RESUMO
Os ribossomos são complexos ribonucleoproteicos conservados formados por duas subunidades assimétricas (40S e 60S em eucariotos) responsáveis pela tradução da informação genética e catálise da síntese proteica. A montagem destes complexos em eucariotos é mais bem descrita em S. cerevisiae, constituindo um processo celular energeticamente dispendioso e com múltiplas etapas. Ela tem origem no nucléolo com a transcrição do pré-rRNA 35S e requer o recrutamento hierárquico e transiente de cerca de 200 fatores de montagem para garantir a formação correta dos centros funcionais aptos à tradução. Neste processo, que se estende no núcleo e citoplasma, 79 proteínas ribossomais associam-se gradativamente à medida que o prérRNA é dobrado, modificado e processado. O processamento do pré-rRNA 35S consiste na remoção progressiva de espaçadores internos (ITS1 e ITS2) e externos (5ETS e 3ETS), que separam e flanqueiam os rRNAs maduros componentes de ambas subunidades ribossomais. A clivagem do ITS1 separa as vias de maturação do pré-60S e do pré-40S. O ITS2, que, em associação a fatores de montagem, forma uma estrutura denominada ITS2-foot, é o último espaçador do pré-60S a ser removido. A composição do ITS2-foot permanece inalterada no nucléolo até a transição entre o estado E nucleolar e a formação da partícula Nog2 nuclear. Nesta etapa, a liberação do fator Erb1 permite o recrutamento do fator de montagem conservado e essencial Nop53. Na base do ITS2-foot, Nop53 recruta o exossomo via RNA helicase Mtr4 para a clivagem 3-5 exonucleolítica de parte do ITS2 levando à desmontagem do ITS2-foot. O fato de Nop53 atuar como ponte entre dois grandes complexos e apresentar uma estrutura flexível e estendida nos levou a aprofundar a caracterização de seu papel durante a maturação do pré60S. Neste trabalho, usando análise proteômica quantitativa label-free baseada em espectrometria de massas, caracterizou-se o interactoma de Nop53, e avaliou-se o impacto da depleção de Nop53 no interactoma da subunidade catalítica do exossomo Rrp6 e na composição de pré-ribossomos representativos de quase todas as etapas de maturação do pré-60S. Em paralelo, foram caracterizados mutantes truncados de Nop53 e avaliada por pull-down a interação de Nop53 com componentes do exossomo. Os resultados obtidos mostraram que Nop53 é capaz de interagir com o cofator do exossomo Mpp6, sugerindo pontos adicionais de interação durante o recrutamento do exossomo ao pré-60S. A análise do interactoma de Rrp6 mostrou uma associação precoce do exossomo aos intermediários pré-ribossomais nucleolares mais iniciais, anteriores aos previamente descritos. Mudanças na composição dos intermediários pré-60S revelaram que a depleção de Nop53 afeta a transição entre o estado E e a partícula Nog2, afetando eventos tardios de maturação como o recrutamento de Yvh1. Comparando-se o efeito da depleção de Nop53 com o de mutantes nop53 desprovidos da região de recrutamento do exossomo, obtivemos evidências bioquímicas do papel estrutural de Nop53 na base do ITS2- foot. Em conjunto, estas observações, à luz de estruturas de intermediários pré-ribossomais recentemente descritas, nos permitiram concluir que o recrutamento de Nop53 ao pré-60S contribui para a estabilização de eventos de remodelamento do rRNA que antecedem a formação da partícula Nog2
Ribosomes are conserved ribonucleoprotein complexes formed by two asymmetric subunits (the 40S and the 60S in eukaryotes) responsible for translating the genetic information and catalyzing protein synthesis. The assembly of these complexes in eukaryotes is best described in S. cerevisiae. It is an energetically demanding, multi-step cellular process, that starts in the nucleolus with the transcription of the 35S pre-rRNA. It requires the hierarchical and transient recruitment of about 200 assembly factors to ensure the correct formation of the functional centers suitable for translation. In this process, which extends into the nucleus and cytoplasm, 79 ribosomal proteins gradually associate as the pre-rRNA is folded, modified, and processed. The 35S pre-rRNA processing happens with the progressive removal of internal (ITS1 and ITS2) and external (5'ETS and 3'ETS) transcribed spacers, which separate and flank the mature rRNA components of both ribosomal subunits. The cleavage at the ITS1 separates the pre-60S and pre40S maturation pathways. The ITS2, which in association with assembly factors constitutes a structure called ITS2-foot, is the last pre-60S spacer