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1.
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
2.
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
3.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205075

RESUMO

Inherited neuropathies known as Charcot-Marie-Tooth (CMT) disease are genetically heterogeneous disorders affecting the peripheral nerves, causing significant and slowly progressive disability over the lifespan. The discovery of their diverse molecular genetic mechanisms over the past three decades has provided the basis for developing a wide range of therapeutics, leading to an exciting era of finding treatments for this, until now, incurable group of diseases. Many treatment approaches, including gene silencing and gene replacement therapies, as well as small molecule treatments are currently in preclinical testing while several have also reached clinical trial stage. Some of the treatment approaches are disease-specific targeted to the unique disease mechanism of each CMT form, while other therapeutics target common pathways shared by several or all CMT types. As promising treatments reach the stage of clinical translation, optimal outcome measures, novel biomarkers and appropriate trial designs are crucial in order to facilitate successful testing and validation of novel treatments for CMT patients.


Assuntos
Doença de Charcot-Marie-Tooth/terapia , Terapia Genética , Proteína P0 da Mielina/genética , Proteínas da Mielina/genética , Doença de Charcot-Marie-Tooth/genética , Inativação Gênica , Humanos , Mutação/genética , Proteína P0 da Mielina/antagonistas & inibidores , Proteínas da Mielina/antagonistas & inibidores , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia
4.
Nat Commun ; 12(1): 4292, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257299

RESUMO

The Microrchidia (MORC) family of ATPases are required for transposable element (TE) silencing and heterochromatin condensation in plants and animals, and C. elegans MORC-1 has been shown to topologically entrap and condense DNA. In Arabidopsis thaliana, mutation of MORCs has been shown to reactivate silent methylated genes and transposons and to decondense heterochromatic chromocenters, despite only minor changes in the maintenance of DNA methylation. Here we provide the first evidence localizing Arabidopsis MORC proteins to specific regions of chromatin and find that MORC4 and MORC7 are closely co-localized with sites of RNA-directed DNA methylation (RdDM). We further show that MORC7, when tethered to DNA by an artificial zinc finger, can facilitate the establishment of RdDM. Finally, we show that MORCs are required for the efficient RdDM mediated establishment of DNA methylation and silencing of a newly integrated FWA transgene, even though morc mutations have no effect on the maintenance of preexisting methylation at the endogenous FWA gene. We propose that MORCs function as a molecular tether in RdDM complexes to reinforce RdDM activity for methylation establishment. These findings have implications for MORC protein function in a variety of other eukaryotic organisms.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Adenosina Trifosfatases/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Metilação de DNA/genética , Metilação de DNA/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Inativação Gênica
5.
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
6.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(3): 805-811, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34105476

RESUMO

OBJECTIVE: To investigate the effect of the tripartite motif containing 31 (TRIM31) gene silencing on the proliferation and apoptosis of multiple myeloma cells and its possible mechanism. METHODS: The normal bone marrow plasma cells (nPCs) were selected as control, and the mRNA and protein expression levels of TRIM31 in human multiple myeloma cell lines (U266, RPMI-8226, NCI-H929 and KMS-11) were detected by RT-qPCR and Western blot. Recombinant lentivirol vector containing shRNA-TRIM31 and its negative control were used to infect U266 cells respectively, and the mRNA expression level of TRIM31 in infected cells was detected by RT-qPCR. Then cell proliferation, colony forming and apoptosis were analyzed by CCK-8, soft agar assay, and flow cytometry, respectively. The protein expression levels of TRIM31, cleaved-caspase-3, BCL-2, Bax, p-Akt (Ser473), Akt and PI3K (p110α) were evaluated by Western blot. In addition, the PI3K/Akt signaling pathway-specific inhibitor LY294002 and TRIM31-shRNA lentivirus were used to interfere with U266 cells, and the cell proliferation, apoptosis, and protein expression of p-Akt (Ser473) and Akt were detected by CCK-8, flow cytometry and Western blot, respectively. RESULTS: Compared with nPCs, the expression levels of TRIM31 mRNA and protein in U266, RPMI-8226, NCI-H929 and KMS-11 cells were significantly increased (P<0.001), especially in U266 cells. After lentivirus infection, the levels of TRIM31 mRNA and protein in U266 cells were significantly decreased (P<0.001). TRIM31 silencing significantly inhibited the proliferation of U266 cells (P<0.05), attenuated the ability of cell cloning, improved cell apoptosis, up-regulated the protein expressions of cleaved-caspase-3 and Bas as well as down-regulated expressions of BCL-2, p-Akt (Ser473) and PI3K (p110α). There was no significant effect on Akt protein. Intervention of LY294002 significantly enhanced the inhibition on cell proliferation and the promotion on apoptosis mediated by TRIM31 gene silencing in U266 cells. CONCLUSION: TRIM31 gene silencing can inhibit U266 cell proliferation and promote its apoptosis, which may be closely related to inhibition of PI3K/Akt signaling pathway.


