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1.
Int J Biol Macromol ; 262(Pt 1): 129951, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38325695

RESUMO

Deoxyribouridine (dU) is an abnormal nucleoside in DNA and plays vital roles in multiple biological and physiological processes. Here, we conducted a mass spectrometry-based screen for dU-binding proteins and found that the heterogeneous nuclear ribonucleoprotein D (HNRNPD) could preferentially bind to dU-containing DNA. We also discovered that HNRNPD engages in the 5-Fluorouracil (5FU)-induced DNA damage response and can modulate the repair of dU in DNA in vitro and in human cells. Moreover, using a shuttle vector- and next-generation sequencing-based method, we unveiled the crucial role of HNRNPD in promoting the replicative bypass of dU in human cells. Taken together, these findings suggested that HNRNPD is a novel dU-bearing DNA-binding protein capable of regulating the removal of dU in DNA, and provided new insights into the molecular mechanisms of dU-associated diseases.


Assuntos
DNA , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Humanos , DNA/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Reparo do DNA , Dano ao DNA
2.
Clin Transl Med ; 13(10): e1451, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37877357

RESUMO

BACKGROUND: Circular RNAs (circRNAs) play a significant role in the initiation and progression of various cancers, including hepatocellular carcinoma (HCC). Circular syntaxin 6 (circSTX6, also known as hsa_circ_0007905) has been identified as a microRNA (miRNA) sponge in pancreatic adenocarcinoma. However, its full range of functions in terms of protein scaffold and translation remain largely unexplored in the context of HCC. METHODS: The expression of circSTX6 and its encoded protein was examined in HCC tumour tissues. N6 -methyladenosine (m6 A) on circSTX6 was verified and quantified by methylated RNA immunoprecipitation (Me-RIP), RIP and dual luciferase reporter assays. The biological functions of circSTX6 and its encoded protein in HCC were clarified by in vitro and in vivo experiments. Mechanistically, the interaction between circSTX6 and heterogeneous nuclear ribonucleoprotein D (HNRNPD) was investigated by RNA pull-down, RIP and fluorescence in situ hybridization (FISH)/IF. The regulatory effects of circSTX6 and HNRNPD on activating transcription factor 3 (ATF3) mRNA were determined by mRNA stability and RIP assays. Furthermore, the presence of circSTX6-encoded protein was verified by mass spectrometry. RESULTS: CircSTX6 and its encoded 144 amino acid polypeptide, circSTX6-144aa, were highly expressed in HCC tumour tissues and served as independent risk factors for overall survival in HCC patients. The expression of circSTX6 was regulated by METTL14 in an m6 A-dependent manner. Functionally, circSTX6 accelerated HCC proliferation and tumourigenicity and reinforced tumour metastasis in vitro and in vivo. Mechanistically, circSTX6 acted as a sponge for HNRNPD protein, facilitating its binding to ATF3 mRNA, consequently promoting ATF3 mRNA decay. Meanwhile, circSTX6-144aa promoted HCC proliferation, migration and invasion independent of circSTX6 itself. CONCLUSION: Collectively, our study reveals that m6 A-modified circSTX6 drives malignancy in HCC through the HNRNPD/ATF3 axis, while its encoded circSTX6-144aa contributes to HCC progression independent of circSTX6. CirSTX6 and its encoded protein hold promise as potential biomarkers and therapeutic targets in HCC.


Assuntos
Fator 3 Ativador da Transcrição , Carcinoma Hepatocelular , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Neoplasias Hepáticas , MicroRNAs , RNA Circular , Humanos , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Aminoácidos , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Hibridização in Situ Fluorescente , Neoplasias Hepáticas/metabolismo , MicroRNAs/genética , RNA Mensageiro , RNA Circular/genética
3.
Biomed Res Int ; 2022: 8610467, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36246972

RESUMO

Coxsackievirus B (CVB) 3C protease (3Cpro) plays a specific cleavage role on AU-rich binding factor (AUF1, also called hnRNP D), which consequently disputes the regulation of AUF1 on downstream molecules. In our study, the iTRAQ approach was first used to quantify the differentially expressed cellular proteins in AUF1-overexpressing HeLa cells, which provides straightforward insight into the role of AUF1 during viral infection. A total of 1,290 differentially expressed proteins (DEPs), including 882 upregulated and 408 downregulated proteins, were identified. The DEPs are involved in a variety of cellular processes via GO terms, protein-protein interactions, and a series of further bioinformatics analyses. Among the DEPs, some demonstrated important roles in cellular metabolism. In particular, DDX5 was further verified to be negatively regulated by AUF1 and increased in CVB-infected cells, which in turn promoted CVB replication. These findings provide potential novel ideas for exploring new antiviral therapy targets.


