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1.
Science ; 373(6551): 231-236, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244417

RESUMO

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.


Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Interferência de RNA , Vírus de RNA/fisiologia , RNA Viral/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células-Tronco/enzimologia , Células-Tronco/virologia , Processamento Alternativo , Animais , Encéfalo/enzimologia , Encéfalo/virologia , Linhagem Celular , RNA Helicases DEAD-box/química , Humanos , Imunidade Inata , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Organoides/enzimologia , Organoides/virologia , Infecções por Vírus de RNA/enzimologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , Vírus de RNA/imunologia , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonuclease III/química , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Replicação Viral , Zika virus/genética , Zika virus/imunologia , Zika virus/fisiologia , Infecção por Zika virus/enzimologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
2.
Nat Commun ; 12(1): 4118, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226542

RESUMO

Living cells actively migrate in their environment to perform key biological functions-from unicellular organisms looking for food to single cells such as fibroblasts, leukocytes or cancer cells that can shape, patrol or invade tissues. Cell migration results from complex intracellular processes that enable cell self-propulsion, and has been shown to also integrate various chemical or physical extracellular signals. While it is established that cells can modify their environment by depositing biochemical signals or mechanically remodelling the extracellular matrix, the impact of such self-induced environmental perturbations on cell trajectories at various scales remains unexplored. Here, we show that cells can retrieve their path: by confining motile cells on 1D and 2D micropatterned surfaces, we demonstrate that they leave long-lived physicochemical footprints along their way, which determine their future path. On this basis, we argue that cell trajectories belong to the general class of self-interacting random walks, and show that self-interactions can rule large scale exploration by inducing long-lived ageing, subdiffusion and anomalous first-passage statistics. Altogether, our joint experimental and theoretical approach points to a generic coupling between motile cells and their environment, which endows cells with a spatial memory of their path and can dramatically change their space exploration.


Assuntos
Movimento Celular/fisiologia , Memória Espacial/fisiologia , Células CACO-2 , Simulação por Computador , Matriz Extracelular/metabolismo , Fibroblastos , Humanos , Modelos Biológicos , RNA Interferente Pequeno
3.
Biomed Pharmacother ; 139: 111633, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243624

RESUMO

Idiopathic pulmonary fibrosis (IPF) is the most common and most deadly form of interstitial lung disease. Osteopontin (OPN), a matricellular protein with proinflammatory and profibrotic properties, plays a major role in several fibrotic diseases, including IPF; OPN is highly upregulated in patients' lung samples. In this study, we knocked down OPN in a bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model using small interfering RNA (siRNA) to determine whether the use of OPN siRNA is an effective therapeutic strategy for IPF. We found that fibrosing areas were significantly smaller in specimens from OPN siRNA-treated mice. The number of alveolar macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid was also reduced in OPN siRNA-treated mice. Regarding the expression of epithelial-mesenchymal transition (EMT)-related proteins, the administration of OPN-siRNA to BLM-treated mice upregulated E-cadherin expression and downregulated vimentin expression. Moreover, in vitro, we incubated the human alveolar adenocarcinoma cell line A549 with transforming growth factor (TGF)-ß1 and subsequently transfected the cells with OPN siRNA. We found a significant upregulation of Col1A1, fibronectin, and vimentin after TGF-ß1 stimulation in A549 cells. In contrast, a downregulation of Col1A1, fibronectin, and vimentin mRNA levels was observed in TGF-ß1-stimulated OPN knockdown A549 cells. Therefore, the downregulation of OPN effectively reduced pulmonary fibrotic and EMT changes both in vitro and in vivo. Altogether, our results indicate that OPN siRNA exerts a protective effect on BLM-induced PF in mice. Our results provide a basis for the development of novel targeted therapeutic strategies for IPF.


Assuntos
Bleomicina/farmacologia , Transição Epitelial-Mesenquimal/genética , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Osteopontina/genética , Células A549 , Animais , Líquido da Lavagem Broncoalveolar , Linhagem Celular Tumoral , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/genética , Regulação para Cima/genética
4.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281265

RESUMO

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5'-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.


