene and target for gene therapy due to the association of high WWOX expression with improved disease free survival.
Assuntos
Genes Supressores de Tumor , Neoplasias/genética , Oxirredutases/genética , Proteínas Supressoras de Tumor/genética , Animais , Neoplasias da Mama/genética , Feminino , Expressão Gênica , Humanos , Masculino , Neoplasias Ovarianas/genética , Neoplasias da Próstata/genética , Oxidorredutase com Domínios WWRESUMO
D-galactosamine (GalN) is a highly selective hepatotoxin that causes liver damage similar to human viral hepatitis via depletion of uridine nucleotides, which subsequently diminishes synthesis of RNA and proteins. Model of galactosamine hepatotoxicity is frequently used in animal experiments in vitro. The purpose of our study was to establish the model of GalN-induced hepatocyte injury in in vitro conditions using primocultures of rat hepatocytes as an important pre-requisite for further experiments in which we would like to study potential hepatoprotective effect of various substances. Rate of hepatocyte injury was evaluated by morphological changes, changes in cell viability, albumin production, mitochondrial membrane potential, activity of mitochondrial dehydrogenases and glutathione content. Marked dose dependent hepatocyte injury was found after 24-hour incubation with GalN. Based on the results we suggest as an optimal model for short-term toxicity test exposure to GalN for 24 hours in dose of 40 mM.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Galactosamina/toxicidade , Hepatócitos/efeitos dos fármacos , Animais , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Masculino , Ratos , Ratos WistarRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. In the last decade, number of evidences showing miRNAs contribution to the regulation of apoptosis, cellular proliferation, differentiation, and other important cellular processes is constantly growing. Specific miRNA expression signatures have been identified in variety of human cancers as well as pathologies of cardiovascular and urinary systems. Our chapter focuses on the potential of urinary miRNAs to serve as biomarkers in uro-oncology, nephrology, and cardiology. We discuss in detail recent knowledge about the origin of urinary miRNAs, their stability, quality control, and their utility as a potential new class of biomarkers in medicine. Finally, we summarize the studies focusing on detection and characterization of urinary miRNAs as potential biomarkers in urologic cancers, nephrology, and cardiology.
Assuntos
Cardiopatias/urina , MicroRNAs/urina , Neoplasias/urina , Doenças Urológicas/urina , Biomarcadores/urina , Feminino , Cardiopatias/diagnóstico , Cardiopatias/genética , Cardiopatias/patologia , Humanos , Masculino , MicroRNAs/genética , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patologia , Estabilidade de RNA , Doenças Urológicas/diagnóstico , Doenças Urológicas/genética , Doenças Urológicas/patologiaRESUMO
Angiotensinogen (AGT), its active fragments and microRNA-31 (miR-31) play an important role in adipocyte differentiation. AGT contains a miR-31 polymorphic binding site. We hypothesize that the rs7079 polymorphism in the miR-31/584 binding site of the AGT gene could influence body fat distribution. A total of 751 subjects (195 men, 556 women) were enrolled in the study. The rs7079 genotypes were determined by qRT-PCR. Anthropometric measurements were taken on all subjects, who were subsequently divided into two groups: obese (>30 kg m(-2)) and non-obese (<30 kg m(-2)). Linear regression models were created to determine the contributions of sex, obesity status and rs7079 to all measured parameters. Adding the rs7079 genotype significantly contributed to the linear regression model for waist circumference (p = 0.013), hip circumference (p = 0.018) and supraspinal skin-fold thickness (p = 1 × 10(-3)). Differences between sexes and between the obese and non-obese groups were observed. Waist circumference was lower in men carrying the A allele (p = 0.022); hip circumference was higher only in obese women carrying the A allele (p = 0.015). While men carrying the A allele had lower supraspinal skin-fold thickness (p = 0.022), this parameter was found to be higher in A allele carrying women (p = 3 × 10(-3)). The higher total sum of skin-fold thickness in A allele carrying women was restricted to obese individuals (p = 0.028). The presence of the A allele was associated with both lower tricipital skin-fold thickness in non-obese women (p = 0.023) and a trend of higher thickness in non-obese men (p = 0.065). Significant associations of rs7079 in the AGT gene and body fat distribution were observed. The distribution followed opposing patterns in both sexes.
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BACKGROUND: Esophageal cancer is the malignant tumor with very poor prognosis and increasing incidence often diagnosed at very late stage, so the prognosis of affected patients is unsatisfactory, despite the development of therapeutic option such as surgery, chemotherapy and radiotherapy. Consequently, there is a great need for biomarkers to allow a tailored multimodality approach with increased efficiency. Altered expression of microRNAs has been reported in wide range of malignancies, including esophageal cancer. The aim of this study was to examine the expression levels of candidate microRNAs in esophageal cancer and evaluate their diagnostic and prognostic potential. FINDINGS: Using quantitative real-time PCR, expression levels of 9 candidate microRNAs were examined in 62 tissue samples, 23 esophageal adenocarcinomas, 22 esophageal squamous cell carcinomas and 17 adjacent esophageal mucosa samples. MicroRNA expression levels were further analyzed in regards to clinico-pathological features of esophageal cancer patients. We observed significantly decreased levels of miR-203 and increased levels of miR-21 in adenocarcinoma tissues when compared to normal mucosa. MiR-29c and miR-148 indicated good ability to distinguish between histological subtypes of esophageal cancer. MiR-203 and miR-148 were linked to disease-free survival and overall survival in esophageal adenocarcinoma patients, and miR-148 also in esophageal squamous cell carcinoma patients. CONCLUSIONS: Our data suggest that altered expression of miR-21, miR-29c, miR-148 and miR-203 are related to neoplastic transformation and progression of the disease and these microRNAs could serve as a potential diagnostic and prognostic biomarkers in esophageal cancer. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4646922201567057.
Assuntos
Adenocarcinoma/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Neoplasias Esofágicas/diagnóstico , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Adenocarcinoma/genética , Idoso , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Background. Sunitinib is a tyrosine kinase inhibitor used in the treatment of metastatic renal cell carcinoma. The main difficulty related to the treatment is the development of drug resistance followed by rapid progression of the disease. We analyzed tumor tissue of sunitinib treated patients in order to find miRNAs associated with therapeutic response. Methods. A total of 79 patients with metastatic renal cell carcinoma were included in our study. miRNA profiling in tumor tissue samples was performed by TaqMan Low Density Arrays and a group of selected miRNAs (miR-155, miR-374-5p, miR-324-3p, miR-484, miR-302c, and miR-888) was further validated by qRT-PCR. Normalized data were subjected to ROC and Kaplan-Meier analysis. Results. We reported decreased tissue levels of miR-155 and miR-484 as significantly associated with increased time to progression (miR-155: median TTP 5.8 versus 12.8 months, miR-484: median TTP 5.8 versus 8.9 months). Conclusion. miR-155 and miR-484 are potentially connected with sunitinib resistance and failure of the therapy. miR-155 is a known oncogene with direct influence on neovascularization. Biological role of miR-484 has to be clarified. Stratification of patients based on miRNA analysis would allow more personalized approach in therapy of metastatic renal cell carcinoma.