to be removed. The composition of the ITS2- foot remains unchanged in the nucleolus until the transition between the nucleolar state E and the nuclear Nog2 particle. At this stage, the release of Erb1 allows the recruitment of the conserved and essential assembly factor Nop53. At the base of the ITS2-foot, Nop53 recruits the exosome via the RNA helicase Mtr4 for the ITS2 3'-5' exonucleolytic cleavage leading to the ITS2-foot disassembly. The fact that Nop53 acts as a bridge between these two large complexes and exhibits a flexible and extended structure led us to further characterize its role in the pre-60S maturation. In this work, using mass spectrometry-based label-free quantitative proteomics, we characterized the interactome of Nop53, as well as the impact of the depletion of Nop53 on the interactome of the exosome catalytic subunit Rrp6 and on the composition of pre-ribosomes representative of almost all pre-60S maturation stages. In parallel, we characterized nop53 truncated mutants and evaluated the interaction of Nop53 with exosome components by pulldown assays. The results showed that Nop53 can interact with the exosome cofactor Mpp6, suggesting the contribution of additional points of interaction during the exosome recruitment to the pre-60S. The analysis of the Rrp6 interactome revealed an early association of the exosome with pre-ribosomal intermediates at very early nucleolar stages, before those previously described. Changes in the composition of pre-60S intermediates revealed that Nop53 depletion affects the transition between the state E and the Nog2 particle, affecting late pre-60S maturation events, such as the Yvh1 recruitment. Comparing the effect of Nop53 depletion with that of nop53 mutants lacking the exosome interacting region, we obtained biochemical evidence of the structural role of Nop53 at the base of the ITS2-foot. Altogether, and in light of recently described structures of pre-ribosomal intermediates, these observations allowed us to conclude that the recruitment of Nop53 to the pre-60S contributes to the stabilization of rRNA remodeling events that precede the formation of the Nog2 particle
Assuntos
Saccharomyces cerevisiae/classificação , Subunidades Ribossômicas/química , Ribonucleoproteínas , Proteínas Ribossômicas , Espectrometria de Massas/métodos , Nucléolo Celular , Subunidades Ribossômicas Maiores , EucariotosRESUMO
OBJECTIVE@#To explore the role of TRIM21 in modulating the invasive phenotype of hepatocellular carcinoma (HCC) cells and its mechanism of action.@*METHODS@#RNA interference technique was used to knock down the expression of TRIM21 and β-catenin, alone or in combination, in HCC cell lines 97H and LM3, and the interfering efficiency and the activity of closely related pathways were determined using Western blotting. The two cells with TRIM21 knockdown (siTRIM21 97H and siTRIM21 LM3 cells) were assessed for their invasion ability in vitro using Transwell invasion assay, and the lung metastasis capacity of siTRIM21 LM3 cells following tail vein injection was evaluated in nude mice. The binding of TRIM21 with β-catenin and the ubiquitylation level of β-catenin in TRIM21-overexpressing HEK293 cells were determined with Western blotting and co-immunoprecipitation assay. We also compared the overall survival of patients with CTNNB1highTRIM21high and CTNNB1highTRIM21low HCC subtypes using Kaplan-Meier method based on filtrated and grouped HCC clinical data from TCGA database.@*RESULTS@#TRIM21 knockdown significantly enhanced the invasion ability of 97H and LM3 cells in vitro (P < 0.01 or 0.05) and the lung metastasis ability of LM3 cells in nude mice (P < 0.01), and simultaneous knockdown of β -catenin obviously suppressed the in vitro invasiveness of the cells (P < 0.0001 or 0.05). Co-immunoprecipitation assay showed that TRIM21 was capable of directly binding with β-catenin protein to accelerate the ubiquitination and degradation of the latter, leading to inhibition of nuclear translocation of β-catenin and hence reduced invasiveness of HCC cells. Bioinformatic analysis showed that compared patients with CTNNB1highTRIM21low HCC subtype where Wnt pathway was activated, the patients with CTNNB1highTRIM21high HCC subtype had a significantly better survival outcomes (P < 0.05).@*CONCLUSION@#A high expression of TRIM21 suppresses the invasion of HCC cells by promoting β-catenin ubiquitylation and degradation, which possibly explains the poor prognosis of CTNNB1highTRIM21low HCC patients.