Assuntos
RNA Helicases DEAD-box , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Proteômica , Antivirais , RNA Helicases DEAD-box/metabolismo , Enterovirus Humano B/metabolismo , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , Replicação Viral
4.
J Biomed Sci ; 29(1): 73, 2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36127734

RESUMO

BACKGROUND: Mesenchymal stem cells (MSCs) exhibit two bidirectional immunomodulatory abilities: proinflammatory and anti-inflammatory regulatory effects. Long noncoding RNAs (lncRNAs) have important functions in the immune system. Previously, we performed high-throughput sequencing comparing lncRNA expression profiles between MSCs cocultured with or without CD14+ monocytes and screened out a new lncRNA termed lncRNA MCP1 regulatory factor (MRF). However, the mechanism of MRF in MSCs is still unknown. METHODS: MRF expression was quantified via qRT-PCR. RNA interference and lentiviruses were used to regulate MRF expression. The immunomodulatory effects of MSCs on monocytes were evaluated via monocyte migration and macrophage polarization assays. RNA pull-down and mass spectrometry were utilized to identify downstream factors of MRF. A dual-luciferase reporter assay was applied to analyze the transcription factors regulating MRF. qRT-PCR, western blotting and ELISAs were used to assess MCP1 expression. A human monocyte adoptive transfer mouse model was applied to verify the function of MRF in vivo. RESULTS: MRF was upregulated in MSCs during coculture with CD14+ monocytes. MRF increased monocyte recruitment by upregulating the expression of monocyte chemotactic protein (MCP1). Knockdown of MRF enhanced the regulatory effect of MSCs on restraining M1 polarization and facilitating M2 polarization. Mechanistically, MRF bound to the downstream protein heterogeneous nuclear ribonucleoprotein D (HNRNPD) to upregulate MCP1 expression, and the transcription factor interferon regulatory factor 1 (IRF1) activated MRF transcription early during coculture. The human monocyte adoptive transfer model showed that MRF downregulation in MSCs inhibited monocyte chemotaxis and enhanced the effects of MSCs to inhibit M1 macrophage polarization and promote M2 polarization in vivo. CONCLUSION: We identified the new lncRNA MRF, which exhibits proinflammatory characteristics. MRF regulates the ability of MSCs to accelerate monocyte recruitment and modulate macrophage polarization through the HNRNPD-MCP1 axis and initiates the proinflammatory regulatory process in MSCs, suggesting that MRF is a potential target to improve the clinical effect of MSC-based therapy or correct MSC-related immunomodulatory dysfunction under pathological conditions.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Células-Tronco Mesenquimais , RNA Longo não Codificante , Animais , Anti-Inflamatórios/farmacologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/farmacologia , Humanos , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 1 de Interferon/farmacologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteínas Quimioatraentes de Monócitos/metabolismo , Proteínas Quimioatraentes de Monócitos/farmacologia , Monócitos/metabolismo , RNA Longo não Codificante/metabolismo
5.
Ecotoxicol Environ Saf ; 243: 113990, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35998476

RESUMO

Arsenic (As) is a natural hepatotoxicity inducer that is ubiquitous in water, soil, coal, and food. Studies have found that arsenite exposure elicits increased mRNA transcription and decreased protein expression of SOD1 in vivo and in vitro; however, the specific mechanisms remain unclear. Here, we established a model of arsenic-induced chronic liver injury by providing rats with drinking water containing different concentrations of sodium arsenite (NaAsO2) and found that NaAsO2 exposure decreased the mRNA and protein levels of AUF1 and the protein level of SOD1 and elevated the mRNA and protein levels of Dicer1 and miR-155 and the mRNA level of SOD1. Overexpression of AUF1 under NaAsO2 stress in vitro induced Dicer1 mRNA and protein expression and decreased miR-155 levels, which could be reversed by AUF1 siRNA. In addition, miR-155 overexpression downregulated SOD1 mRNA and protein levels, although this change was inhibited after transfection with an miR-155 inhibitor. Taken together, our findings showed that NaAsO2 could upregulate Dicer1 mRNA and protein, thereby increasing miR-155 expression by downregulating AUF1 mRNA and protein expression. A dual-luciferase reporter assay indicated that miR-155 decreased the mRNA and protein levels of SOD1 by targeting the SOD1 3'UTR, resulting in liver injury. This study provides an important research basis for further understanding the factors underlying arsenic-induced liver injury to improve the prevention and control strategies for arsenism.


