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2.
J Int Med Res ; 49(9): 3000605211041466, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34586953

RESUMO

OBJECTIVE: This study explored the functional interactions between the long non-coding RNA DICER-AS1 and the cellular stress response 1 (CSR1) gene in gastric cancer. METHODS: Quantitative polymerase chain reaction (qPCR) and western blotting were used to measure DICER-AS1, CSR1, and miR-650 expression levels. Gastric cancer cell line proliferation and migration abilities were analyzed using the MTT and transwell migration and invasion assays, respectively. Bioinformatic analysis and dual luciferase reporter assays were employed to study the functional interactions among miR-650, DICER-AS1, and CSR1. RESULTS: DICER-AS1 and CSR1 expression levels were significantly decreased in gastric cancer tissues compared with normal tissues, and qPCR analysis showed that miR-650 was upregulated in gastric cancer tissues. Bioinformatic analysis and dual luciferase reporter assays revealed that DICER-AS1 functioned as a competing endogenous RNA that sponged miR-650, which in turn regulated CSR1 expression. Importantly, ectopic DICER-AS1 and CSR1 expression inhibited cell proliferation and migration in vitro and suppressed xenograft tumorgenicity in vivo. CONCLUSIONS: These results suggest that DICER-AS1 functions as a competing endogenous RNA that regulates miR-650 to suppress proliferation and migration of gastric cancer cells by targeting CSR1. These findings indicate that targeting DICER-AS1 and miR-650 could be a novel treatment for gastric cancer.


Assuntos
MicroRNAs , Neoplasias Gástricas , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , RNA Helicases DEAD-box , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico , Humanos , MicroRNAs/genética , Invasividade Neoplásica/genética , Ribonuclease III , Receptores Depuradores Classe A , Neoplasias Gástricas/genética
3.
Int J Mol Sci ; 22(18)2021 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-34576018

RESUMO

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.


Assuntos
RNA Helicases DEAD-box/fisiologia , Células Endoteliais/fisiologia , Neovascularização Fisiológica , Ribonuclease III/fisiologia , Animais , Humanos
5.
Front Cell Infect Microbiol ; 11: 725392, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34485180

RESUMO

Previous studies have shown that DEAD (Glu-Asp-Ala-Glu)-box RNA helicases play important roles in viral infection, either as cytosolic sensors of pathogenic molecules or as essential host factors against viral infection. In the current study, we found that DDX6, an RNA helicase belonging to the DEAD-box family of helicase, exhibited anti-Enterovirus 71 activity through augmenting RIG-I-mediated type-I IFN response. Moreover, DDX6 binds viral RNA to form an RNA-protein complex to positively regulate the RIG-I-mediated interferon response; however, EV71 has evolved a strategy to antagonize the antiviral effect of DDX6 by proteolytic degradation of the molecule through its non-structural protein 2A, a virus-encoded protease.


Assuntos
RNA Helicases DEAD-box , Infecções por Enterovirus/imunologia , Interferon Tipo I , Proteínas Proto-Oncogênicas , Proteína DEAD-box 58 , Enterovirus Humano A , Humanos , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon , RNA Viral , Receptores Imunológicos
6.
Cell Stem Cell ; 28(11): 1966-1981.e6, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34473945

RESUMO

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.


Assuntos
RNA Helicases DEAD-box , Síndromes Mielodisplásicas , Animais , RNA Helicases DEAD-box/genética , Células Germinativas , Hematopoese/genética , Camundongos , Mutação/genética , Síndromes Mielodisplásicas/genética
7.
Int J Mol Sci ; 22(18)2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34575922

RESUMO

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA binding protein involved in diverse cell processes; it is also a p53 coregulator that initiates apoptosis under DNA damage conditions. However, the upregulation of hnRNPK is correlated with cancer transformation, progression, and migration, whereas the regulatory role of hnRNPK in cancer malignancy remains unclear. We previously showed that arginine methylation of hnRNPK attenuated the apoptosis of U2OS osteosarcoma cells under DNA damage conditions, whereas the replacement of endogenous hnRNPK with a methylation-defective mutant inversely enhanced apoptosis. The present study further revealed that an RNA helicase, DDX3, whose C-terminus preferentially binds to the unmethylated hnRNPK and could promote such apoptotic enhancement. Moreover, C-terminus-truncated DDX3 induced significantly less apoptosis than full-length DDX3. Notably, we also identified a small molecule that docks at the ATP-binding site of DDX3, promotes the DDX3-hnRNPK interaction, and induces further apoptosis. Overall, we have shown that the arginine methylation of hnRNPK suppresses the apoptosis of U2OS cells via interfering with DDX3-hnRNPK interaction. On the other hand, DDX3-hnRNPK interaction with a proapoptotic role may serve as a target for promoting apoptosis in osteosarcoma cells.