Assuntos
Mieloma Múltiplo , Fosfatidilinositol 3-Quinases , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Inativação Gênica , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética
7.
Int J Mol Sci ; 22(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066899

RESUMO

Plant NAC (NAM, ATAF1/2, and CUC2) family is involved in various development processes including Programmed Cell Death (PCD) associated development. However, the relationship between NAC family and PCD-associated cotton pigment gland development is largely unknown. In this study, we identified 150, 153 and 299 NAC genes in newly updated genome sequences of G. arboreum, G. raimondii and G. hirsutum, respectively. All NAC genes were divided into 8 groups by the phylogenetic analysis and most of them were conserved during cotton evolution. Using the vital regulator of gland formation GhMYC2-like as bait, expression correlation analysis screened out 6 NAC genes which were low-expressed in glandless cotton and high-expressed in glanded cotton. These 6 NAC genes acted downstream of GhMYC2-like and were induced by MeJA. Silencing CGF1(Cotton Gland Formation1), another MYC-coding gene, caused almost glandless phenotype and down-regulated expression of GhMYC2-like and the 6 NAC genes, indicating a MYC-NAC regulatory network in gland development. In addition, predicted regulatory mechanism showed that the 6 NAC genes were possibly regulated by light, various phytohormones and transcription factors as well as miRNAs. The interaction network and DNA binding sites of the 6 NAC transcription factors were also predicted. These results laid the foundation for further study of gland-related genes and gland development regulatory network.


Assuntos
Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Gossypium/anatomia & histologia , Gossypium/genética , Pigmentação/genética , Proteínas de Plantas/genética , Cromossomos de Plantas/genética , Diploide , Duplicação Gênica , Perfilação da Expressão Gênica , Inativação Gênica , Genes de Plantas , Modelos Biológicos , Família Multigênica , Filogenia , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Sintenia/genética
8.
Anticancer Res ; 41(6): 2817-2828, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34083271

RESUMO

BACKGROUND/AIM: Epigenetic alterations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). To obtain further insight into the GIST epigenome, we analyzed genome-wide histone modification and DNA methylation in GIST cells. MATERIALS AND METHODS: To reverse epigenetic silencing, GIST-T1 cells were treated with a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, and subsequently H3K4me3 levels, the DNA methylome, and the transcriptome were analyzed. RESULTS: Treatment with epigenetic inhibitors not only up-regulated genes with DNA methylation, but also genes related to interferon signaling. ChIP-seq analysis revealed that drug treatment up-regulated H3K4me3 levels in retrotransposons, including endogenous retroviruses (ERV). Finally, utilizing the omics data, we found that hypermethylation of MEG3 is a frequent event and an indicator of poorer prognosis in GIST patients. CONCLUSION: Epigenetic inhibitors may activate interferon signaling via viral mimicry in GIST cells. Moreover, epigenome data could be a useful resource to identify novel GIST-related genes.


Assuntos
Epigênese Genética/efeitos dos fármacos , Epigenoma , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Inibidores de Histona Desacetilases/farmacologia , Transcriptoma , Linhagem Celular Tumoral , Metilação de DNA , Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Histonas/metabolismo , Humanos
9.
Science ; 372(6547)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112668

RESUMO

X-inactive specific transcript (Xist) RNA directs the process of X chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual-color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, which enabled us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we find that the protein SPEN, a key factor for Xist-mediated gene silencing, has a distinct function in Xist RNA localization, stability, and coupling behaviors. Our results provide insights toward understanding the distinct dynamic properties of Xist RNA.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Inativação do Cromossomo X , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias , Inativação Gênica , Camundongos , Microscopia , Proteínas Nucleares/genética , Estabilidade de RNA , RNA Longo não Codificante/biossíntese , Proteínas de Ligação a RNA/genética , Análise Espacial , Transcrição Genética , Cromossomo X/metabolismo
10.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066408