Assuntos
Arsênio , Arsenitos , Doença Hepática Crônica Induzida por Substâncias e Drogas , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , MicroRNAs , Regiões 3' não Traduzidas/genética , Animais , Arsênio/metabolismo , Arsênio/toxicidade , Arsenitos/metabolismo , Arsenitos/toxicidade , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ribonuclease III/genética , Ribonuclease III/metabolismo , Compostos de Sódio , Superóxido Dismutase-1/genética
6.
Breast Cancer Res ; 24(1): 46, 2022 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-35821051

RESUMO

BACKGROUND: Locally advanced breast cancer (LABC), the most aggressive form of the disease, is a serious threat for women's health worldwide. The AU-rich RNA-binding factor 1 (AUF1) promotes the formation of chemo-resistant breast cancer stem cells. Thereby, we investigated the power of AUF1 expression, in both cancer cells and their stromal fibroblasts, as predictive biomarker for LABC patients' clinical outcome following neoadjuvant treatment. METHODS: We have used immunohistochemistry to assess the level of AUF1 on formalin-fixed paraffin-embedded tissues. Immunoblotting was utilized to show the effect of AUF1 ectopic expression in breast stromal fibroblasts on the expression of various genes both in vitro and in orthotopic tumor xenografts. Cytotoxicity was evaluated using the WST1 assay, while a label-free real-time setting using the xCELLigence RTCA technology was utilized to assess the proliferative, migratory and invasive abilities of cells. RESULTS: We have shown that high AUF1 immunostaining (≥ 10%) in both cancer cells and their adjacent cancer-associated fibroblasts (CAFs) was significantly associated with higher tumor grade. Kaplan-Meier univariate analysis revealed a strong correlation between high AUF1 level in CAFs and poor patient's survival. This correlation was highly significant in patients with triple negative breast cancer, who showed poor disease-free survival (DFS) and overall survival (OS). High expression of AUF1 in CAFs was also associated with poor OS of ER+/Her2- patients. Similarly, AUF1-positive malignant cells tended to be associated with shorter DFS and OS of ER+/Her2+ patients. Interestingly, neoadjuvant therapy downregulated AUF1 to a level lower than 10% in malignant cells in a significant number of patients, which improved both DFS and OS. In addition, ectopic expression of AUF1 in breast fibroblasts activated these cells and enhanced their capacity to promote, in an IL-6-dependent manner, the epithelial-to-mesenchymal transition and stemness processes. Furthermore, these AUF1-expressing cells enhanced the chemoresistance of breast cancer cells and their growth in orthotopic tumor xenografts. CONCLUSIONS: The present findings show that the CAF-activating factor AUF1 has prognostic/predictive value for breast cancer patients and could represent a great therapeutic target in order to improve the precision of cancer treatment.


Assuntos
Neoplasias da Mama , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinogênese/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fibroblastos/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Prognóstico
7.
Molecules ; 27(10)2022 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-35630659

RESUMO

The study on the mechanism and kinetics of mRNA degradation provides a new vision for chemical intervention on protein expression. The AU enrichment element (ARE) in mRNA 3'-UTR can be recognized and bound by the ARE binding protein (AU-rich Element factor (AUF1) to recruit RNase for degradation. In the present study, we proposed a novel strategy for expression regulation that interferes with the AUF1-RNA binding. A small-molecule compound, JNJ-7706621, was found to bind AUF1 protein and inhibit mRNA degradation by screening the commercial compound library. We discovered that JNJ-7706621 could inhibit the expression of AUF1 targeted gene IL8, an essential pro-inflammatory factor, by interfering with the mRNA homeostatic state. These studies provide innovative drug design strategies to regulate mRNA homeostasis.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D , Regiões 3' não Traduzidas , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Cell Rep ; 30(4): 1117-1128.e5, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995753

RESUMO

Prion-like proteins form multivalent assemblies and phase separate into membraneless organelles. Heterogeneous ribonucleoprotein D-like (hnRNPDL) is a RNA-processing prion-like protein with three alternative splicing (AS) isoforms, which lack none, one, or both of its two disordered domains. It has been suggested that AS might regulate the assembly properties of RNA-processing proteins by controlling the incorporation of multivalent disordered regions in the isoforms. This, in turn, would modulate their activity in the downstream splicing program. Here, we demonstrate that AS controls the phase separation of hnRNPDL, as well as the size and dynamics of its nuclear complexes, its nucleus-cytoplasm shuttling, and amyloidogenicity. Mutation of the highly conserved D378 in the disordered C-terminal prion-like domain of hnRNPDL causes limb-girdle muscular dystrophy 1G. We show that D378H/N disease mutations impact hnRNPDL assembly properties, accelerating aggregation and dramatically reducing the protein solubility in the muscle of Drosophila, suggesting a genetic loss-of-function mechanism for this muscular disorder.


