RESUMEN
The aberrant expression of microRNAs (miRNAs) is always associated with tumor development and progression. Microvascular proliferation is one of the unique pathologic features of glioblastoma (GBM) . In this study, the microvasculature from GBM or normal brain tissue derived from neurosurgeries was purified and total RNA was isolated from purified microvasculature. The difference of miRNA expression profiles between glioblastoma microvasculature and normal brain capillaries was investigated. It was found that miR-7-5p in GBM microvessels was significantly reduced compared with that in normal brain capillaries. In the in vitro experiments, overexpression of miR-7-5p significantly inhibited human umbilical vein endothelial cell proliferation. Forced expression of miR-7-5p in human umbilical vein endothelial cells in vitro significantly reduced the protein level of RAF1 and repressed the activity of the luciferase, a reporter vector carrying the 3'-untranslated region of RAF1. These findings indicate that RAF1 is one of the miR-7-5p target genes. Furthermore, a significant inverse correlation between miR-7-5p expression and RAF1 protein level in GBM microvasculature was found. These data suggest that miR-7-5p functions as a tumor suppressor gene to regulate GBM microvascular endothelial cell proliferation potentially by targeting the RAF1 oncogene, implicating an important role for miR-7-5p in the pathogenesis of GBM. It may serve as a guide for the antitumor angiogenesis drug development.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , MicroARNs/genética , Microvasos/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Adulto , Anciano , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/genética , Proliferación Celular , Regulación hacia Abajo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor , Glioblastoma/irrigación sanguínea , Glioblastoma/genética , Humanos , Masculino , Microvasos/patología , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-raf/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , TranscriptomaRESUMEN
BACKGROUND: Cancer-associated fibroblasts (CAFs) play important roles in tumor microenvironments. Pyrroline-5-carboxylate reductase 1 (PYCR1) is a potential cancer therapy target. This study aimed to explore the expression of PYCR1 in glioma-associated CAFs and analyze the effects of PYCR1 expression in CAFs on the proliferation of C6 glioma. METHODS: A rat glioma model was induced by injecting C6 cells in the right caudate putamen via a microliter syringe. After 14 days, tumor tissues were collected, and the levels of COL1A1 and PYCR1 were measured by immunohistochemistry. The colocalization of fibroblast activation protein α (FAP) and PYCR1 in tissues was measured by double-immunofluorescence. The CAFs were labeled by FAP and isolated from the tumor tissues using a fluorescence-activated cell sorting (FACS) machine. The isolated CAFs were further separated into CAFs with different PYCR1 expressions using the FACS machine. CAFs with different PYCR1 expressions were respectively cocultured with C6 cells or MUVECs for 48h using a Transwell permeable support. The invasion and proliferation of C6 cells were measured using a Transwell assay and colony formation assay, and the angiogenesis of MUVECs was measured using a Tube formation assay. The expression of COL1A1 and PYCR1 proteins in C6 cells and VEGF-A and EGF proteins in MUVECs was measured by western blotting. PYCR1 silencing in C6 cells was induced by PYCR1 siRNA transfection, the effects of which on the proliferation of C6 cells were measured using a wound healing assay, a Transwell assay, and western blotting. RESULTS: The PYCR1 and COL1A1 upregulation co-occurred in the rat glioma, and PYCR1 was expressed in CAFs. The CAF coculture enhanced the invasion and proliferation of C6 cells and the angiogenesis of MUVECs. Meanwhile, the levels of COL1A1 protein in C6 cells, and the levels of VEGF-A and EGF proteins in MUVECs were increased after CAF coculture. Moreover, the effects of CAF coculture were increased with PYCR1 expression in the CAF. Silencing PYCR1 suppressed the migration and invasion of C6 cells, and decreased the levels of COL1A1 and VEGF-A proteins in C6 cells. CONCLUSIONS: PYCR1 is expressed in glioma-associated CAFs, and promotes the proliferation of C6 cells and angiogenesis of MUVECs, suggesting that targeting PYCR1 may be a therapeutic strategy for glioma.
RESUMEN
Chronic traumatic encephalopathy (CTE) is a distinct neurodegenerative disease that associated with repetitive head trauma. CTE is neuropathologically defined by the perivascular accumulation of abnormally phosphorylated tau protein in the depths of the sulci in the cerebral cortices. In advanced CTE, hyperphosphorylated tau protein deposits are found in widespread regions of brain, however the mechanisms of the progressive neurodegeneration in CTE are not fully understood. In order to identify which proteomic signatures are associated with CTE, we prepared RIPA-soluble fractions and performed quantitative proteomic analysis of postmortem brain tissue from individuals neuropathologically diagnosed with CTE. We found that axonal guidance signaling pathwayrelated proteins were most significantly decreased in CTE. Immunohistochemistry and Western blot analysis showed that axonal signaling pathway-related proteins were down regulated in neurons and oligodendrocytes and neuron-specific cytoskeletal proteins such as TUBB3 and CFL1 were reduced in the neuropils and cell body in CTE. Moreover, oligodendrocyte-specific proteins such as MAG and TUBB4 were decreased in the neuropils in both gray matter and white matter in CTE, which correlated with the degree of axonal injury and degeneration. Our findings indicate that deregulation of axonal guidance proteins in neurons and oligodendrocytes is associated with the neuropathology in CTE. Together, altered axonal guidance proteins may be potential pathological markers for CTE.