RESUMO

WUSCHEL-related homeobox (WOX) transcription factors (TFs) are well known for their role in plant development but are rarely studied in citrus. In this study, we identified 11 putative genes from the sweet orange genome and divided the citrus WOX genes into three clades (modern/WUSCHEL(WUS), intermediate, and ancient). Subsequently, we performed syntenic relationship, intron-exon organization, motif composition, and cis-element analysis. Co-expression analysis based on RNA-seq and tissue-specific expression patterns revealed that CsWOX gene expression has multiple intrinsic functions. CsWUS homolog of AtWUS functions as a transcriptional activator and binds to specific DNA. Overexpression of CsWUS in tobacco revealed dramatic phenotypic changes, including malformed leaves and reduced gynoecia with no seed development. Silencing of CsWUS in lemon using the virus-induced gene silencing (VIGS) system implied the involvement of CsWUS in cells of the plant stem. In addition, CsWUS was found to interact with CsCYCD3, an ortholog in Arabidopsis (AtCYCD3,1). Yeast one-hybrid screening and dual luciferase activity revealed that two TFs (CsRAP2.12 and CsHB22) bind to the promoter of CsWUS and regulate its expression. Altogether, these results extend our knowledge of the WOX gene family along with CsWUS function and provide valuable findings for future study on development regulation and comprehensive data of WOX members in citrus.


Assuntos
Citrus sinensis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Família Multigênica , Proteínas de Plantas/genética , Simulação por Computador , Sequência Conservada/genética , Éxons/genética , Flores/genética , Inativação Gênica , Íntrons/genética , Motivos de Nucleotídeos/genética , Fenótipo , Filogenia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Mapas de Interação de Proteínas/genética , Frações Subcelulares/metabolismo , Sintenia/genética , Tabaco/genética , Água
11.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073989

RESUMO

(1) Background: The transforming growth factor (TGF)-ß plays a dual role in liver carcinogenesis. At early stages, it inhibits cell growth and induces apoptosis. However, TGF-ß expression is high in advanced stages of hepatocellular carcinoma (HCC) and cells become resistant to TGF-ß induced suppressor effects, responding to this cytokine undergoing epithelial-mesenchymal transition (EMT), which contributes to cell migration and invasion. Metabolic reprogramming has been established as a key hallmark of cancer. However, to consider metabolism as a therapeutic target in HCC, it is necessary to obtain a better understanding of how reprogramming occurs, which are the factors that regulate it, and how to identify the situation in a patient. Accordingly, in this work we aimed to analyze whether a process of full EMT induced by TGF-ß in HCC cells induces metabolic reprogramming. (2) Methods: In vitro analysis in HCC cell lines, metabolomics and transcriptomics. (3) Results: Our findings indicate a differential metabolic switch in response to TGF-ß when the HCC cells undergo a full EMT, which would favor lipolysis, increased transport and utilization of free fatty acids (FFA), decreased aerobic glycolysis and an increase in mitochondrial oxidative metabolism. (4) Conclusions: EMT induced by TGF-ß in HCC cells reprograms lipid metabolism to facilitate the utilization of FFA and the entry of acetyl-CoA into the TCA cycle, to sustain the elevated requirements of energy linked to this process.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Metaboloma/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Células Hep G2 , Humanos , Metaboloma/genética , Metabolômica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transcriptoma/genética
12.
Nat Commun ; 12(1): 3423, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103507

RESUMO

Chromatin architecture plays an important role in gene regulation. Recent advances in super-resolution microscopy have made it possible to measure chromatin 3D structure and transcription in thousands of single cells. However, leveraging these complex data sets with a computationally unbiased method has been challenging. Here, we present a deep learning-based approach to better understand to what degree chromatin structure relates to transcriptional state of individual cells. Furthermore, we explore methods to "unpack the black box" to determine in an unbiased manner which structural features of chromatin regulation are most important for gene expression state. We apply this approach to an Optical Reconstruction of Chromatin Architecture dataset of the Bithorax gene cluster in Drosophila and show it outperforms previous contact-focused methods in predicting expression state from 3D structure. We find the structural information is distributed across the domain, overlapping and extending beyond domains identified by prior genetic analyses. Individual enhancer-promoter interactions are a minor contributor to predictions of activity.