Assuntos
Proteínas Amiloidogênicas/metabolismo , Núcleo Celular/metabolismo , Drosophila/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Agregação Patológica de Proteínas/metabolismo , Processamento Alternativo , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/ultraestrutura , Animais , Núcleo Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Dactinomicina/farmacologia , Drosophila/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/ultraestrutura , Humanos , Cinética , Microscopia Eletrônica de Transmissão , Células Musculares/metabolismo , Células Musculares/patologia , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Mutação , Agregação Patológica de Proteínas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura
9.
J Neurol ; 266(10): 2524-2534, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31267206

RESUMO

Autosomal dominant limb girdle muscular dystrophy D3 HNRNPDL-related is a rare dominant myopathy caused by mutations in HNRNPDL. Only three unrelated families have been described worldwide, a Brazilian and a Chinese carrying the mutation c.1132G>A p.(Asp378Asn), and one Uruguayan with the mutation c.1132G>C p. (Asp378His), both mutations occurring in the same codon. The present study enlarges the clinical, morphological and muscle MRI spectrum of AD-HNRNPDL-related myopathies demonstrating the significant particularities of the disease. We describe two new unrelated Argentinean families, carrying the previously reported c.1132G>C p.(Asp378His) HNRNPDL mutation. There was a wide phenotypic spectrum including oligo-symptomatic cases, pure limb girdle muscle involvement or distal lower limb muscle weakness. Scapular winging was the most common finding, observed in all patients. Muscle MRIs of the thigh, at different stages of the disease, showed particular involvement of adductor magnus and vastus besides a constant preservation of the rectus femoris and the adductor longus muscles, defining a novel MRI pattern. Muscle biopsy findings were characterized by the presence of numerous rimmed vacuoles, cytoplasmic bodies, and abundant autophagic material at the histochemistry and ultrastructural levels. HNRNPDL-related LGMD D3 results in a wide range of clinical phenotypes from the classic proximal form of LGMD to a more distal phenotype. Thigh MRI suggests a specific pattern. Codon 378 of HNRNPDL gene can be considered a mutation hotspot for HNRNPDL-related myopathy. Pathologically, the disease can be classified among the autophagic rimmed vacuolar myopathies as with the other multisystem proteinopathies.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Distrofia Muscular do Cíngulo dos Membros , Idoso , Argentina , Feminino , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Distrofia Muscular do Cíngulo dos Membros/fisiopatologia , Mutação , Linhagem , Fenótipo
10.
Mol Cell ; 74(6): 1189-1204.e6, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31226278

RESUMO

RNA-binding proteins (RBPs) regulate post-transcriptional gene expression by recognizing short and degenerate sequence motifs in their target transcripts, but precisely defining their binding specificity remains challenging. Crosslinking and immunoprecipitation (CLIP) allows for mapping of the exact protein-RNA crosslink sites, which frequently reside at specific positions in RBP motifs at single-nucleotide resolution. Here, we have developed a computational method, named mCross, to jointly model RBP binding specificity while precisely registering the crosslinking position in motif sites. We applied mCross to 112 RBPs using ENCODE eCLIP data and validated the reliability of the discovered motifs by genome-wide analysis of allelic binding sites. Our analyses revealed that the prototypical SR protein SRSF1 recognizes clusters of GGA half-sites in addition to its canonical GGAGGA motif. Therefore, SRSF1 regulates splicing of a much larger repertoire of transcripts than previously appreciated, including HNRNPD and HNRNPDL, which are involved in multivalent protein assemblies and phase separation.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/química , Modelos Moleculares , RNA/química , Fatores de Processamento de Serina-Arginina/química , Sequência de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , Expressão Gênica , Células HeLa , Células Hep G2 , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Células K562 , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA/genética , RNA/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
11.
RNA Biol ; 16(7): 960-971, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30951406