Assuntos
Apoptose , Arginina/metabolismo , RNA Helicases DEAD-box/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Motivos de Aminoácidos , Apoptose/genética , Linhagem Celular Tumoral , RNA Helicases DEAD-box/química , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Metilação , Modelos Moleculares , Mutação , Osteossarcoma/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
8.
Nature ; 596(7871): 296-300, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349264

RESUMO

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1-4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Spliceossomos/enzimologia , Actinas/genética , Adenosina/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , RNA Helicases DEAD-box/ultraestrutura , Modelos Moleculares , Mutação , Domínios Proteicos , Precursores de RNA/metabolismo , Precursores de RNA/ultraestrutura , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Spliceossomos/química , Spliceossomos/metabolismo
9.
FEBS Lett ; 595(19): 2447-2462, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34387860

RESUMO

The NLRP3 inflammasome, a critical component of the innate immune system, induces caspase-1 activation and interleukin-1ß maturation and drives cell fate toward pyroptosis. However, the mechanism of NLRP3 inflammasome activation still remains elusive. Here we provide evidence that AKT regulates NLRP3 inflammasome activation. Upon NLRP3 activation, AKT activity is inhibited by second stimulus-induced reactive oxygen species. In contrast, AKT activation leads to NLRP3 inhibition and improved mitochondrial fitness. Mechanistically, AKT induces the phosphorylation of the DDX3X (DEAD-box helicase 3, X-linked), a recently identified NLRP3 inflammasome component, and impairs the interaction between DDX3X and NLRP3. Furthermore, an AKT agonist reduces NLRP3-dependent inflammation in two in vivo models of LPS-induced sepsis and Alum-induced peritonitis. Altogether, our study highlights an important role of AKT in controlling NLRP3 inflammasome activation.


Assuntos
RNA Helicases DEAD-box/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora) , Humanos , Camundongos
10.
J Cell Sci ; 134(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34435633

RESUMO

mRNA decay is a key step in regulating the cellular proteome. Processing bodies (P-bodies) are thought to be sites of mRNA decay and/or storage. P-body units assemble into P-body granules under stress conditions. How this assembly is regulated, however, remains poorly understood. Here, we show, in the yeast Saccharomyces cerevisiae, that the translational repressor Scd6 and the decapping stimulator Edc3 act partially redundantly in P-body assembly by sequestering the Dcp1-Dcp2 (denoted Dcp1/2) decapping complex in the cytoplasm and preventing it from becoming imported into the nucleus by the karyopherin ß protein Kap95. One of two nuclear localization signals in Dcp2 overlaps with the RNA-binding site, suggesting an additional mechanism to regulate Dcp1/2 localization. Nuclear Dcp1/2 does not drive mRNA decay and might be stored there as a readily releasable pool, indicating a dynamic equilibrium between cytoplasmic and nuclear Dcp1/2. Cytoplasmic Dcp1/2 is linked to Dhh1 via Edc3. Functional P-bodies are present at the endoplasmic reticulum where Dcp2 potentially acts to increase the local concentration of Dhh1 through interaction with Edc3 to drive phase separation and hence P-body formation.


Assuntos
Endorribonucleases , Proteínas de Saccharomyces cerevisiae , RNA Helicases DEAD-box , Endorribonucleases/genética , Endorribonucleases/metabolismo , Estabilidade de RNA/genética , Ribonucleoproteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Nucleic Acids Res ; 49(15): 8743-8756, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352092

RESUMO

Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential. Here, we investigated the proposed role of the major initiation scaffold protein eIF4G in reinitiation and its prospective interactions with uORF's cis-acting features in yeast. In analogy to the eIF3 complex, we found that eIF4G and eIF4A but not eIF4E (all constituting the eIF4F complex) are preferentially retained on ribosomes elongating and terminating on reinitiation-permissive uORFs. The loss of the eIF4G contact with eIF4A specifically increased this retention and, as a result, increased the efficiency of reinitiation on downstream initiation codons. Combining the eIF4A-binding mutation with that affecting the integrity of the eIF4G1-RNA2-binding domain eliminated this specificity and produced epistatic interaction with a mutation in one specific cis-acting feature. We conclude that similar to humans, eIF4G is retained on ribosomes elongating uORFs to control reinitiation also in yeast.