Assuntos
Animais , Humanos , Camundongos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Neoplasias Hepáticas/patologia , Camundongos Nus , Ribonucleoproteínas/genética , Ubiquitinação , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
OBJECTIVE: To determine the role of RNA-binding protein with serine-rich domain 1 (RNPS1) in uterine corpus endometrial carcinoma (UCEC), the role of RNPS1 knockdown in UCEC development in vitro and in vivo, and the relationship between RNPS1 and mismatch repair (MMR) in UCEC. METHODS: We predicted the potential function of RNPS1 using bioinformatics systems. The expression of RNPS1 in tissues and cell lines was analyzed by western blotting and immunohistochemistry. The expression of RNPS1 in MMR was assessed using bioinformatics and western blotting. The proliferation and apoptosis of UCEC cells were assessed under RNPS1 knockdown conditions, and RNPS1 regulation in MMR was detected by suppressing Notch signaling. Associations between RNPS1 and gene mutations in UCEC and prognosis were analyzed. RESULTS: The RNPS1 level was higher in UCEC tumors than in normal tissues and tumors or RL952 cells. Prognostic outcomes were worse when UCEC showed abundant RNPS1 expression. Lentiviral RNPS1 knockdown weakened tumor cell proliferation and suppressed biomarker expression, reduced the tumor volume, promoted apoptosis in vitro and in vivo, and inhibited UCEC development. Increased MutS homolog 2 (MSH2) and MutS homolog 6 (MSH6) levels in MMR after RNPS1 knockdown were reversed by inhibiting Notch signaling. Furthermore, RNPS1 was associated with mutations in NAA11, C2orf57, NUPR1, and other genes involved in UCEC prognosis. CONCLUSION: RNPS1 may regulate the expression levels of MSH2 and MSH6 in MMR, enhancing the proliferation, development, and prognosis of UCEC through a Notch signaling pathway in UCEC. Our study offers a new method and strategy for delaying UCEC development through modulating MMR.
Assuntos
Humanos , Feminino , Ribonucleoproteínas/genética , Neoplasias do Endométrio/genética , Carcinoma Endometrioide/congênito , Serina , Proteínas de Ligação a RNA , Linhagem Celular Tumoral , Instabilidade de MicrossatélitesRESUMO
OBJECTIVE@#To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD).@*METHODS@#ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting.@*RESULTS@#We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6.@*CONCLUSIONS@#7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.
Assuntos
Humanos , Proteínas de Ciclo Celular , Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Células HeLa , Proteínas Nucleares , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA , Ribonucleoproteínas , Fatores de TranscriçãoRESUMO
ABSTRACT Pubertal timing in humans is determined by complex interactions including hormonal, metabolic, environmental, ethnic, and genetic factors. Central precocious puberty (CPP) is defined as the premature reactivation of the hypothalamic-pituitary-gonadal axis, starting before the ages of 8 and 9 years in girls and boys, respectively; familial CPP is defined by the occurrence of CPP in two or more family members. Pioneering studies have evidenced the participation of genetic factors in pubertal timing, mainly identifying genetic causes of CPP in sporadic and familial cases. In this context, rare activating mutations were identified in genes of the kisspeptin excitatory pathway (KISS1R and KISS1 mutations). More recently, loss-of-function mutations in two imprinted genes (MKRN3 and DLK1) have been identified as important causes of familial CPP, describing novel players in the modulation of the hypothalamic-pituitary-gonadal axis in physiological and pathological conditions. MKRN3 mutations are the most common cause of familial CPP, and patients with MKRN3 mutations present clinical features indistinguishable from idiopathic CPP. Meanwhile, adult patients with DLK1 mutations present high frequency of metabolic alterations (overweight/obesity, early onset type 2 diabetes and hyperlipidemia), indicating that DLK1 may be a novel link between reproduction and metabolism. Arch Endocrinol Metab. 2019;63(4):438-44
Assuntos
Humanos , Puberdade Precoce/genética , Fenótipo , Puberdade Precoce/etiologia , Ribonucleoproteínas/genética , Proteínas de Ligação ao Cálcio , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Kisspeptinas/genética , Receptores de Kisspeptina-1/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metilação , MutaçãoRESUMO
BACKGROUND: Chronic inflammation has been linked to insulin resistance and type 2 diabetes mellitus (T2DM). High-fat diet (HFD)-derived fatty acid is associated with the activation of chronic inflammation in T2DM. PF-04620110, which is currently in phase 1 clinical trials as a selective acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) inhibitor, is a potent anti-diabetic agent that may be important for the regulation of chronic inflammation in T2DM. However, the mechanisms by which PF-04620110 regulates fatty acid-induced chronic inflammation remain unclear. METHODS: PF-04620110 was used in vitro and in vivo. DGAT1-targeting gRNAs were used for deletion of mouse DGAT1 via CRISPR ribonucleoprotein (RNP) system. The activation of NLRP3 inflammasome was measured by immunoblot or cytokine analysis in vitro and in vivo. RESULTS: Here we show that PF-04620110 suppressed fatty acid-induced nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation in macrophages. In contrast, PF-04620110 did not change the activation of the NLR family, CARD-domain-containing 4 (NLRC4), or the absent in melanoma 2 (AIM2) inflammasomes. Moreover, PF-04620110 inhibited K⁺ efflux and the NLRP3 inflammasome complex formation, which are required for NLRP3 inflammasome activation. PF-04620110 reduced the production of interleukin 1β (IL-1β) and IL-18 and blood glucose levels in the plasma of mice fed HFD. Furthermore, genetic inhibition of DGAT1 suppressed fatty acid-induced NLRP3 inflammasome activation. CONCLUSION: Our results suggest that PF-04620110 suppresses fatty acid-induced NLRP3 inflammasome activation.