Assuntos
DNA/genética , Aprendizado Profundo , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Transcrição Genética , Algoritmos , Animais , Cromatina/genética , Simulação por Computador , Regulação da Expressão Gênica , Inativação Gênica , Genoma de Inseto , Família Multigênica , Redes Neurais de Computação
13.
Plant Sci ; 309: 110934, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34134841

RESUMO

With the discovery of essential genes regulating tillering, such as MONOCULM 1 (MOC1) in rice and LATERAL SUPPRESSOR (LAS in Arabidopsis, LS in tomato), research on tillering mechanisms has made great progress; however, the study of tillering in non-heading Chinese cabbage (NHCC) is rare. Here, we report that BcLAS, as a member of the GRAS family, plays an important role in the tillering of NHCC during its vegetative growth. BcLAS was almost not expressed in other examed parts except leaf axils throughout life. When the expression of BcLAS was silenced utilizing virus-induced gene silencing (VIGS) technology, we found that the tiller number of 'Maertou' decreased sharply. In 'Suzhouqing', overexpression of BcLAS significantly promoted tillering. BcCCS52, the orthologue to CELL CYCLE SEITCH 52 (CCS52), interacts with BcLAS. Downregulation of the expression of BcCCS52 promoted tillering of 'Suzhouqing'; therefore, we conclude that BcCCS52 plays a negative role in tillering regulation. Our findings reveal the tillering regulation mechanism of NHCCs at the vegetative stage and report an orthologue of CCS52 regulating tillering in NHCC.


Assuntos
Brassica rapa/genética , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassica rapa/crescimento & desenvolvimento , Brassica rapa/fisiologia , Ciclo Celular , Proteínas de Ciclo Celular/genética , Expressão Gênica , Inativação Gênica , Filogenia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Domínios Proteicos
14.
Plant Sci ; 309: 110937, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34134844

RESUMO

Small GTP-binding proteins, also known as ROPs (Rho of Plants), are a subfamily of the Ras superfamily of signaling G-proteins and are required for numerous signaling processes, ranging from growth and development to biotic and abiotic signaling. In this study, we cloned and characterized wheat TaRop10, a homolog of Arabidopsis ROP10 and member of the class II ROP, and uncovered a role for TaRop10 in wheat response to Puccinia striiformis f. sp. tritici (Pst). TaRop10 was downregulated by actin depolymerization and was observed to be differentially induced by abiotic stress and the perception of plant hormones. A combination of yeast two-hybrid and bimolecular fluorescence complementation assays revealed that TaRop10 interacted with a h-type thioredoxin (TaTrxh9). Knocking-down of TaRop10 and TaTrxh9 was performed using the BSMV-VIGS (barley stripe mosaic virus-based virus-induced gene silencing) technique and revealed that TaRop10 and TaTrxh9 play a role in the negative regulation of defense signaling in response to Pst infection. In total, the data presented herein further illuminate our understanding of how intact plant cells accommodate fungal infection structures, and furthermore, support the function of TaRop10 and TaTrxh9 in negative modulation of defense signaling in response to stripe rust infection.


Assuntos
Proteínas de Arabidopsis/metabolismo , Resistência à Doença/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Puccinia/fisiologia , Triticum/enzimologia , Proteínas de Arabidopsis/genética , Regulação para Baixo , Proteínas de Ligação ao GTP/genética , Inativação Gênica , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Triticum/genética
15.
Nat Commun ; 12(1): 3582, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117224

RESUMO

In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse development.