Assuntos
RNA Helicases DEAD-box/genética , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 4G em Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Códon de Iniciação/genética , Fator de Iniciação 4E em Eucariotos/genética , Humanos , Fases de Leitura Aberta/genética , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética
12.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445396

RESUMO

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Deleção de Sequência , Pareamento de Bases , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , RNA/química , Ribonuclease III/genética
13.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445801

RESUMO

The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon (IFN) production and antiviral gene expression in response to RNA virus infection. Consequently, RLR signalling is tightly regulated by both host and viral factors. Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade, playing both ubiquitination-dependent and -independent roles in RIG-I-mediated IFN induction. However, additional regulatory roles are emerging. Here, we show a novel interaction between TRIM25 and another protein in the RLR pathway that is essential for type I IFN induction, DEAD-box helicase 3X (DDX3X). In vitro assays and knockdown studies reveal that TRIM25 ubiquitinates DDX3X at lysine 55 (K55) and that TRIM25 and DDX3X cooperatively enhance IFNB1 induction following RIG-I activation, but the latter is independent of TRIM25's catalytic activity. Furthermore, we found that the influenza A virus non-structural protein 1 (NS1) disrupts the TRIM25:DDX3X interaction, abrogating both TRIM25-mediated ubiquitination of DDX3X and cooperative activation of the IFNB1 promoter. Thus, our results reveal a new interplay between two RLR-host proteins that cooperatively enhance IFN-ß production. We also uncover a new and further mechanism by which influenza A virus NS1 suppresses host antiviral defence.


Assuntos
Antivirais/imunologia , Proteína DEAD-box 58/imunologia , RNA Helicases DEAD-box/imunologia , Imunidade/imunologia , Receptores Imunológicos/imunologia , Fatores de Transcrição/imunologia , Proteínas com Motivo Tripartido/imunologia , Ubiquitina-Proteína Ligases/imunologia , Linhagem Celular , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Vírus da Influenza A/imunologia , Interferons/imunologia , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/imunologia , Transdução de Sinais/imunologia , Ubiquitinação/imunologia
14.
Toxicol Lett ; 351: 53-64, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34454013

RESUMO

Benzo[a]pyrene(B[a]P) is a known human carcinogen. The ability of B[a]P to form stable DNA adducts has been repeatedly demonstrated. However, the relationship between DNA adduct formation and cell damage and its underlying molecular mechanisms are less well understood. In this study, we determined the cytotoxicity of benzo[a]pyrenediolepoxide, a metabolite of B[a]P, in human bronchial epithelial cells (BEAS-2B). The formation of BPDE-DNA adducts was quantified using a dot blot. DNA damage resulting from the formation of BPDE-DNA adducts was detected by chromatin immuneprecipitation sequencing (ChIP-Seq), with minor modifications, using specific antibodies against BPDE. In total, 1846 differentially expressed gene loci were detected between the treatment and control groups. The distribution of the BPDE-bound regions indicated that BPDE could covalently bind with both coding and non-coding regions to cause DNA damage. However, the majority of binding occurred at protein-coding genes. Furthermore, among the BPDE-bound genes, we found 16 protein-coding genes related to DNA damage repair. We explored the response to BPDE exposure at the transcriptional level using qRT-PCR and observed a strong inhibition of EIF4A3. We then established an EIF4A3 overexpression cell model and performed comet assays, which revealed that the levels of DNA damage in EIF4A3-overexpressing cells were lower than those in normal cells following BPDE exposure. This suggests that the BPDE-DNA adduct-induced reduction in EIF4A3 expression contributed to the DNA damage induced by BPDE exposure in BEAS-2B cells. These novel findings indicate that ChIP-Seq combined with BPDE specific antibody may be used for exploring the underlying mechanism of DNA adduct-induced genomic damage.