Assuntos
Animais , Humanos , Camundongos , Glicemia , Ensaios Clínicos Fase I como Assunto , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Diabetes Mellitus Tipo 2 , Diacilglicerol O-Aciltransferase , Dieta Hiperlipídica , Ácidos Graxos , Técnicas In Vitro , Inflamassomos , Inflamação , Resistência à Insulina , Interleucina-18 , Interleucinas , Macrófagos , Melanoma , Plasma , RibonucleoproteínasRESUMO
<p><b>OBJECTIVE</b>Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21 (TRIM21) in osteosarcoma cell proliferation.</p><p><b>METHODS</b>3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation (BIFC) were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.</p><p><b>RESULTS</b>TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However, overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.</p><p><b>CONCLUSION</b>Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.</p>
Assuntos
Humanos , Proteínas 14-3-3 , Genética , Metabolismo , Proliferação de Células , Genética , Osteossarcoma , Genética , Ribonucleoproteínas , Genética , Metabolismo , Células Tumorais CultivadasRESUMO
The valproic acid (VPA)-induced animal model is one of the most widely utilized environmental risk factor models of autism. Autism spectrum disorder (ASD) remains an insurmountable challenge among neurodevelopmental disorders due to its heterogeneity, unresolved pathological pathways and lack of treatment. We previously reported that VPA-exposed rats and cultured rat primary neurons have increased Pax6 expression during post-midterm embryonic development which led to the sequential upregulation of glutamatergic neuronal markers. In this study, we provide experimental evidence that telomerase reverse transcriptase (TERT), a protein component of ribonucleoproteins complex of telomerase, is involved in the abnormal components caused by VPA in addition to Pax6 and its downstream signals. In embryonic rat brains and cultured rat primary neural progenitor cells (NPCs), VPA induced the increased expression of TERT as revealed by Western blot, RT-PCR, and immunostainings. The HDAC inhibitor property of VPA is responsible for the TERT upregulation. Chromatin immunoprecipitation revealed that VPA increased the histone acetylation but blocked the HDAC1 binding to both Pax6 and Tert genes. Interestingly, the VPA-induced TERT overexpression resulted to sequential upregulations of glutamatergic markers such as Ngn2 and NeuroD1, and inter-synaptic markers such as PSD-95, α-CaMKII, vGluT1 and synaptophysin. Transfection of Tert siRNA reversed the effects of VPA in cultured NPCs confirming the direct involvement of TERT in the expression of those markers. This study suggests the involvement of TERT in the VPA-induced autistic phenotypes and has important implications for the role of TERT as a modulator of balanced neuronal development and transmission in the brain.
Assuntos
Animais , Feminino , Gravidez , Ratos , Acetilação , Transtorno do Espectro Autista , Transtorno Autístico , Western Blotting , Encéfalo , Imunoprecipitação da Cromatina , Desenvolvimento Embrionário , Histonas , Modelos Animais , Transtornos do Neurodesenvolvimento , Neurônios , Fenótipo , Características da População , Ribonucleoproteínas , Fatores de Risco , RNA Interferente Pequeno , Células-Tronco , Sinaptofisina , Telomerase , Transfecção , Regulação para Cima , Ácido ValproicoRESUMO
PURPOSE: Splicing factors play important roles in tumorigenesis. Serine/arginine-rich splicing factors 2 (SRSF2) and SRSF4 proteins, the members of SR family proteins, are dysregulated in various cancers. However, their protein expression levels and diagnostic values are unclear in colorectal cancer.METHODS: We quantified the protein levels of SRSF2, SRSF4, and previously known colon cancer markers (heterogeneous nuclear ribonucleoprotein A1 [HNRNPA1] and carcinoembryonic antigen [CEA]) in tumor compared with adjacent normal-looking areas (non-tumor) of the colon in Korean patients with colon cancer using immunoblot analysis.RESULTS: The protein levels of HNRNPA1 and CEA were remarkably increased in tumor compared to non-tumor tissue and up-regulated in all of the tumor samples. However, the protein levels of SRSF2 and SRSF4 in tumor tissue were reduced in contrast with those of non-tumor tissue.CONCLUSION: None of the SRSF proteins were significantly different between the low (≤II) and high (>II) stages.