Assuntos
Ilhas de CpG/genética , Fator de Transcrição E2F6/genética , Fator de Transcrição E2F6/metabolismo , Desenvolvimento Embrionário/genética , Epigênese Genética , Células Germinativas/metabolismo , Animais , Sítios de Ligação , Sistemas CRISPR-Cas , Diferenciação Celular , Metilação de DNA , Inativação Gênica , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Complexo Repressor Polycomb 1/metabolismo , RNA Interferente Pequeno
16.
Cancer Sci ; 112(7): 2803-2820, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34109710

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the most chemoresistant cancers. An understanding of the molecular mechanism by which PDAC cells have a high chemoresistant potential is important for improvement of the poor prognosis of patients with PDAC. Here we show for the first time that disruption of heat shock protein 47 (HSP47) enhances the efficacy of the therapeutic agent gemcitabine for PDAC cells and that the efficacy is suppressed by reconstituting HSP47 expression. HSP47 interacts with calreticulin (CALR) and the unfolded protein response transducer IRE1α in PDAC cells. Ablation of HSP47 promotes both the interaction of CALR with sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase 2 and interaction of IRE1α with inositol 1,4,5-triphosphate receptor, which generates a condition in which an increase in intracellular Ca2+ level is prone to be induced by oxidative stimuli. Disruption of HSP47 enhances NADPH oxidase-induced generation of intracellular reactive oxygen species (ROS) and subsequent increase in intracellular Ca2+ level in PDAC cells after treatment with gemcitabine, resulting in the death of PDAC cells by activation of the Ca2+ /caspases axis. Ablation of HSP47 promotes gemcitabine-induced suppression of tumor growth in PDAC cell-bearing mice. Overall, these results indicated that HSP47 confers chemoresistance on PDAC cells and suggested that disruption of HSP47 may improve the efficacy of chemotherapy for patients with PDAC.


Assuntos
Calreticulina/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Resistencia a Medicamentos Antineoplásicos , Endorribonucleases/metabolismo , Proteínas de Choque Térmico HSP47/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Cálcio/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Caspases/metabolismo , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Técnicas de Inativação de Genes , Inativação Gênica , Proteínas de Choque Térmico HSP47/genética , Xenoenxertos , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , NADPH Oxidases/metabolismo , Transplante de Neoplasias , Neoplasias Pancreáticas/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Resposta a Proteínas não Dobradas
17.
Nat Commun ; 12(1): 3638, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131144

RESUMO

To ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is initiated during early development through upregulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and gene silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cell RNA-sequencing. Specifically, we assess the onset of random XCI in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify putative Xist regulators. Moreover, we show that transient Xist upregulation from both X chromosomes results in biallelic gene silencing right before transitioning to the monoallelic state, confirming a prediction of the stochastic model of XCI. Finally, we show that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known X-controlling element (Xce) effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.


Assuntos
RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Regulação para Cima , Inativação do Cromossomo X , Alelos , Animais , Compensação de Dosagem (Genética) , Feminino , Inativação Gênica , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas , Análise de Sequência de RNA , Cromossomo X , Inativação do Cromossomo X/genética , Inativação do Cromossomo X/fisiologia
18.
Nat Commun ; 12(1): 3953, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172725

RESUMO

The systemic therapeutic utilisation of RNA interference (RNAi) is limited by the non-specific off-target effects, which can have severe adverse impacts in clinical applications. The accurate use of RNAi requires tumour-specific on-demand conditional activation to eliminate the off-target effects of RNAi, for which conventional RNAi systems cannot be used. Herein, a tumourous biomarker-activated RNAi platform is achieved through the careful design of RNAi prodrugs in extracellular vesicles (EVs) with cancer-specific recognition/activation features. These RNAi prodrugs are assembled by splitting and reconstituting the principal siRNAs into a hybridisation chain reaction (HCR) amplification machine. EVs facilitate the specific and efficient internalisation of RNAi prodrugs into target tumour cells, where endogenous microRNAs (miRNAs) promote immediate and autonomous HCR-amplified RNAi activation to simultaneously silence multiantenna hypoxia-related genes. With multiple guaranteed cancer recognition and synergistic therapy features, the miRNA-initiated HCR-promoted RNAi cascade holds great promise for personalised theranostics that enable reliable diagnosis and programmable on-demand therapy.