RESUMEN
Glioma, one of the most common cancers in human, is classified to different grades according to the degrees of malignancy. Glioblastoma (GBM) is known to be the most malignant (Grade IV) whereas low-grade astrocytoma (LGA, Grade II) is relatively benign. The mechanism underlying the pathogenesis and progression of glioma malignancy remains unclear. Here we report a quantitative proteomic study to elucidate the differences between GBM and LGA using liquid chromatography and tandem mass spectrometry followed by label-free quantification. A total of 136 proteins were differentially expressed in GBM for at least five folds in comparison with LGA. Ontological analysis revealed a close correlation between GBM-associated proteins and RNA processing. Interaction network analysis indicated that the GBM-associated proteins in the RNA processing were linked to crucial signaling transduction modulators including epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 1 (STAT1), and mitogen-activated protein kinase 1 (MAPK1), which were further connected to the proteins important for neuronal structural integrity, development and functions. Upregulation of 40S ribosomal protein S5 (RPS5), Ferritin Heavy chain (FTH1) and STAT1, and downregulation of tenascin R (TNR) were validated as representatives by immune assays. In summary, we revealed a panel of GBM-associated proteins and the important modulators centered at the RNA-processing network in glioma malignancy that may become novel biomarkers and help elucidate the underlying mechanism.
Asunto(s)
Glioblastoma/genética , Glioma/genética , Proteómica , ARN/genética , Adulto , Astrocitoma/genética , Astrocitoma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioma/patología , Humanos , Masculino , Persona de Mediana Edad , Proteómica/métodos , Transducción de Señal/genéticaRESUMEN
Lysine acetylation is known as a post translational modification (PTM) by histone acetyltransferases (HAT) that modifies histones and non-histone proteins to regulate gene expression. Serine acetylation, however, is reported in mammalian hosts by serine acetyltransferase of Yersinia pestis (YopJ) during infection. The protein target and cellular function of bacterial YopJ in mammalian systems are not fully addressed. Here we report dual acetylation at the serine and lysine residues by transiently expressed serine acetyltransferase YopJ mimicking Y. pestis infection in HeLa cells. Using shotgun proteomics followed by label-free quantification, we demonstrate an increase of dual acetylation in YopJ transfected human cells, including 10 Ser- (YopJ/non-YopJ 1.3-fold, p = 0.02) and 8 Lys- (YopJ/non-YopJ 3.5-fold, p = 0.00003) acetylation sites. Specifically, YopJ expression augments acetylation of membrane-associated E3 ubiquitin ligase MARCH8 at the serine residue Sac44, Sac71 and Sac253, and the lysine residue Kac247 and Kac252. YopJ-mediated Ser- and Lys-acetylation of MARCH8 is further confirmed by Western blotting using the specific antibodies against MARCH8 Sac71 and pan-acetyl lysine. Functional study demonstrates that YopJ-mediated Ser- and Lys-acetylation affects the auto-ubiquitination of MARCH8. The mutant C172A of YopJ previously shown to abolish the acetyltransferase activity also reduces Ser- and Lys-acetylation and diminishes the auto-ubiquitination of MARCH8. In support, MARCH8 is indeed acetylated at serine and lysine in vitro by purified YopJ but the activity is reduced by the C172A mutant in YopJ. Our study provides evidence that bacterial serine acetyltransferase YopJ mediates Ser- and Lys-acetylation and affects auto-ubiquitination of ubiquitin ligase MARCH8 in human cells.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Lisina/metabolismo , Serina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Acetilación , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Biocatálisis , Células HeLa , Humanos , Inmunoensayo , Péptidos/química , Péptidos/metabolismo , Reproducibilidad de los Resultados , Ubiquitina-Proteína Ligasas/química , Yersinia pestisRESUMEN
ADAR1 is a double-stranded RNA (dsRNA) editing enzyme that specifically converts adenosine to inosine. ADAR1 is ubiquitously expressed in eukaryotes and participate in various cellular processes such as differentiation, proliferation and immune responses. We report here a new proteomics study of HEK293T cells with and without ADAR1 overexpression. The up- and down-regulated proteins by ADAR1 overexpression are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by label-free protein quantification. Totally 1,495 proteins (FDR < 0.01) are identified, among which 211 are up- and 159 are down-regulated for at least 1.5-fold (n = 3, p < 0.05). Gene ontology analysis reveals that these ADAR1-regulated proteins are involved in protein translation and cell cycle regulation. Bioinformatics analysis identifies a closely related network consistent for the protein translation machinery and a tightly connected network through proliferating cell nuclear antigen (PCNA)-interactions. Up-regulation of the proteins in the PCNA-mediated cell proliferation network is confirmed by Western blotting. In addition, ADAR1 overexpression is confirmed to increase cell proliferation in HEK293T cells and A549 cells. We conclude that ADAR1 overexpression modulates the protein translation and cell cycle networks through PCNA-mediated protein-protein interaction to promote cell proliferation in HEK293 cells.