Assuntos
Humanos , Antígeno Carcinoembrionário , Carcinogênese , Colo , Neoplasias do Colo , Neoplasias Colorretais , RibonucleoproteínasRESUMO
Enterovirus 71 (EV71) is a neurotropic pathogen that can induce hand, foot and mouth disease in children. There is an appreciable mortality rate after EV71 infections. The mechanism of action of EV71 replication is not known. Recent work has identified some of cell factors of the host that participate in the synthesis of the RNA and proteins of EV71 (e.g., hnRNP K, reticulon 3 (RTN 3)). In that work, researchers used a competitive assay to show that hnRNP K can interact with EV71 5' UTR, which is required for efficient synthesis of viral RNA. Using a yeast two-hybrid system, other researchers demonstrated that RTN 3 interacts with the N-terminal domain of EV71 2C, which is crucial for replication of viral RNA. Here, we discuss recent work focusing on the molecular mechanisms of hnRNP K and RTN 3 in the synthesis of the RNA and proteins of EV71.
Assuntos
Animais , Humanos , Proteínas de Transporte , Genética , Metabolismo , Enterovirus Humano A , Genética , Fisiologia , Infecções por Enterovirus , Genética , Metabolismo , Virologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Interações Hospedeiro-Patógeno , Proteínas de Membrana , Genética , Metabolismo , Proteínas do Tecido Nervoso , Genética , Metabolismo , Ribonucleoproteínas , Genética , Metabolismo , Proteínas Virais , Genética , Metabolismo , Replicação ViralRESUMO
BACKGROUND: Recurrent somatic SET-binding protein 1 (SETBP1) and splicing pathway gene mutations have recently been found in atypical chronic myeloid leukemia and other hematologic malignancies. These mutations have been comprehensively analyzed in adult AML, but not in childhood AML. We investigated possible alteration of the SETBP1, splicing factor 3B subunit 1 (SF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and serine/arginine-rich splicing factor 2 (SRSF2) genes in childhood AML. METHODS: Cytogenetic and molecular analyses were performed to reveal chromosomal and genetic alterations. Sequence alterations in the SETBP1, SF3B1, U2AF1, and SRSF2 genes were examined by using direct sequencing in a cohort of 53 childhood AML patients. RESULTS: Childhood AML patients did not harbor any recurrent SETBP1 gene mutations, although our study did identify a synonymous mutation in one patient. None of the previously reported aberrations in the mutational hotspot of SF3B1, U2AF1, and SRSF2 were identified in any of the 53 patients. CONCLUSIONS: Alterations of the SETBP1 gene or SF3B1, U2AF1, and SRSF2 genes are not common genetic events in childhood AML, implying that the mutations are unlikely to exert a driver effect in myeloid leukemogenesis during childhood.
Assuntos
Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Proteínas de Transporte/genética , Estudos de Coortes , Análise Citogenética , Análise Mutacional de DNA , Frequência do Gene , Genótipo , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Polimorfismo de Nucleotídeo Único , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteínas/genéticaRESUMO
This study aims to investigate whether the nucleoprotein (NP) of severe fever with thrombocytopenia syndrome virus (SFTSV) can impact the cellular immunity of host cells. Gene segments that encode the NP and non-structural protein (NSs) of SFTSV were inserted into eukaryotic expression vector VR1012. Host proteins that interact with NP and affect immunity were identified with co-immunoprecipitation (IP), SDS-PAGE, mass spectrometry (MS), and Western blot. Co-localization of NP and the identified host proteins was confirmed by confocal microscopy. A 60kD SSA/Ro, a protein related to immunity, interacted with NP, as found by IP and MS. Confocal microscopy showed that NP and SSA/Ro were co-localized in cytoplasm. These results indicated that SFTSV NP may specifically bind to 60kD SSA/Ro and cause a series of immune responses and clinical symptoms.