Assuntos
Benzo(a)pireno/toxicidade , RNA Helicases DEAD-box/metabolismo , Adutos de DNA , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Fator de Iniciação 4A em Eucariotos/metabolismo , Linhagem Celular , Clonagem Molecular , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mucosa Respiratória/citologia
15.
J Int Med Res ; 49(8): 3000605211031776, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34369193

RESUMO

The probability of rhabdomyosarcoma occurring in the cervix is less than 0.5% and may be associated with a pathogenic dicer 1, ribonuclease III (DICER1) gene variation. Tumour-induced hypercoagulability and high levels of cancer antigen (CA) 125 are risk factors for cerebral venous sinus thrombosis (CVST). In addition, although nonpuerperal uterine inversion is very rare and is usually caused by leiomyomas from the uterus, large cervical masses can also be the cause. This case report describes a 24-year-old woman with uterine inversion caused by an embryonic cervical rhabdomyosarcoma that presented with CVST as her first symptom. The patient underwent laparoscopic total uterus and bilateral salpingectomy, during which the uterus was found to be completely inverted. Postoperative pathology confirmed embryonic cervical rhabdomyosarcoma. The patient quickly developed lung and para-aortic lymph node metastases. Two months later, the patient died of complications. When coagulation indices in patients with tumours are abnormal, especially when the levels of D-dimer and CA125 increase, it is recommended that anticoagulant therapy is administered in a timely manner to prevent the occurrence of CVST. Furthermore, for large cervical tumours, physicians should also be alert to the occurrence of uterine inversion.


Assuntos
Rabdomiossarcoma , Trombose dos Seios Intracranianos , Neoplasias do Colo do Útero , Inversão Uterina , Adulto , RNA Helicases DEAD-box , Feminino , Humanos , Gravidez , Ribonuclease III , Trombose dos Seios Intracranianos/complicações , Trombose dos Seios Intracranianos/diagnóstico por imagem , Neoplasias do Colo do Útero/complicações , Adulto Jovem
16.
Nat Commun ; 12(1): 5043, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413292

RESUMO

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5'-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5' UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5' UTR of target mRNAs.


Assuntos
Regiões 5' não Traduzidas , RNA Helicases DEAD-box/metabolismo , Quadruplex G , Músculos/citologia , Regeneração/fisiologia , Células-Tronco/citologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Modelos Animais de Doenças , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Músculos/metabolismo , Mioblastos/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Células-Tronco/metabolismo
17.
Cells ; 10(8)2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34440801

RESUMO

Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/farmacologia , Células Germinativas/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Testículo/citologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Colágeno/metabolismo , Meios de Cultura/química , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Combinação de Medicamentos , Gelatina/metabolismo , Perfilação da Expressão Gênica/métodos , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Laminina/metabolismo , Masculino , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteoglicanas/metabolismo , RNA-Seq/métodos , Análise de Célula Única/métodos , Células-Tronco/citologia , Células-Tronco/metabolismo , Testículo/embriologia , Vitronectina/metabolismo
18.
Biol Psychiatry ; 90(11): 742-755, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34344536

RESUMO

BACKGROUND: Mutations in the X-linked gene DDX3X account for approximately 2% of intellectual disability in females, often comorbid with behavioral problems, motor deficits, and brain malformations. DDX3X encodes an RNA helicase with emerging functions in corticogenesis and synaptogenesis. METHODS: We generated a Ddx3x haploinsufficient mouse (Ddx3x+/- females) with construct validity for DDX3X loss-of-function mutations. We used standardized batteries to assess developmental milestones and adult behaviors, as well as magnetic resonance imaging and immunostaining of cortical projection neurons to capture early postnatal changes in brain development. RESULTS: Ddx3x+/- females showed physical, sensory, and motor delays that evolved into behavioral anomalies in adulthood, including hyperactivity, anxiety-like behaviors, cognitive impairments in specific tasks (e.g., contextual fear memory but not novel object recognition memory), and motor deficits. Motor function declined with age but not if mice were previously exposed to behavioral training. Developmental and behavioral changes were associated with a reduction in brain volume, with some regions (e.g., cortex and amygdala) disproportionally affected. Cortical thinning was accompanied by defective cortical lamination, indicating that Ddx3x regulates the balance of glutamatergic neurons in the developing cortex. CONCLUSIONS: These data shed new light on the developmental mechanisms driving DDX3X syndrome and support construct and face validity of this novel preclinical mouse model.