RESUMO

The RNA-binding protein AUF1 regulates post-transcriptional gene expression by affecting the steady state and translation levels of numerous target RNAs. Remodeling of RNA structures by the largest isoform AUF1 p45 was recently demonstrated in the context of replicating RNA viruses, and involves two RNA remodeling activities, i.e. an RNA chaperone and an RNA annealing activity. AUF1 contains two non-identical RNA recognition motifs (RRM) and one RGG/RG motif located in the C-terminus. In order to determine the functional significance of each motif to AUF1's RNA-binding and remodeling activities we performed a comprehensive mutagenesis study and characterized the wildtype AUF1, and several variants thereof. We demonstrate that each motif contributes to efficient RNA binding and remodeling by AUF1 indicating a tight cooperation of the RRMs and the RGG/RG motif. Interestingly, the data identify two distinct roles for the arginine residues of the RGG/RG motif for each RNA remodeling activity. First, arginine-mediated stacking interactions promote AUF1's helix-destabilizing RNA chaperone activity. Second, the electropositive character of the arginine residues is the major driving force for the RNA annealing activity. Thus, we provide the first evidence that arginine residues of an RGG/RG motif contribute to the mechanism of RNA annealing and RNA chaperoning.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , RNA/metabolismo , Motivos de Aminoácidos , Arginina/metabolismo , Sequência de Bases , Ribonucleoproteína Nuclear Heterogênea D0 , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA/química , RNA/genética , Relação Estrutura-Atividade , Termodinâmica
12.
J Exp Clin Cancer Res ; 38(1): 161, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987669

RESUMO

BACKGROUND: Long non-coding RNAs (lncRNAs) have been identified to play an important role in the development and progression of various tumors, including colorectal cancer (CRC). However, the regulatory molecular mechanism by lncRNA in CRC initiation and progression has not been fully clarified. METHODS: TCGA database was used to identify the involvement of LINC01354 in CRC. qRT-PCR and western blot were used to determine RNA and protein expression. The gain- and loss-of-function assays were conducted to explore the function of LINC01354 in the progression of CRC. In order to investigate the LINC01354-mediated mRNA in CRC tumorigenesis, we applied the profiling analysis as well as GO and KEGG analysis. Pulldown and RIP assays were applied to detect the interaction of hnRNP-D with LINC01354 and ß-catenin. RESULTS: The upregulation of LINC01354 in CRC and its prognostic significance were identified by TCGA database and confirmed in CRC tissues. Functionally, forced expression of LINC01354 promoted, while knockdown of LINC01354 inhibited cell proliferation, migration and EMT phenotype formation of CRC cells. A significant enrichment of the Wnt/ß-catenin signaling pathway genes under LINC01354 overexpression. In addition, LINC01354 modulated the mRNA stability of ß-catenin through interacting with hnRNP-D, thereby activating Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our investigations proposed novel regulatory axis of LINC01354/hnRNP-D/Wnt/ß-catenin, which might be in favor of exploring novel therapeutic regimens for the clinical treatment of CRC.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , RNA Longo não Codificante/genética , Via de Sinalização Wnt , Adulto , Idoso , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Colorretais/patologia , Biologia Computacional/métodos , Bases de Dados Genéticas , Transição Epitelial-Mesenquimal , Feminino , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Metástase Neoplásica , Estadiamento de Neoplasias , Conformação de Ácido Nucleico , Ligação Proteica , Estabilidade Proteica , RNA Longo não Codificante/química , Carga Tumoral , beta Catenina/metabolismo
13.
Autophagy ; 15(8): 1419-1437, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30870073