Assuntos
Humanos , Infecções por Bunyaviridae , Genética , Metabolismo , Virologia , Células HEK293 , Nucleoproteínas , Genética , Metabolismo , Phlebovirus , Genética , Metabolismo , Ligação Proteica , Ribonucleoproteínas , Genética , Metabolismo , Proteínas Virais , Genética , MetabolismoRESUMO
<p><b>OBJECTIVE</b>To determine the association between U2 small nuclear ribonucleoprotein auxiliary factor 35/65 (U2AF35 and U2AF65) and pancreatic cancer (PC).</p><p><b>METHODS</b>A two-stage analysis case-control study was conducted. Four candidate tag single nucleotide polymorphisms (tagSNPs) were genotyped by Taqman Openarray assay in a screening population living in Central China (298 PC cases and 525 controls). Thereafter, rs310445 in U2AF65 was genotyped by TaqMan real-time polymerase chain reaction (RT-PCR) in a validation Chinese Han population from Beijing (413 cases and 557 controls).</p><p><b>RESULTS</b>rs310445 in U2AF65 gene was significantly associated with PC in both screened population and combined population. Subjects with C allele had a higher risk of PC compared to those with the TT genotype, with OR of 1.31 (95%CI:1.07-1.60, P = 0.010) for the combined population. A synergic effect of smoking and C allele of rs310445 was also observed in the combined population, with Synergic Index of 2.08 (95% CI:1.37-2.78) in the combined population.</p><p><b>CONCLUSION</b>Our findings suggested the interaction between smoking and U2AF65 might play a role in PC. These findings should be confirmed by further independently large-scale population studies.</p>
Assuntos
Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos de Casos e Controles , Genótipo , Proteínas Nucleares , Genética , Neoplasias Pancreáticas , Genética , Polimorfismo de Nucleotídeo Único , Ribonucleoproteínas , Genética , Fatores de Risco , Fumar , Fator de Processamento U2AFRESUMO
<p><b>BACKGROUND</b>Spliceosome mutations have been recently identified and associated with hematological malignancies. SRSF2, one of components of the splicing machinery, has a high mutation frequency during chronic myelomonocytic leukemia, according to previous reports. However, the relevance of this finding in Chinese populations remains unknown.</p><p><b>METHODS</b>We recruited 50 Chinese patients with chronic myelomonocytic leukemia to analyze the state of SRSF2 and to assess the corresponding clinical features by polymerase chain reaction followed by direct sequencing.</p><p><b>RESULTS</b>Ten of 50 patients (20%) harbored SRSF2 mutations, including five P95R, two 95H, and three P95L point mutations. The patient group was older than the wild type group (P < 0.01). No significant statistical differences were observed with regard to the other clinical characteristics (sex, peripheral blood count, serum lactate dehydrogenase, karyotype, World Health Organization classification, etc.) between these two groups. Two of the patients showed an early evolution to acute myeloid leukemia.</p><p><b>CONCLUSIONS</b>SRSF2 mutations are frequent in chronic myelomonocytic leukemia patients, but show a relatively lower incidence in Chinese patients. Moreover, the mutation can be related to old age and an unfavorable prognosis. Our results provide valuable insights for the development of a diagnostic marker, or for the identification of a therapeutic target for chronic myelomonocytic leukemia.</p>
Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Mutacional de DNA , Predisposição Genética para Doença , Genética , Leucemia Mielomonocítica Crônica , Genética , Mutação , Proteínas Nucleares , Genética , Ribonucleoproteínas , Genética , Fatores de Processamento de Serina-ArgininaRESUMO
PURPOSE: To investigate the relationship between plasma TDRD7 mRNA and lens transparency, and to evaluate plasma TDRD7 mRNA as a potential marker for cataracts and its sub-type by quantitatively analyzing human peripheral blood. METHODS: Plasma RNA was extracted from 40 patients with cataracts, and 30 normal controls of matched age and gender. Blood cholesterol and fasting glucose were measured, and the RNA extracted from the sample was synthesized into cDNA. After polymerase chain reaction, the results were compared after quantifying the TDRD7 mRNA using ABL1 mRNA for normalization. We analyzed the relative gene expression data via the DeltaDeltaCt method. RESULTS: The normalized 2(-DeltaDeltaCt) of plasma TDRD7 mRNA based on ABL1 mRNA was 1.52 ± 0.63 in the case of the control group and 1.05 ± 0.34 in the case of the cataract patients, and the TDRD7 expression level of the cataract patients was lower than that of the control group (p = 0.048). The comparison of the genetic values of different types of cataracts demonstrated that the TDRD7 expression level of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017). CONCLUSIONS: Human cataracts and the TDRD7 gene loss-of-function mutations are strongly causally related, as the expression level of plasma TDRD7 mRNA in patients with cataracts was statistically significantly lower than in the normal control group.
Assuntos
Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Catarata/sangue , Regulação da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas c-abl/genética , RNA Mensageiro/sangue , Reação em Cadeia da Polimerase em Tempo Real , Ribonucleoproteínas/genéticaRESUMO
Myelodysplastic syndrome (MDS) is highly heterogeneous clonal hematological malignancy, having a high rate of progression to acute myeloid leukemia (AML). With the rapid development of molecular biological techniques, plenty of gene mutations were found to have close relationships with the transformation from MDS to AML. SRSF2 is a RNA splicing-related gene, which mutation may prompt a poor prognosis, and have a higher rate of progressing to AML. DNMT3A plays an important role in DNA methylation, its mutation often indicate a worse overall survival and a more rapid progression to AML. ASXL1 regulates the synthesis of histone, which frameshift mutations are molecular marks of an adverse outcome. IDH contains IDH1 and IDH2, which are related with the Krebs cycle. Patients with IDH1 mutation have a shorter overall survival and a higher risk of AML transformation than that of patients with wild-type IDH1, while IDH2 was a poor prognostic factor for overall survival in patients with lower-risk MDS. Another gene related with DNA methylation is TET2, which is the most frequently mutated gene in MDS known so far and it may act as tumor-suppressor gene, but the opinions on its impact on patients' outcomes are still controversial. Some studies show that its mutations relate to a shorter time to progression to AML. Because of the differentiations in patients' races, regions and clinical characteristics, the results of different studies are varied. In this review, the recent advances on these related genes are summarized.
