RESUMO
OBJECTIVE: Collagen type IV alpha 1 (COL4A1) exerts tumor-promoting functions in several tumors. However, its role in liver cancer remains not fully understood. Hence, this study aims to investigate the role of COL4A1 in regulating liver cancer cell behaviors and to validate its upstream regulatory mechanism. METHODS: Expression of xeroderma pigmentosum D (XPD) and COL4A1 was examined by qRT-PCR and western blot. Cell proliferation, migration, and invasion were evaluated. The protein levels of N-cadherin, vimentin, and E-cadherin were determined by western blot to evaluate epithelial-mesenchymal transition (EMT). The interaction between miR-29a-3p and COL4A1 was analyzed by luciferase reporter assay. RESULTS: COL4A1 overexpression significantly promoted cell proliferation, migration, invasion, and EMT in Hep3B cells. In contrast, COL4A1 silencing yielded the opposite effects in HepG2 cells. Expression of COL4A1 was increased, whereas expression of XPD and miR-29a-3p was decreased in HCC tissues compared to controls. COL4A1 mRNA level was negatively correlated with expression of XPD and miR-29a-3p in HCC tissues. Furthermore, XPD silencing-mediated up-regulation of COL4A1 expression was attenuated by miR-29a-3p mimic. Moreover, miR-29a-3p mimic inhibited Hep3B cell proliferation, migration, and invasion by directly targeting COL4A1. CONCLUSION: COL4A1 is negatively regulated by XPD-miR-29a-3p axis and promotes liver cancer progression in vitro.
Assuntos
Carcinoma Hepatocelular/metabolismo , Colágeno Tipo IV/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Antígenos CD/análise , Caderinas/análise , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colágeno Tipo IV/genética , Transição Epitelial-Mesenquimal , Feminino , Inativação Gênica , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Vimentina/análiseRESUMO
BACKGROUND: Alport syndrome (AS) is a disease caused by mutations in COL4A3, COL4A4 or COL4A5, the genes that encode distinct chains of type IV collagen. The vast majority of cases present as an inherited disorder, although de novo mutations are present in around 10% of the cases. METHODS: This non-systematic review summarizes recent evidence on AS. We discuss the genetic and pathophysiology of AS, clinical manifestations, histopathology, diagnostic protocols, conventional treatment and prognostic markers of the disease. In addition, we summarize experimental findings with novel therapeutic perspectives for AS. RESULTS: The deficient synthesis of collagen heterotrimers throughout the organism leads to impaired basement membranes (BM) in several organs. As a result, the disease manifests in a wide range of conditions, particularly renal, ocular and auricular alterations. Moreover, leiomyomatosis and vascular abnormalities may also be present as atypical presentations. In this framework, diagnosis can be performed based on clinical evaluation, skin or renal biopsy and genetic screening, the latter being the gold standard. There are no formally approved treatments for AS, even though therapeutic options have been described to delay disease progression and increase life expectancy. Novel therapeutic targets under pre-clinical investigation included paricalcitol, sodium-glucose co-transporter- 2 inhibitors, bardoxolone methyl, anti-microRNA-21 oligonucleotides, recombinant human pentraxin-2, lysyl oxidase-like-2 blockers, hydroxypropyl-b-cyclodextrin, sodium 4-phenylbutyrate and stem cell therapy. CONCLUSION: AS is still a greatly under and misdiagnosed disorder. The pathophysiology is still not fully understood and genetics of the disease also have some gaps. Up to know, there is no specific and effective treatment for AS. Further studies are necessary to establish novel and effective therapeutic protocols.
Assuntos
Nefrite Hereditária , Membrana Basal , Colágeno Tipo IV/genética , Humanos , Rim , Mutação , Nefrite Hereditária/genéticaRESUMO
BACKGROUND: Previous study showed that purinergic P2X7 receptors (P2X7R) reach the highest expression in the first week after unilateral ureteral obstruction (UUO) in mice, and are involved in the process of inflammation, apoptosis and fibrosis of renal tissue. We, herein, document the role of purinergic P2X7 receptors activation on the third day of UUO, as assessed by means of BBG as its selective inhibitor. METHODS: We investigated the effects of brilliant blue G (BBG), a P2X7R antagonist, in the third day of kidney tissue response to UUO in rats. For this purpose, male Wistar rats submitted to UUO or sham operated, received BBG or vehicle (V), comprising four groups: UUO-BBG, UUO-V, sham-BBG and sham-V. The kidneys were harvested on day 3 UUO and prepared for histology, immunohistochemistry (P2X7R, PCNA, CD-68, α-sma, TGF-ß1, Heat-shock protein-47, TUNEL assay), quantitative real-time PCR (IL-1ß, procollagens type I, III, and IV) for mRNA quantification. RESULTS: The group UUO-V presented an enhancement in tubular cell P2X7-R expression, increase influx of macrophages and myofibroblasts, HSP-47 and TGF- ß1 expression. Also, upregulation of procollagen types I, III, and IV, and IL-1ß mRNAs were seen. On the other hand, group UUO-BBG showed lower expression of procollagens and IL-1ß mRNAs, as well as less immunoreactivity of HSP-47, TGF-ß, macrophages, myofibroblasts, and tubular apoptosis. This group also presented increased epithelial cell proliferation. CONCLUSION: BBG, a known highly selective inhibitor of P2X7R, attenuated renal inflammation, collagen synthesis, renal cell apoptosis, and enhanced renal cell proliferation in the early phase of rat model of UUO.