RESUMO

N6-methyladenosine (m6A) mRNA modifications play critical roles in various biological processes. However, no study addresses the role of m6A in macroautophagy/autophagy. Here, we show that m6A modifications are increased in H/R-treated cardiomyocytes and ischemia/reperfusion (I/R)-treated mice heart. We found that METTL3 (methyltransferase like 3) is the primary factor involved in aberrant m6A modification. Silencing METTL3 enhances autophagic flux and inhibits apoptosis in H/R-treated cardiomyocytes. However, overexpression of METTL3 or inhibition of the RNA demethylase ALKBH5 has an opposite effect, suggesting that METTL3 is a negative regulator of autophagy. Mechanistically, METTL3 methylates TFEB, a master regulator of lysosomal biogenesis and autophagy genes, at two m6A residues in the 3'-UTR, which promotes the association of the RNA-binding protein HNRNPD with TFEB pre-mRNA and subsequently decreases the expression levels of TFEB. Further experiments show that autophagic flux enhanced by METTL3 deficiency is TFEB dependent. In turn, TFEB regulates the expression levels of METTL3 and ALKBH5 in opposite directions: it induces ALKBH5 and inhibits METTL3. TFEB binds to the ALKBH5 promoter and activates its transcription. In contrast, inhibition of METTL3 by TFEB does not involve transcriptional repression but rather downregulation of mRNA stability, thereby establishing a negative feedback loop. Together, our work uncovers a critical link between METTL3-ALKBH5 and autophagy, providing insight into the functional importance of the reversible mRNA m6A methylation and its modulators in ischemic heart disease. Abbreviations: ACTB, actin beta; ALKBH5, alkB homolog 5, RNA demethylase; ANXA5, annexin A5; ATG, autophagy-related; BafA, bafilomycin A1; CASP3, caspase 3; ELAVL1, ELAV like RNA binding protein 1; FTO, FTO, alpha-ketoglutarate dependent dioxygenase; GFP, green fluorescent protein; GST, glutathione S-transferase; HNRNPD, heterogeneous nuclear ribonucleoprotein D; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LAD, left anterior descending; m6A, N6-methyladenosine; MEFs, mouse embryo fibroblasts; Mer, mutated estrogen receptor domains; METTL3, methyltransferase like 3; METTL14, methyltransferase like 14; mRFP, monomeric red fluorescent protein; MTORC1, mechanistic target of rapamycin kinase complex 1; NMVCs, neonatal mouse ventricular cardiomyocytes; PCNA, proliferating cell nuclear antigen; PE, phosphatidylethanolamine; PI, propidium iodide; PTMs, post-translational modifications; PVDF, polyvinylidenedifluoride; RIP, RNA-immunoprecipitation; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TFEB, transcription factor EB; TUBA: tublin alpha; WTAP, WT1 associated protein; YTHDF, YTH N6-methyladenosine RNA binding protein.


Assuntos
Adenosina/análogos & derivados , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Metiltransferases/metabolismo , Miócitos Cardíacos/metabolismo , Oxigênio/farmacologia , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Hipóxia Celular/efeitos dos fármacos , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Metilação , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Precursores de RNA/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ativação Transcricional/genética , Regulação para Cima/efeitos dos fármacos
14.
Nucleic Acids Res ; 47(8): 4068-4085, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30799487

RESUMO

DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced: the ssDNA fraction upon camptothecin treatment; AsiSI-induced DSB resection; and RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage. Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.


Assuntos
Cromatina/metabolismo , Genoma Humano , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Reparo de DNA por Recombinação , Proteína de Replicação A/genética , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Instabilidade Genômica , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Proteína de Replicação A/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
15.
Med Sci Monit ; 25: 730-738, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30681073

RESUMO

BACKGROUND Berberine, a natural isoquinoline alkaloid derived from Berberis genus plants, has been reported to have anti-cancer effects. While cell behavior can be modulated by long non-coding RNAs (lncRNAs), the contributions of lncRNAs in progression and berberine effects on colorectal cancer are largely unknown. Therefore, the present study investigated the involvement and regulatory function of lncRNA cancer susceptibility candidate 2 (CASC2) during the treatment of human colorectal cancer using berberine. MATERIAL AND METHODS Reverse transcription-quantitative PCR (RT-qPCR) was performed to detect the expression levels of lncRNA CASC2 and Bcl-2 mRNA in colorectal cancer cells. MTT assay was performed to evaluate cell viability. Flow cytometry and TUNEL assay were used to analyze the apoptosis of cancer cells. RNA immunoprecipitation (RIP) assay was done to verify the interaction between lncRNA CASC2 and (AU-binding factor 1) AUF1, or AUF1 and B-cell CLL/lymphoma 2 (Bcl-2). RESULTS Treatment with berberine suppressed cell viability of colorectal cancer by promoting apoptosis level. LncRNA CASC2 was upregulated in cells treated with berberine, and knockdown of lncRNA CASC2 reversed the berberine-induced apoptosis. In addition, anti-apoptotic gene Bcl-2 was suppressed by berberine treatment and lncRNA CASC2, inducing the pro-apoptotic effects. Moreover, lncRNA CASC2 binds to AUF1, which sequestered AUF1 from binding to Bcl-2 mRNA, thus inducing the inactivation of Bcl-2 translation. CONCLUSIONS Our study reveals that lncRNA CASC2 mediates the berberine-induced pro-apoptotic effect via inhibition of Bcl-2 expression at the post-transcriptional level.