Assuntos
Humanos , DNA (Citosina-5-)-Metiltransferases , Genética , Proteínas de Ligação a DNA , Genética , Genótipo , Isocitrato Desidrogenase , Genética , Leucemia Mieloide Aguda , Genética , Patologia , Síndromes Mielodisplásicas , Genética , Patologia , Proteínas Nucleares , Genética , Oncogenes , Proteínas Proto-Oncogênicas , Genética , Proteínas Repressoras , Genética , Ribonucleoproteínas , Genética , Fatores de Processamento de Serina-ArgininaRESUMO
O linfoma de Hodgkin clássico (LHc) caracteriza-se por apresentar 1-2% de células tumorais (Hodgkin e Reed-Sternberg, H-RS), em meio de um infiltrado inflamatório. As células H-RS são caracterizadas por alterações simultâneas e ainda não bem entendidas no ciclo celular e apoptose, e uma alta dependência das células imunes infiltrantes, tendo inclusive estas alterações sido associadas ao desfecho clínico. O objetivo deste trabalho foi investigar alterações no ciclo celular e apoptose das células H-RS e a sua relação com a resposta imune local, definida pela composição do microambiente tumoral, em casos de LHc pediátricos, visando identificar cross-talks potenciais entre os dois componentes do tumor. Para isto, foram analisadas por imunohistoquímica (IHQ) os marcadores p53, p21, MDM2, nucleofosmina (NPM) H2AX, ATM, a cinética populacional (Ki-67, Histona H3, número de H-RS, uni e multinucleação), BCL2, caspase-3 (CASP3) e SURVIVINA e a fragmentação de DNA (ensaio de TUNEL) nas células H-RS, em um grupo de 87 pacientes com LHc pediátrico. Estas alterações foram analisadas em relação ao status do vírus Epstein-Barr (EBV), as sub-populações celulares do MAT e a sobrevida livre de progressão (SLP) e global (SG). Uma alta taxa de proliferação (IPC; Ki-67>50% nas H-RS) e taxa mitótica (IM; histona H3>25% nas H-RS) foram observados em 75,9% e 37,5% dos casos, sem associação com o número de células tumorais, enquanto que a expressão de p53, p21, MDM2, ATM ,H2AX, BCL2, CASP3 e SURVIVINA foram observadas em 13,4%, 69%, 83,6%, 59%, 72,7%, 56,6%, 71,4% e 52% dos casos, respectivamente. Quanto a NPM, foi identificada a expressão citoplasmática da proteína (NPMc+) em 66,2% dos casos, posteriormente confirmada por imunoflorescência e por microscopia confocal a laser...
Classical Hodgkin lymphoma (cHL) is a B-cell neoplasm characterized by two components, the malignant (Hodgkin-Reed-Sternberg, H-RS) cells which comprise 1-2% of the tumor mass, and an intense inflammatory infiltrate, the tumor microenvironment. H-RS cells exhibit simultaneous alterations in cell cycle and apoptosis and a strong dependence on infiltrating immune cells. The objective of this study was to investigate alterations of H-RS cell cycle and apoptosis and their relationship with the local immune response, defined by the TME composition, aiming to identify a potential cross-talk between the two components of the tumor, in a series of pediatric cHL. For this, different markers were analyzed by immunohistochemistry (IHC), such as H-RS population kinetics (Ki-67, histone H3, number of H-RS, uni and multinucleation), p53, p21, MDM2, nucleophosmin (NPM), pH2AX, pATM, BCL2, caspase-3 (CASP3), survivin and DNA fragmentation (TUNEL assay) in H-RS cells. These results were analyzed in respect of Epstein-Barr virus (EBV) status, TME cell subpopulations and survival. A high proliferation rate (IPC; Ki-67>50% in H- RS) and mitotic rate (IM; histone H3>25% in H-RS) were seen in 75.9% and 37.5% of cases, without association with the number of tumor cells, while the expression of p53, p21, MDM2, ATM, H2AX, BCL2, CASP3, and SURVIVIN were seen in 13.4%, 69%, 83.6 %, 59%, 72.7%, 56.6%, 71.4% and 52% of cases, respectively. NPM was aberrantly localized to the cytoplasm (NPMc+) in 66.2% of cases, confirmed by immunofluorescence and confocal laser scanning microscopy. In order to disclose the causes of the aberrant localization, exon 12 mutation analysis was performed from H-RS microdissected cells (2 NPMc+ cases) and by subcloning of total DNA (4 NPMc+ cases), and no mutation were identified. FISH analysis with non-commercial probes did not identify any chromosomal translocation in the NPM1 gene...