Assuntos
Proliferação de Células/efeitos dos fármacos , Rim/patologia , Nefrite/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Corantes de Rosanilina/uso terapêutico , Obstrução Ureteral/complicações , Actinas/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Apoptose/efeitos dos fármacos , Movimento Celular , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Colágeno Tipo IV/genética , Fibrose , Proteínas de Choque Térmico HSP47/metabolismo , Interleucina-1beta/genética , Rim/metabolismo , Túbulos Renais/patologia , Macrófagos/fisiologia , Masculino , Miofibroblastos/fisiologia , Nefrite/etiologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Corantes de Rosanilina/farmacologia , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismo , Regulação para CimaRESUMO
Prognostic biomarkers for recurrence of Oral Squamous Cell Carcinoma (OSCC) are urgently needed. We aimed to independently validate a 4-gene expression signature (MMP1, COL4A1, P4HA2, THBS2) predictive of OSCC recurrence risk. Gene expression was measured using Nanostring nCounter® in 245 histologically normal surgical resection margins from 62 patients. Association between risk scores for individual patients and recurrence was assessed by Kaplan-Meier analysis. Signature performance was quantified by concordance index (CI), hazard ratio (HR) and the area under receiver operating characteristics (AUC). Risk scores for recurrence were significantly higher than recurrence-free patients (p = 9.58e-7, Welch's t-test). A solid performance of the 4-gene signature was determined: CI = 0.64, HR = 3.38 (p = 1.4E-4; log-rank test), AUC = 0.71. We showed that three margins per patient are sufficient to preserve predictive performance (CI = 0.65; HR = 2.92; p = 2.94e-3; AUC = 0.71). Association between the predicted risk scores and recurrence was assessed and showed HR = 2.44 (p = 9.6E-3; log-rank test, N = 62). Signature performance analysis was repeated using an optimized threshold (70th percentile of risks), resulting in HR = 3.38 (p = 1.4E-4; log-rank test, N = 62). The 4-gene signature was validated as predictive of recurrence risk in an independent cohort of patients with resected OSCC and histologically negative margins, and is potentially applicable for clinical decision making on adjuvant treatment and disease monitoring.
Assuntos
Carcinoma de Células Escamosas/diagnóstico , Colágeno Tipo IV/genética , Metaloproteinase 1 da Matriz/genética , Neoplasias Bucais/diagnóstico , Prolil Hidroxilases/genética , Trombospondinas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Feminino , Seguimentos , Humanos , Masculino , Margens de Excisão , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Recidiva Local de Neoplasia , Prognóstico , TranscriptomaRESUMO
Background: This study was undertaken to analyze if different preparation and exposure periods of eluates from ocular prosthesis acrylic resin influence the cytotoxicity for conjunctival cells. Methods: Twenty-four acrylic resin specimens were divided, according to the period of eluate exposure to Chang conjunctival cells (24 and 72 hours). Eluates were prepared in four different ways: 24, 48, and 72 hours of resin specimen immersion in medium and 24 hours of immersion in water, followed by 24 hours of immersion in medium. MTT assay was used to evaluate the cytotoxic effect. The production of IL-1ß, IL-6, TNF-α, and chemokine macrophage inflammatory protein 1α was evaluated by ELISA, while the mRNA expression of type IV collagen (COL IV), transforming growth factor ß (TGF-ß), and matrix metalloproteinase 9 (MMP9) were evaluated by real-time RT-PCR technique. The statistical analysis was carried out using ANOVA with Bonferroni post-hoc test and the student's t-test (p < 0.05). Results: Significant quantities of IL-6 (4.594 pg/mL) and mRNA expression of COL IV (1.58) were verified at 72 hours of eluate exposure to cells, as compared to 24 hours. After the 72-hour exposure of eluates to cells, lower cell proliferation (88.4%) and higher IL-6 quantities (12.374 pg/mL), as well as mRNA expression of COL IV (2.21), TGF-ß (2.02), and MMP9 (5.75) were observed, which corresponded to 72 hours of a specimen immersed in medium. Conclusion: Longer periods of eluate preparation and exposure from the acrylic resin to cells are related to higher production of proinflammatory cytokines and extracellular matrix proteins.