Assuntos
Proteção de Cultivos/métodos , Hordeum/genética , Hordeum/microbiologia , RNA Interferente Pequeno/administração & dosagem , Família 3 do Citocromo P450/genética , Resistência à Doença/genética , Vesículas Extracelulares/genética , Vesículas Extracelulares/microbiologia , Inativação Gênica , Interações entre Hospedeiro e Microrganismos/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Interferência de RNA , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , RNA Interferente Pequeno/isolamento & purificação
5.
BMC Infect Dis ; 21(1): 655, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34233649

RESUMO

BACKGROUND: Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages. METHODS: We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student's t-test from at least four independent experiments. RESULTS: We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production. CONCLUSIONS: We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.


Assuntos
Apoptose/genética , Proteínas de Fluorescência Verde , Infecções por HIV , Macrófagos , RNA Interferente Pequeno , Linfócitos T CD4-Positivos/virologia , Estudo de Associação Genômica Ampla , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Linfócitos T
6.
Int J Nanomedicine ; 16: 4451-4470, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234436

RESUMO

Background: Liver fibrosis is a chronic liver disease with excessive production of extracellular matrix proteins, leading to cirrhosis, hepatocellular carcinoma, and death. Purpose: This study aimed at the development of a novel derivative of polyethyleneimine (PEI) that can effectively deliver transforming growth factor ß (TGFß) siRNA and inhibit chemokine receptor 4 (CXCR4) for TGFß silencing and CXCR4 Inhibition, respectively, to treat CCl4-induced liver fibrosis in a mouse model. Methods: Cyclam-modified PEI (PEI-Cyclam) was synthesized by incorporating cyclam moiety into PEI by nucleophilic substitution reaction. Gel electrophoresis confirmed the PEI-Cyclam polyplex formation and stability against RNAase and serum degradation. Transmission electron microscopy and zeta sizer were employed for the morphology, particle size, and zeta potential, respectively. The gene silencing and CXCR4 targeting abilities of PEI-Cyclam polyplex were evaluated by luciferase and CXCR4 redistribution assays, respectively. The histological and immunohistochemical staining determined the anti-fibrotic activity of PEI-Cyclam polyplex. The TGFß silencing of PEI-Cyclam polyplex was authenticated by Western blotting. Results: The 1H NMR of PEI-Cyclam exhibited successful incorporation of cyclam content onto PEI. The PEI-Cyclam polyplex displayed spherical morphology, positive surface charge, and stability against RNAse and serum degradation. Cyclam modification decreased the cytotoxicity and demonstrated CXCR4 antagonistic and luciferase gene silencing efficiency. PEI-Cyclam/siTGFß polyplexes decreased inflammation, collagen deposition, apoptosis, and cell proliferation, thus ameliorating liver fibrosis. Also, PEI-Cyclam/siTGFß polyplex significantly downregulated α-smooth muscle actin, TGFß, and collagen type III. Conclusion: Our findings validate the feasibility of using PEI-Cyclam as a siRNA delivery vector for simultaneous TGFß siRNA delivery and CXCR4 inhibition for the combined anti-fibrotic effects in a setting of CCl4-induced liver fibrosis.


Assuntos
Tetracloreto de Carbono/efeitos adversos , Compostos Heterocíclicos/química , Cirrose Hepática/genética , Polietilenoimina/química , RNA Interferente Pequeno/genética , Fator de Crescimento Transformador beta/genética , Animais , Apoptose/efeitos dos fármacos , Portadores de Fármacos/química , Inativação Gênica , Humanos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Camundongos , Tamanho da Partícula , RNA Interferente Pequeno/química , Receptores CXCR4/genética , Fator de Crescimento Transformador beta/deficiência
7.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204950

RESUMO

The dysregulation of autophagy is important in the development of many cancers, including thyroid cancer, where V600EBRAF is a main oncogene. Here, we analyse the effect of V600EBRAF inhibition on autophagy, the mechanisms involved in this regulation and the role of autophagy in cell survival of thyroid cancer cells. We reveal that the inhibition of V600EBRAF activity with its specific inhibitor PLX4720 or the depletion of its expression by siRNA induces autophagy in thyroid tumour cells. We show that V600EBRAF downregulation increases LKB1-AMPK signalling and decreases mTOR activity through a MEK/ERK-dependent mechanism. Moreover, we demonstrate that PLX4720 activates ULK1 and increases autophagy through the activation of the AMPK-ULK1 pathway, but not by the inhibition of mTOR. In addition, we find that autophagy blockade decreases cell viability and sensitize thyroid cancer cells to V600EBRAF inhibition by PLX4720 treatment. Finally, we generate a thyroid xenograft model to demonstrate that autophagy inhibition synergistically enhances the anti-proliferative and pro-apoptotic effects of V600EBRAF inhibition in vivo. Collectively, we uncover a new role of AMPK in mediating the induction of cytoprotective autophagy by V600EBRAF inhibition. In addition, these data establish a rationale for designing an integrated therapy targeting V600EBRAF and the LKB1-AMPK-ULK1-autophagy axis for the treatment of V600EBRAF-positive thyroid tumours.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Apoptose/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia
8.
Science ; 373(6550)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210850