Assuntos
Deficiência Intelectual , Animais , RNA Helicases DEAD-box/genética , Modelos Animais de Doenças , Feminino , Camundongos , Neurogênese , Fenótipo , Síndrome
19.
J Transl Med ; 19(1): 333, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34353336

RESUMO

BACKGROUND: In recent years, long non-coding RNAs (lncRNAs) have attracted much attention because of its regulatory role in occurrence and progression of tumors, including triple-negative breast cancer (TNBC). LncRNA PITPNA antisense RNA 1 (PITPNA-AS1) has been explored in some cancers, whereas its function and molecular mechanism in TNBC remain unclear. METHODS: PITPNA-AS1 expression in TNBC tissues and cells was determined by RT-qPCR. TNBC cell viability, proliferation, migration, invasion were assessed with CCK-8, colony formation, wound healing, transwell assays. Cell apoptosis was evaluated by flow cytometry. Expression of EMT-related markers was detected by western blot analyses. The molecular mechanism of PITPNA-AS1 was explored by RNA pull down, luciferase reporter, RIP and ChIP assays. RESULTS: PITPNA-AS1 showed high expression levels in TNBC tissues and cells. PITPNA-AS1 knockdown suppressed TNBC cell viability, proliferation, migration, invasion in vitro and inhibited xenograft tumor growth in mice. Mechanistically, PITPNA-AS1 upregulated SIK2 expression by sponging miR-520d-5p and recruiting DDX54 protein. Results of rescue assays suggested that the inhibitive effects of silenced PITPNA-AS1 on TNBC cellular processes were partially rescued by overexpressing SIK2 or combination of miR-520d-5p inhibition and DDX54 overexpression. More importantly, we found that the upregulation of PITPNA-AS1 in TNBC cells was attributed to transcription factor MYBL2. CONCLUSION: PITPNA-AS1 activated by MYBL2 plays an oncogenic role in TNBC through upregulating SIK2.


Assuntos
MicroRNAs , RNA Longo não Codificante , Neoplasias de Mama Triplo Negativas , Animais , Proteínas de Ciclo Celular , Proliferação de Células , RNA Helicases DEAD-box/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , Proteínas de Neoplasias , RNA Longo não Codificante/genética , Transativadores , Neoplasias de Mama Triplo Negativas/genética
20.
J Transl Med ; 19(1): 334, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362383

RESUMO

BACKGROUND: Although the rapid development of diagnosis and treatment has improved prognosis in early breast cancer, challenges from different therapeutic response remain due to breast cancer heterogeneity. DEAD-box helicase 27 (DDX27) had been proved to influence ribosome biogenesis and identified as a promoter in gastric and colorectal cancer associated with stem cell-like properties, while the impact of DDX27 on breast cancer prognosis and biological functions is unclear. We aimed to explore the influence of DDX27 on stem cell-like properties and prognosis in breast cancer. METHODS: The expression of DDX27 was evaluated in 24 pairs of fresh breast cancer and normal tissue by western blot. We conducted Immunohistochemical (IHC) staining in paraffin sections of 165 breast cancer patients to analyze the expression of DDX27 and its correlation to stemness biomarker. The Cancer Genome Atlas-Breast Cancer (TCGA-BRCA) database and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database were used to analyze the expression of DDX27 in breast cancer. Kaplan-Meier survival analysis were used to investigate the implication of DDX27 on breast cancer prognosis. Western blot, CCK-8 assay, Transwell assay and wound-healing assay were carried out to clarify the regulation of DDX27 on stem cell-like properties in breast cancer cells. Gene Set Enrichment Analysis (GSEA) was performed to analyze the potential molecular mechanisms of DDX27 in breast cancer. RESULTS: DDX27 was significantly high expressed in breast cancer compared with normal tissue. High expression of DDX27 was related to larger tumor size (p = 0.0005), positive lymph nodes (p = 0.0008), higher histological grade (p = 0.0040), higher ki-67 (p = 0.0063) and later TNM stage (p < 0.0001). Patients with high DDX27 expression turned out a worse prognosis on overall survival (OS, p = 0.0087) and disease-free survival (DFS, p = 0.0235). Overexpression of DDX27 could enhance the expression of biomarkers related to stemness and promote stem cell-like activities such as proliferation and migration in breast cancer cells. CONCLUSION: DDX27 can enhance stem cell-like properties and cause poor prognosis in breast cancer, also may be expected to become a potential biomarker for breast cancer therapy.


Assuntos
Neoplasias da Mama , Biomarcadores Tumorais , Neoplasias da Mama/genética , RNA Helicases DEAD-box/genética , Feminino , Humanos , Prognóstico , Proteômica , Células-Tronco
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