Assuntos
Humanos , Masculino , Feminino , Apoptose , Ciclo Celular , Doença de Hodgkin , Microambiente Tumoral , RibonucleoproteínasRESUMO
OBJECTIVE: To analyze the prevalence of myositis-specific and myositis-associated autoantibodies and their clinical correlations in a large series of patients with dermatomyositis/polymyositis. METHOD: This cross-sectional study enrolled 127 dermatomyositis cases and 95 polymyositis cases. The disease-related autoantibody profiles were determined using a commercially available blood testing kit. RESULTS: The prevalence of myositis-specific autoantibodies in all 222 patients was 34.4%, whereas myositis-associated autoantibodies were found in 41.4% of the patients. The most frequently found autoantibody was anti-Ro-52 (36.9%), followed by anti-Jo-1 (18.9%), anti-Mi-2 (8.1%), anti-Ku (4.1%), anti-SRP (3.2%), anti-PL-7 (3.2%), anti-PL-12 (2.7%), anti-PM/Scl75 (2.7%), and anti-PM/Scl100 (2.7%). The distributions of these autoantibodies were comparable between polymyositis and dermatomyositis, except for a higher prevalence of anti-Jo-1 in polymyositis. Anti-Mi-2 was more prevalent in dermatomyositis. Notably, in the multivariate analysis, anti-Mi-2 and anti-Ro-52 were associated with photosensitivity and pulmonary disorders, respectively, in dermatomyositis. Anti-Jo-1 was significantly correlated with pulmonary disorders in polymyositis. Moreover, anti-Ro-52 was associated with anti-Jo-1 in both diseases. No significant correlation was observed between the remaining autoantibodies and the clinical and/or laboratory findings. CONCLUSIONS: Our data are consistent with those from other published studies involving other populations, although certain findings warrant consideration. Anti-Ro-52 and anti-Jo-1 were strongly associated with one another. Anti-Ro-52 was correlated with pulmonary disorders in dermatomyositis, whereas anti-Jo-1 was correlated with pulmonary alterations in polymyositis. .
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Autoanticorpos/sangue , Miosite/imunologia , Idade de Início , Estudos Transversais , Dermatomiosite/sangue , Dermatomiosite/imunologia , Modelos Logísticos , Pneumopatias/sangue , Pneumopatias/imunologia , Força Muscular , Miosite/sangue , Ribonucleoproteínas/sangue , Estatísticas não Paramétricas , Fatores de TempoRESUMO
<p><b>OBJECTIVE</b>To investigate SRSF2 mutations in patients with chronic myelomonocytic leukemia (CMML) and the clinical characteristics of patients with SRSF2 mutants.</p><p><b>METHODS</b>In this study, the frequency of SRSF2 mutation in a cohort of 20 patients with CMML was detected by polymerase chain reaction (PCR) followed by direct sequencing to couple with their clinical features.</p><p><b>RESULTS</b>Of 20 patients, 4 patients were found harboring SRSF2 mutations, including 2 P95L, 1 P95H and 1 P95R point mutations. There were no significantly statistical differences in terms of their clinical characteristics between mutant and wild type group.</p><p><b>CONCLUSION</b>SRSF2 mutation was not frequently occurred in CMML patients and might associated with poor prognosis. It might be a practically diagnostic maker and therapeutic target in CMML.</p>
Assuntos
Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Mutacional de DNA , Genótipo , Leucemia Mielomonocítica Crônica , Genética , Mutação , Proteínas Nucleares , Genética , Prognóstico , Ribonucleoproteínas , Genética , Fatores de Processamento de Serina-ArgininaRESUMO
<p><b>OBJECTIVE</b>To investigate the expression of La protein in cervical cancer tissues and explore its role in the occurrence and progression of cervical cancer.</p><p><b>METHODS</b>The expression of La protein in cervical cancer and normal cervical tissues was detected by immunohistochemical staining. RNA interference technology was used to silence La protein expression in HeLa cells and the changes in cell proliferation, tumor sphere formation and cell cycles were investigated.</p><p><b>RESULTS</b>The expression of La protein was significantly higher in cervical cancer tissues than in normal cervical tissues (61% vs 9%, P<0.05). Silencing La protein expression in HeLa cells caused significantly reduced the cell proliferation and lowered the tumor sphere formation rate from the control level of (17.1=1.92)% to (6.3=0.45)% (P<0.05), resulting also in G0/G1 cell cycle arrest and reduced cyclin D1 protein expression.</p><p><b>CONCLUSION</b>The RNA binding protein La can promote the development of cervical cancer and may play a critical role in the carcinogenesis and progression of cervical cancer.</p>