Assuntos
Berberina/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , RNA Longo não Codificante/genética , Proteínas Supressoras de Tumor/genética , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Progressão da Doença , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima
16.
Mol Carcinog ; 58(5): 777-793, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30604907

RESUMO

Although overexpression of the non-canonical NFκB subunit p52 has been observed in several tumors, the function and mechanism of p52 in bladder cancer (BC) are less well understood. Here, we aimed at understanding the role and mechanism underlying p52 regulation of BC invasion. Human p52 was stably knockdown with shRNA targeting p52 in two bladder cancer cell lines (T24 and UMUC3). Two constitutively expressing constructs, p52 and p100, were stably transfected in to T24 or UMUC3, respectively. The stable transfectants were used to determine function and mechanisms responsible for p52 regulation of BC invasion. We demonstrate that p52 mediates human BC invasion. Knockdown of p52 impaired bladder cancer invasion by reduction of rhogdiß mRNA stability and expression. Positively regulation of rhogdiß mRNA stability was mediated by p52 promoting AUF1 protein degradation, consequently resulting in reduction of AUF1 binding to rhogdiß mRNA. Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1. Moreover, we found that p52 upregulated miR-145, which directly bound to the 3'-UTR of sp1 mRNA, leading to downregulation of Sp1 protein translation. Our results reveal a comprehensive pathway that p52 acts as a positive regulator of BC invasion by initiating a novel miR-145/Sp1/USP8/AUF1/RhoGDIß axis. These findings provide insight into the understanding of p52 in the pathology of human BC invasion and progression, which may be useful information in the development of preventive and therapeutic approaches for using p52 as a potential target.


Assuntos
Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , MicroRNAs/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Estabilidade de RNA , Fator de Transcrição Sp1/metabolismo , Ubiquitina Tiolesterase/metabolismo , Neoplasias da Bexiga Urinária/patologia , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , MicroRNAs/genética , Subunidade p52 de NF-kappa B/genética , Biossíntese de Proteínas , Proteólise , Fator de Transcrição Sp1/genética , Células Tumorais Cultivadas , Ubiquitina Tiolesterase/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/química , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/genética
17.
Biochem J ; 476(2): 333-352, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30578289

RESUMO

Type III interferons (IFNs) are the latest members of the IFN family. They play an important role in immune defense mechanisms, especially in antiviral responses at mucosal sites. Moreover, they control inflammatory reactions by modulating neutrophil and dendritic cell functions. Therefore, it is important to identify cellular mechanisms involved in the control of type III IFN expression. All IFN family members contain AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTR) of their mRNAs that determine mRNA half-life and consequently the expressional level of these cytokines. mRNA stability is controlled by different proteins binding to these AREs leading to either stabilization or destabilization of the respective target mRNA. The KH-type splicing regulatory protein KSRP (also named KHSRP) is an important negative regulator of ARE-containing mRNAs. Here, we identify the interferon lambda 3 (IFNL3) mRNA as a new KSRP target by pull-down and immunoprecipitation experiments, as well as luciferase reporter gene assays. We characterize the KSRP-binding site in the IFNL3 3'-UTR and demonstrate that KSRP regulates the mRNA half-life of the IFNL3 transcript. In addition, we detect enhanced expression of IFNL3 mRNA in KSRP-/- mice, establishing a negative regulatory function of KSRP in type III IFN expression also in vivo Besides KSRP the RNA-binding protein AUF1 (AU-rich element RNA-binding protein 1) also seems to be involved in the regulation of type III IFN mRNA expression.


Assuntos
Regiões 3' não Traduzidas , Interferons/biossíntese , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Interferons/genética , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Transativadores/genética
18.
J Immunol ; 201(12): 3617-3629, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30429285

RESUMO

Tissue repair is a complex process that necessitates an interplay of cellular processes, now known to be dictated by epigenetics. Intriguingly, macrophages are testimony to a large repertoire of evolving functions in this process. We identified a role for BMP signaling in regulating macrophage responses to Candida albicans infection during wound repair in a murine model. In this study, the RNA binding protein, AU-rich element-binding factor 1, was posttranslationally destabilized to bring about ubiquitin ligase, NEDD4-directed activation of BMP signaling. Concomitantly, PI3K/PKCδ mobilized the rapid phosphorylation of BMP-responsive Smad1/5/8. Activated BMP pathway orchestrated the elevated recruitment of EZH2 at promoters of genes assisting timely wound closure. In vivo, the repressive H3K27 trimethylation was observed to persist, accompanied by a robust upregulation of BMP pathway upon infection with C. albicans, culminating in delayed wound healing. Altogether, we uncovered the signaling networks coordinated by fungal colonies that are now increasingly associated with the infected wound microbiome, resulting in altered wound fate.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Candida albicans/fisiologia , Candidíase/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Macrófagos/fisiologia , Cicatrização , Animais , Candidíase/metabolismo , Modelos Animais de Doenças , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Processamento de Proteína Pós-Traducional , Células RAW 264.7 , Transdução de Sinais
19.
PLoS One ; 13(11): e0206823, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30418981