Assuntos
Hipóxia/genética , Medicina de Precisão , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/uso terapêutico , Vesículas Extracelulares/química , Vesículas Extracelulares/transplante , Inativação Gênica , Humanos , Camundongos , MicroRNAs/genética , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/uso terapêutico , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética
19.
J Cancer Res Clin Oncol ; 147(8): 2309-2322, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34080067

RESUMO

PURPOSE: Our study was aimed to understand the importance of LIMD1-VHL-HIF1α pathway in development of bladder carcinoma (BlCa) in association with arsenic prevalence. METHODS: At first, the mRNA expression pattern of the genes of this pathway (LIMD1, VHL and HIF1α) was checked in GEO datasets and in our samples. Next, genetic and epigenetic profiling of LIMD1 and VHL was done in our sample pool, validated in T24 BlCa cell line. The results were next correlated with various clinico-pathological parameters. RESULTS: Differential under-expression of LIMD1 and VHL genes was found in muscle-invasive BlCa (MIBC) in comparison to non-muscle-invasive BlCa (NMIBC). However, HIF1α protein, but mRNA, was found to be overexpressed among the MIBC samples; depicting the probability of HIF1α protein stabilization. Analysis of genetic and epigenetic profiles of LIMD1 and VHL exposed a frequent promoter methylation of LIMD1 gene in MIBC samples. Further, in-depth look into the results unveiled that the high nuclear expression of HIF1α was significantly correlated with genetic alterations of LIMD1, alone or in combination with VHL. Moreover, treating the T24 cells with a de-methylating agent (5-aza-2'-deoxycytidine) re-expressed the methylated LIMD1 and VHL genes, which in turn, reduced the HIF1α protein level significantly. Additionally, patients with high arsenic content (> 112 ng/g, AsH) seemed to have recurrent promoter methylation in LIMD1, as well as co-methylation/alteration of LIMD1 and VHL gene. Lastly, high nuclear expression of HIF1α in association with co-alteration of VHL and LIMD1 showed the worst overall survival (OS) among the patients. CONCLUSION: To conclude, MIBC samples portrayed higher alterations in VHL and LIMD1, thereby, stabilizing HIF1α protein and lowering the OS of patients.


Assuntos
Carcinoma de Células de Transição/diagnóstico , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Neoplasias da Bexiga Urinária/diagnóstico , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Intoxicação por Arsênico/diagnóstico , Intoxicação por Arsênico/epidemiologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células de Transição/epidemiologia , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Comorbidade , Metilação de DNA , Conjuntos de Dados como Assunto , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Prevalência , Prognóstico , Análise de Sobrevida , Neoplasias da Bexiga Urinária/epidemiologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
20.
Aging (Albany NY) ; 13(11): 15638-15658, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34077394

RESUMO

Dendritic cell-derived exosomes have been proven to be efficient adjuvant options for anti-tumor vaccines in cancer immunotherapy. However, their potency in atherosclerosis remains unclear. Here we summarize the association of microRNA-203-3p (miR-203-3p) with dendritic cell-derived exosomes and atherosclerosis. Firstly, dendritic cell-derived exosomes and bone marrow-derived macrophages were isolated, after which expression of miR-203-3p and cathepsin S was determined. After the establishment of atherosclerosis mouse models, gain- and loss-of-function experiments were conducted for the analysis of effects of miR-203-3p and cathepsin S on foam-cell formation, lipid accumulation, collagen deposition and serum total cholesterol. The results found high expression of cathepsin S in atherosclerosis mice and downregulation of miR-203-3p in the serum of atherosclerosis patients and ox-LDL-simulated bone marrow-derived macrophages. Cathepsin S was the target gene of miR-203-3p. miR-203-3p transporting from exosomes to bone marrow-derived macrophages resulted in inhibition of cathepsin S expression and atherosclerosis-related phenotypes in bone marrow-derived macrophages, thus alleviating atherosclerosis in mice, and this process was found to involve the p38/MAPK signaling pathway. These findings provided evidence that the transfer of miR-203-3p by dendritic cell-derived exosomes targeted cathepsin S in bone marrow-derived macrophages to attenuate atherosclerosis progression in mice, serving as a promising clinical target for atherosclerosis.


Assuntos
Aterosclerose/genética , Catepsinas/genética , Células Dendríticas/metabolismo , Regulação para Baixo/genética , Exossomos/genética , Macrófagos/metabolismo , MicroRNAs/metabolismo , Animais , Sequência de Bases , Catepsinas/metabolismo , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Feminino , Inativação Gênica , Humanos , Lipoproteínas LDL/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Pessoa de Meia-Idade , Modelos Biológicos , Fenótipo , Transporte de RNA/efeitos dos fármacos , Reprodutibilidade dos Testes
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