Assuntos
Resinas Acrílicas/toxicidade , Túnica Conjuntiva/patologia , Olho Artificial , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Anomalous histoarchitecture with increased levels of type-V collagen (Col V) in lungs of human idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM) airway-centered interstitial fibrosis suggest that this collagen can be a possible trigger involved in the pathogenesis of these diseases. Butylated hydroxytoluene (BHT) injury model revealed a distal involvement of lung parenchyma with significant endothelial injury and fibrotic response, contrasting with the BLM airway-centered insult. We undertook this study to analyze whether BHT alters distal airway/alveolar epithelial cells (AECs) and extracellular matrix (ECM) signaling involved in the initiation and progression of pulmonary fibrosis in a different pathway concerning overexpression of Col V. Female mice C57BL/6 (n=6) were instilled intraperitoneally with 400 mg/kg of BHT dissolved in 1 mL of corn oil and euthanized at day 14 or 21 after BHT administration. Morphometry, immunohistochemistry and transmission electron microscopy were performed to characterize microscopic and submicroscopic changes of AECs and endothelial cells through transforming growth factor beta (TGF-ß) basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) expression. Immunofluorescence and immunogold electron microscopy were performed to characterize Col V. Quantitative polymerase chain reaction (qPCR) was used to confirm differential levels of RNA messenger. BHT lungs showed marked fibrotic areas and hyperplastic AECs. The alveolar damage caused destruction of elastic fibers and a critical increase of Col V in ECM of distal lung parenchyma. Fibrogenesis-promoting markers TGF-ß, bFGF and VEGF were also overexpressed in situ, coinciding with up-regulation in remodeling enzymes, growth factors, cytokines, transduction and transcription genes. BHT alters distal lung parenchyma signaling involved in pulmonary fibrosis highlighted similarities to human IPF in a pathway involving Col V arising as a promissory model to identify effective therapeutic targets.
Assuntos
Hidroxitolueno Butilado , Colágeno Tipo IV/metabolismo , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Animais , Colágeno Tipo IV/genética , Modelos Animais de Doenças , Células Epiteliais/ultraestrutura , Matriz Extracelular/genética , Matriz Extracelular/ultraestrutura , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Pulmão/ultraestrutura , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Alport syndrome (AS) is a disorder of collagen IV, a component of glomerular basement membrane (GBM). The association of AS and immunocomplex nephropathies is uncommon. This is a case of a 37-year-old woman with family history of X-linked AS, including 4 affected sons. This patient developed full-blown nephrotic syndrome along a 3-month period, a presentation not consistent with AS progression. This scenario suggested an alternative diagnosis. A kidney biopsy was therefore performed, showing membranous nephropathy (MN) in addition to GBM structural alterations compatible with AS. Whole exome sequencing also confirmed the diagnosis of X-linked AS, revealing a heterozygous pathogenic mutation in COL4A5. While a negative serum anti-phospholipase A2 receptor did not rule out a primary form of MN, it was also uncertain whether positive serologic tests for syphilis could represent a secondary factor. It is currently unknown whether this unusual association represents AS susceptibility to immunocomplex-mediated diseases or simply an association of 2 disorders.
Assuntos
Glomerulonefrite Membranosa/complicações , Nefrite Hereditária/complicações , Adulto , Colágeno Tipo IV/genética , Progressão da Doença , Suscetibilidade a Doenças , Exoma , Feminino , Glomerulonefrite Membranosa/genética , Glomerulonefrite Membranosa/patologia , Humanos , Rim/patologia , Mutação , Nefrite Hereditária/genética , Nefrite Hereditária/patologia , Síndrome Nefrótica/etiologia , Síndrome Nefrótica/genética , LinhagemRESUMO
The aim of this study was to evaluate the influence of pigment incorporation on the cytotoxicity of ocular prosthesis N1 color acrylic resin. Nine samples were manufactured by heat-polymerization in water bath and divided into 3 groups: acrylic resin without pigment incorporation (group R), acrylic resin with pigment incorporation (group RP), and acrylic pigment (group P). Eluates formed after 72h of sample immersion in medium were incubated with conjunctival cell line (Chang conjunctival cells) for 72h. The negative control group consisted in medium without samples (group C). The cytotoxic effect from the eluates was evaluated using MTT assay (cell proliferation), ELISA assay (quantification of IL1ß, IL6, TNF α and CCL3/MIP1α) and RT-PCR assay (mRNA expression of COL IV, TGF ß and MMP9). Data were submitted to ANOVA with Bonferroni post-tests (p<0.05). All groups were considered non-cytotoxic based on cell proliferation. However, resin with pigment incorporation showed significant IL6 quantity increase. Resin without pigment incorporation exhibited higher mRNA expression of COL IV, MMP9 and TGF ß, however it was also observed for the negative control group. The materials exhibited divergent biological behavior. Despite the pigment incorporation that resulted in an increase of IL6, no cytotoxicity was observed based on cell proliferation.