RESUMO

Regulation of mRNA stability by RNA-protein interactions contributes significantly to quantitative aspects of gene expression. We have identified potential mRNA targets of the AU-rich element binding protein AUF1. Myc-tagged AUF1 p42 was induced in mouse NIH/3T3 cells and RNA-protein complexes isolated using anti-myc tag antibody beads. Bound mRNAs were analyzed with Affymetrix microarrays. We have identified 508 potential target mRNAs that were at least 3-fold enriched compared to control cells without myc-AUF1. 22.3% of the enriched mRNAs had an AU-rich cluster in the ARED Organism database, against 16.3% of non-enriched control mRNAs. The enrichment towards AU-rich elements was also visible by AREScore with an average value of 5.2 in the enriched mRNAs versus 4.2 in the control group. Yet, numerous mRNAs were enriched without a high ARE score. The enrichment of tetrameric and pentameric sequences suggests a broad AUF1 p42-binding spectrum at short U-rich sequences flanked by A or G. Still, some enriched mRNAs were highly unstable, as those of TNFSF11 (known as RANKL), KLF10, HES1, CCNT2, SMAD6, and BCL6. We have mapped some of the instability determinants. HES1 mRNA appeared to have a coding region determinant. Detailed analysis of the RANKL and BCL6 3'UTR revealed for both that full instability required two elements, which are conserved in evolution. In RANKL mRNA both elements are AU-rich and separated by 30 bases, while in BCL6 mRNA one is AU-rich and 60 bases from a non AU-rich element that potentially forms a stem-loop structure.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Ligante RANK/genética , Estabilidade de RNA/genética , Regiões 3' não Traduzidas/genética , Elementos Ricos em Adenilato e Uridilato/genética , Animais , Sítios de Ligação/genética , Células HEK293 , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Humanos , Camundongos , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Ligante RANK/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
Int J Chron Obstruct Pulmon Dis ; 13: 3173-3190, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30349226

RESUMO

PURPOSE: Inflammatory gene expression is modulated by posttranscriptional regulation via RNA-binding proteins (RBPs), which regulate mRNA turnover and translation by binding to conserved mRNA sequences. Their role in COPD is only partially defined. This study evaluated RBPs tristetraprolin (TTP), human antigen R (HuR), and AU-rich element-binding factor 1 (AUF-1) expression using lung tissue from COPD patients and control subjects and probed their function in epithelial responses in vitro. PATIENTS AND METHODS: RBPs were detected by immunohistochemistry in bronchial and peripheral lung samples from mild-to-moderate stable COPD patients and age/smoking history-matched controls; RBPs and RBP-regulated genes were evaluated by Western blot, ELISA, protein array, and real-time PCR in human airway epithelial BEAS-2B cell line stimulated with hydrogen peroxide, cytokine combination (cytomix), cigarette smoke extract (CSE), and following siRNA-mediated silencing. Results were verified in a microarray database from bronchial brushings of COPD patients and controls. RBP transcripts were measured in peripheral blood mononuclear cell samples from additional stable COPD patients and controls. RESULTS: Specific, primarily nuclear immunostaining for the RBPs was detected in structural and inflammatory cells in bronchial and lung tissues. Immunostaining for AUF-1, but not TTP or HuR, was significantly decreased in bronchial epithelium of COPD samples vs controls. In BEAS-2B cells, cytomix and CSE stimulation reproduced the RBP pattern while increasing expression of AUF-1-regulated genes, interleukin-6, CCL2, CXCL1, and CXCL8. Silencing expression of AUF-1 reproduced, but not enhanced, target upregulation induced by cytomix compared to controls. Analysis of bronchial brushing-derived transcriptomic confirmed the selective decrease of AUF-1 in COPD vs controls and revealed significant changes in AUF-1-regulated genes by genome ontology. CONCLUSION: Downregulated AUF-1 may be pathogenic in stable COPD by altering posttranscriptional control of epithelial gene expression.


Assuntos
Brônquios/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Inflamação/metabolismo , Doença Pulmonar Obstrutiva Crônica , Fumar/metabolismo , Idoso , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Inativação Gênica/fisiologia , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Mucosa Respiratória/patologia , Transdução de Sinais , Tristetraprolina/genética
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