RESUMO

The plant male germline undergoes DNA methylation reprogramming, which methylates genes de novo and thereby alters gene expression and regulates meiosis. Here, we reveal the molecular mechanism underlying this reprogramming. We demonstrate that genic methylation in the male germline, from meiocytes to sperm, is established by 24-nucleotide small interfering RNAs (siRNAs) transcribed from transposons with imperfect sequence homology. These siRNAs are synthesized by meiocyte nurse cells (tapetum) through activity of CLSY3, a chromatin remodeler absent in other anther cells. Tapetal siRNAs govern germline methylation throughout the genome, including the inherited methylation patterns in sperm. Tapetum-derived siRNAs also silence germline transposons, safeguarding genome integrity. Our results reveal that tapetal siRNAs are sufficient to reconstitute germline methylation patterns and drive functional methylation reprogramming throughout the male germline.


Assuntos
Arabidopsis/citologia , Arabidopsis/genética , Epigênese Genética , Herança Paterna , Pólen/genética , RNA Interferente Pequeno/genética , Metilação de DNA , Meiose/genética , Mitose/genética
9.
Anticancer Res ; 41(7): 3317-3326, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230127

RESUMO

BACKGROUND/AIM: We evaluated the impact of FosL1, a member of the activated protein-1 family, on the pathways leading to regional metastasis of head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: We examined the influence of small interfering RNA (siRNA) and short heparin RNA (shRNA) mediated knockdown of FosL1 on cell migration, invasion, and proliferation in vitro as well as on regional metastasis in vivo. The prognostic significance of FosL1 was also analyzed using the Kaplan- Meier plotter using data from an HNSCC patient database. RESULTS: Down-regulation of FosL1 inhibited cell migration, invasion, and proliferation in vitro, decreased the incidence of regional metastases, and prolonged the survival of mice in vivo. We also determined that HNSCC patients with higher expression levels of FosL1 had a significantly shorter survival time than those with low expression of FosL1. CONCLUSION: FosL1 plays a crucial role in promoting cell migration, invasion, and proliferation in HNSCC.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Invasividade Neoplásica/genética , Proteínas Proto-Oncogênicas c-fos/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica/patologia , Prognóstico , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
10.
Cells ; 10(6)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201214

RESUMO

In late 2019, the betacoronavirus SARS-CoV-2 was identified as the viral agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. Coronaviruses Spike proteins are responsible for their ability to interact with host membrane receptors and different proteins have been identified as SARS-CoV-2 interactors, among which Angiotensin-converting enzyme 2 (ACE2), and Basigin2/EMMPRIN/CD147 (CD147). CD147 plays an important role in human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, Kaposi's sarcoma-associated herpesvirus, and severe acute respiratory syndrome coronavirus infections. In particular, SARS-CoV recognizes the CD147 receptor expressed on the surface of host cells by its nucleocapsid protein binding to cyclophilin A (CyPA), a ligand for CD147. However, the involvement of CD147 in SARS-CoV-2 infection is still debated. Interference with both the function (blocking antibody) and the expression (knock down) of CD147 showed that this receptor partakes in SARS-CoV-2 infection and provided additional clues on the underlying mechanism: CD147 binding to CyPA does not play a role; CD147 regulates ACE2 levels and both receptors are affected by virus infection. Altogether, these findings suggest that CD147 is involved in SARS-CoV-2 tropism and represents a possible therapeutic target to challenge COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2/fisiologia , Basigina/fisiologia , SARS-CoV-2/fisiologia , Internalização do Vírus , Células A549 , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Basigina/antagonistas & inibidores , Basigina/genética , COVID-19/patologia , COVID-19/prevenção & controle , COVID-19/virologia , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Células Hep G2 , Interações Hospedeiro-Patógeno , Humanos , Terapia de Alvo Molecular , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Receptores Virais/metabolismo , Receptores Virais/fisiologia , SARS-CoV-2/metabolismo , Células Vero , Tropismo Viral/fisiologia
11.
Nan Fang Yi Ke Da Xue Xue Bao ; 41(6): 931-936, 2021 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-34238747

RESUMO

OBJECTIVE: To investigate the regulatory role of the long non-coding RNA (lncRNA) small nucleolar host gene 3 (SNHG3) in proliferation, migration and invasion of human cervical cancer cell line SiHa. OBJECTIVE: Array data were retrieved from GEO database to analyze the expression levels of SNHG3 in cervical cancer and adjacent normal tissues. SiHa cells were transfected with a small interfering RNA (siRNA) targeting SNHG3, and the changes in the transcriptional levels of lncRNA SNHG3 and the epithelial-mesenchymal transition (EMT) markers N-cadherin, Snail, vimentin and E-cadherin were detected using real-time quantitative PCR; the protein expressions of N-cadherin, Snail, vimentin and E-cadherin were determined using Western blotting. Cell counting kit-8 (CCK8) assay was utilized to assess the proliferation capacity of the transfected cells. Wound healing assay and Transwell assay were performed to evaluate the transversal and longitudinal migration and invasion abilities of the cells. OBJECTIVE: SNHG3 was over-expressed in cervical cancer tissues and SiHa cells. In SiHa cells, knocking down SNHG3 significantly inhibited the proliferation (P < 0.001), migration (P < 0.01) and invasion abilities (P < 0.001) of the cells, down-regulated the expression levels of N-cadherin, Snail and vimentin (P < 0.001) and up-regulated the expression of E-cadherin (P < 0.001). OBJECTIVE: SNHG3 may promote the proliferation, migration and invasion of SiHa cells by activating the EMT signaling pathway.


Assuntos
RNA Longo não Codificante , Neoplasias do Colo do Útero , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Neoplasias do Colo do Útero/genética
12.
Nat Commun ; 12(1): 4061, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210982

RESUMO

PIWI proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two PIWI proteins, Aub and Ago3. Here we show that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimper uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. Our results reveal that post-translational modifications in unstructured regions of PIWI proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Proteínas de Transporte/química , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Masculino , Metilação , Modelos Moleculares , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Domínios Proteicos , Proteína-Arginina N-Metiltransferases , RNA Interferente Pequeno/química
13.
Nat Commun ; 12(1): 4212, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244496

RESUMO

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Interferência de RNA , Espermatogênese/genética , Animais , Animais Geneticamente Modificados , Arginina/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Éxons/genética , Feminino , Masculino , Metilação , Oogênese/genética , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Processos de Determinação Sexual/genética
14.
BMC Genomics ; 22(1): 560, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34284724

RESUMO

BACKGROUND: Root knot nematodes (RKN) are plant parasitic nematodes causing major yield losses of widely consumed food crops such as rice (Oryza sativa). Because non-coding RNAs, including small interfering RNAs (siRNA), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are key regulators of various plant processes, elucidating their regulation during this interaction may lead to new strategies to improve crop protection. In this study, we aimed to identify and characterize rice siRNAs, miRNAs and lncRNAs responsive to early infection with RKN Meloidogyne graminicola (Mg), based on sequencing of small RNA, degradome and total RNA libraries from rice gall tissues compared with uninfected root tissues. RESULTS: We found 425 lncRNAs, 3739 siRNAs and 16 miRNAs to be differentially expressed between both tissues, of which a subset was independently validated with RT-qPCR. Functional prediction of the lncRNAs indicates that a large part of their potential target genes code for serine/threonine protein kinases and transcription factors. Differentially expressed siRNAs have a predominant size of 24 nts, suggesting a role in DNA methylation. Differentially expressed miRNAs are generally downregulated and target transcription factors, which show reduced degradation according to the degradome data. CONCLUSIONS: To our knowledge, this work is the first to focus on small and long non-coding RNAs in the interaction between rice and Mg, and provides an overview of rice non-coding RNAs with the potential to be used as a resource for the development of new crop protection strategies.


Assuntos
MicroRNAs , Oryza , RNA Longo não Codificante , Tylenchoidea , Animais , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Oryza/genética , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Tylenchoidea/genética
15.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204592

RESUMO

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Potencial da Membrana Mitocondrial , Mitocôndrias/genética , Mitocôndrias/metabolismo , NADH Desidrogenase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , NADH Desidrogenase/genética , Transporte Proteico , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
16.
J Cardiothorac Surg ; 16(1): 194, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34233689

RESUMO

OBJECTIVE: C-erbB-2 has been confirmed to be an oncogene that participates in cell growth, differentiation and division of tumors. We are wondered if its silenced expression can exert an anti-tumor effect. Therefore, this study is conducted to investigate the mechanism of C-erbB-2 silencing and IGF-1 pathway on esophageal carcinoma (EC) cell biological behaviors. METHODS: The objects of study were 84 EC patients from Heping Hospital Affiliated to Changzhi Medical College, with the collection of EC tissue and adjacent normal tissue (> 5 cm away from cancer tissue). C-erbB-2 protein expression in EC tissues was detected by immunohistochemistry. Human EC cell line Eca-109 was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Based on different transfection protocols, EC cells with logarithmic growth phase of 3-5 passages were divided into blank control group, oe-C-erbB-2 NC group, siRNA C-erbB-2 NC group, oe-C-erbB-2 group, siRNA C-erbB-2 group, OSI-906 group, Rg5 group, Rg5 + siRNA C-erbB-2 NC group and Rg5 + siRNA C-erbB-2 group. Cell proliferation was detected by MTT assay; cell cycle distribution and apoptosis by flow cytometry; C-erbB-2, IGF-1, IGF-1R and Akt mRNA and protein expressions by qRT-PCR and western blot; and cell invasion and migration by Transwell assay and scratch test. Tumor growth was observed in male BALB/c nude mice (Shanghai Experimental Animal Center) based on Eca109 cell implantation, raising, and measurement. RESULTS: C-erbB-2, IGF-1, IGF-1R and Akt expression were higher in EC tissues than those in adjacent tissues (all P < 0.05). Compared with blank control group, both si-C-erbB-2 and OSI-906 groups had decreased IGF-1, IGF-1R and Akt mRNA and protein expressions, decreased cell proliferation, migration and invasion, prolonged G0/G1 phase, shortened S phase, increased cell apoptosis, and inhibited tumor growth (all P < 0.05); while opposite trends were detected in C-erbB-2 vector and Rg5 groups (all P < 0.05), without statistical differences in siRNA C-erbB-2 + Rg5 group (all P > 0.05). CONCLUSION: Silencing C-erbB-2 expression may inhibit EC cell proliferation, promote cell apoptosis and block cell cycle progression by inhibiting IGF-1 pathway activation. The beneficial effect of silencing C-erbB-2 expression can be reversed by promoting the activation of IGF-1 pathway. Findings in our study may provide potential reference for understanding the molecular mechanism of EC and supply possible axis for preventing the development of EC from the perspective of molecular biology.


Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Inativação Gênica/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Receptor ErbB-2/genética , Adulto , Idoso , Animais , Apoptose/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor IGF Tipo 1 , Transfecção
18.
Pestic Biochem Physiol ; 177: 104906, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34301367

RESUMO

The response of insects to orally delivered double-stranded RNA ranges widely among taxa studied to date. Long dsRNA does elicit a response in stink bugs but the dose required to achieve an effect is relatively high compared to other insects such Colorado potato beetle or western corn rootworm. Improving the delivery of dsRNA to stink bugs will improve the likelihood of using RNA-based biocontrols for the management of these economically important pests. Short hairpin RNA (shRNA) is a useful molecule with which to test improvements in the delivery of double stranded RNA in the neotropical brown stink bug, Euschistus heros, since shRNA alone does not elicit a clear effect like that for long dsRNA. Here, we show for the first time the oral delivery of shRNA triggering RNA interference (RNAi) in E. heros using 4 nm cerium oxide nanoparticles (CeO2 NPs) coated with diethylamioethyl dextran (Dextran-DEAE) as a carrier. We identified particle properties (coating composition and degree of substitution, hydrodynamic diameter, and zeta potential) and shRNA loading rates (Ce:shRNA mass ratio) that resulted in successful transcript reduction or RNAi. When the Z-average diameter of CeO2 Dextran-DEAE-shRNA NP complex was less than 250 nm and the zeta potential was in the 15-25 mV range (Ce:shRNA mass ratio of 0.7:1), significant mortality attributed to RNAi was observed with a shRNA concentration in feeding solution of 250 ng/µl. The degradation of the targeted troponin transcript by NP-delivered shRNA was equivalent to that observed with long dsRNA, while naked shRNA transcript reduction was not statistically significant. Elemental mapping by synchrotron X-ray fluorescence microprobe confirmed uptake and distribution of Ce throughout the body with the highest concentrations found in gut tissue. Taken together, our results suggest that a nanoparticle delivery system can improve the delivery of RNA-based biocontrols to E. heros, and therefore its attractiveness as an application in the management of this important pest in soybean production.


Assuntos
Heterópteros , Nanoestruturas , Animais , Heterópteros/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética
19.
Breast Cancer Res Treat ; 189(1): 49-61, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34196902

RESUMO

PURPOSE: Breast cancer remains a prominent global disease affecting women worldwide despite the emergence of novel therapeutic regimens. Metastasis is responsible for most cancer-related deaths, and acquisition of a mesenchymal and migratory cancer cell phenotypes contributes to this devastating disease. The utilization of kinase targets in drug discovery have revolutionized the field of cancer research but despite impressive advancements in kinase-targeting drugs, a large portion of the human kinome remains understudied in cancer. NEK5, a member of the Never-in-mitosis kinase family, is an example of such an understudied kinase. Here, we characterized the function of NEK5 in breast cancer. METHODS: Stably overexpressing NEK5 cell lines (MCF7) and shRNA knockdown cell lines (MDA-MB-231, TU-BcX-4IC) were utilized. Cell morphology changes were evaluated using immunofluorescence and quantification of cytoskeletal components. Cell proliferation was assessed by Ki-67 staining and transwell migration assays tested cell migration capabilities. In vivo experiments with murine models were necessary to demonstrate NEK5 function in breast cancer tumor growth and metastasis. RESULTS: NEK5 activation altered breast cancer cell morphology and promoted cell migration independent of effects on cell proliferation. NEK5 overexpression or knockdown does not alter tumor growth kinetics but promotes or suppresses metastatic potential in a cell type-specific manner, respectively. CONCLUSION: While NEK5 activity modulated cytoskeletal changes and cell motility, NEK5 activity affected cell seeding capabilities but not metastatic colonization or proliferation in vivo. Here we characterized NEK5 function in breast cancer systems and we implicate NEK5 in regulating specific steps of metastatic progression.


Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Humanos , Camundongos , Quinases Relacionadas a NIMA/genética , Fenótipo , RNA Interferente Pequeno
20.
Neuroscience ; 469: 91-102, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34216695

RESUMO

The transport mechanism of intestinal α-synuclein to the central nervous system has become a new hot topic in Parkinson's disease (PD) research. It is worth noting that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been reported to be involved in the pathogenesis of PD. After silencing GAPDH expression by GAPDH siRNA, the normal human intestinal epithelial crypt-like (HIEC) and human SH-SY5Y neuroblastoma cell lines were co-cultured with Escherichia coli cells which were transfected with an α-synuclein overexpression plasmid. The levels of autophagy-related proteins (BECN1, ATG5, LC3A/B and p62) were determined by Western blot analysis. Changes in pro-apoptosis protein levels and flow cytometry analysis were used to assess cell apoptosis and relative intracellular ATP concentration was measured. Oxidative stress was assessed by measuring the levels of reactive oxygen species (ROS) using 2',7'-dichlorofluorescein diacetate (DCFH-DA), thiobarbituric acid-reactive substances (TBARS), and antioxidant capacity was assessed by measuring the glutathione (GSH) levels and superoxide dismutase (SOD) activity. The silencing of the expression of GAPDH pre-knockdown was found to reduce the intracellular levels of ROS and lipid peroxidation, enhance autophagy activity, thereby reducing the cell injury, apoptosis and necrosis induced by exogenous α-synuclein protein in SH-SY5Y cells. This study identifies a new therapeutic target of exogenous α-synuclein protein induced SH-SY5Y cell injury and improves our understanding of the pathophysiological role of GAPDH in vitro.


Assuntos
Apoptose , alfa-Sinucleína , Linhagem Celular Tumoral , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Estresse Oxidativo , RNA Interferente Pequeno , alfa-Sinucleína/metabolismo
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