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1.
Arch Virol ; 164(11): 2747-2759, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31502079

RESUMO

RNA silencing is a major antiviral mechanism in plants, which is counteracted by virus-encoded proteins with silencing suppression activity. ORFs encoding putative silencing suppressor proteins that share no structural or sequence homology have been identified in the genomes of four criniviruses. In this study, we investigated the RNA silencing suppression activity of several proteins encoded by the RNA1 (RdRp, p22) and RNA2 (CP, CPm and p26) of cucurbit chlorotic yellows virus (CCYV) using co-agroinfiltration assays on Nicotiana benthamiana plants. Our results indicate that p22 is a suppressor of local RNA silencing that does not interfere with cell-to-cell movement of the RNA silencing signal or with systemic silencing. Furthermore, comparisons of the suppression activity of CCYV p22 with that of two other well-known crinivirus suppressors (CYSDV p25 and ToCV p22) revealed that CCYV p22 is a weaker suppressor of local RNA silencing than the other two proteins. Finally, a comparative sequence analysis of the p22 genes of seven Greek CCYV isolates was performed, revealing a high level of conservation. Taken together, our research advances our knowledge about plant-virus interactions of criniviruses, an emergent group of pathogens that threatens global agriculture.


Assuntos
Crinivirus/genética , Interferência de RNA/fisiologia , RNA Viral/genética , Tabaco/virologia , Proteínas do Core Viral/genética , Genoma Viral/genética , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/virologia
2.
Pestic Biochem Physiol ; 159: 118-126, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400773

RESUMO

In the plant-insect arms race, plants synthesize toxic compounds to defend against herbivorous insects, whereas insects employ cytochrome P450 monooxygenases (P450s) to detoxify these phytotoxins. As ubiquitous environmental contaminants, heavy metals can be easily absorbed by plants and further accumulated in herbivorous insects through the food chains, resulting in tangible consequences for plant-insect interactions. However, whether heavy metals can influence P450 activities and thereby cause further effects on larval tolerance to phytotoxins remains unknown. In this study, we shown that prior exposure to copper (Cu) enhanced larval tolerance to xanthotoxin in Spodoptera litura, a major polyphagous pest of agriculture. P450 activities were induced in larvae exposed to Cu or xanthotoxin, and a midgut specific expressed P450 gene, CYP6B50 was cross-induced after exposure to these two toxic xenobiotics. Knocking down CYP6B50 by RNA interference (RNAi) rendered the larvae more sensitive to xanthotoxin. As defense against oxidative stress following metal exposure has been demonstrated to affect insecticide resistance, the reactive oxygen species (ROS) generation and antioxidant enzyme activities were assessed. Cu exposure caused the accumulation of hydrogen peroxide (H2O2) and enhanced the activities of superoxide dismutase (SOD) and peroxidase (POD) in larval midgut. In addition, two antioxidant response elements (AREs) were identified from the CYP6B50 promoter, indicating that Cu-induced CYP6B50 expression may be related to the ROS burst. Application of ROS scavenger N-acetylcysteine (NAC) effectively suppressed CYP6B50 expression, inhibited P450 activities and impaired larval tolerance to xanthotoxin that had been induced by Cu. These results indicate that the increase in CYP6B50 expression regulated by Cu-induced H2O2 generation contributed to the enhancement of larval tolerance to xanthotoxin in S. litura. Ingestion of heavy metals from their host plants can inadvertently boost the counter-defense system of herbivorous insects to protect themselves against plant defensive toxins.


Assuntos
Cobre/farmacologia , Peróxido de Hidrogênio/metabolismo , Metoxaleno/farmacologia , Spodoptera/efeitos dos fármacos , Spodoptera/metabolismo , Animais , Elementos de Resposta Antioxidante/genética , Elementos de Resposta Antioxidante/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Interferência de RNA , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
3.
Pestic Biochem Physiol ; 159: 85-90, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400788

RESUMO

RNA interference (RNAi) is a potentially useful pest control method because of its high specificity. Silencing the expression of important RNAi target genes of pests will block important biological processes and reduce pest damage. Ecdysone is a unique arthropod hormone and the ecdysone receptor (EcR) is a key factor in molting pathway. We investigated the possibility that dsRNA targeting of the EcR of Tetranychus cinnabarinus (TcEcR) could effectively block development from larvae to adults. The mRNA level of TcEcR was highest in the larva stage, and 73.1% of the mites failed to survive the larva stage when TcEcR expression was silenced. Only 11.7% of T. cinnabarinus ingesting dsRNA successfully developed into adults, while 86.7% in the control succeeded in molting across each stage. RNAi significantly increased the developmental intervals of T. cinnabarinus. Under the effects of dsRNA, development times for the larva and first nymph doubled. Phenotype of body size change and death were observed during the development of T. cinnabarinus ingesting dsRNA. These findings suggest that RNAi is a potential means for the control of T. cinnabarinus. Genes in hormone pathways such as EcR are possible RNAi targets.


Assuntos
Larva/metabolismo , Interferência de RNA/fisiologia , Receptores de Esteroides/metabolismo , Tetranychidae/metabolismo , Animais , Tamanho Corporal , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , RNA de Cadeia Dupla/genética , Receptores de Esteroides/genética , Tetranychidae/crescimento & desenvolvimento
4.
Adv Exp Med Biol ; 1155: 543-553, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468430

RESUMO

Taurine transporter (TauT) has been identified as a target gene of p53 tumor suppressor. TauT is also found to be overexpressed in variety type of human cancers, such as leukemia. This study showed that expression of TauT was upregulated by c-Myc and c-Jun oncogenes. To explore whether blocking of TauT inhibits tumor development, the RNA interference (RNAi) and immune targeting approaches were tested in tumor cells in vitro and in p53 mutant mice in vivo. Knockdown of TauT expression by RNAi resulted in cell cycle G2 arrest and suppressed human breast cancer MCF-7 cells proliferation determined by colonies production and cell migration assays. Knockdown of TauT also rendered MCF-7 cells more susceptible to chemotherapeutic drug-induced apoptosis. An antibody specifically against TauT blocked taurine uptake and induced cell cycle G2 arrest leading to cell death of variety type of tumor cells without affecting the viability of normal mammalian cells. TauT peptide vaccination significantly increased median lifespan (1.5-fold) of the p53 null mice and rescued p53+/- mice by extending the median lifespan from 315 days to 621 days. Furthermore, single dose treatment of tumor-bearing (thymic lymphoma) p53 null mice with TauT peptide reduced tumor size by about 50% and significantly prolonged survival of these mice from average 7 days (after observing the thymic lymphoma) to 21 days. This finding demonstrates that a novel TauT peptide vaccine can delay, inhibit, and/or treat p53 mutation related spontaneous tumorigenesis in vivo. Therefore, TauT peptide may be used as a universal cancer vaccine to prevent and/or treat patients with p53 mutation-mediated cancers.


Assuntos
Vacinas Anticâncer , Imunoterapia , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Interferência de RNA , Proteína Supressora de Tumor p53/genética , Animais , Anticorpos Monoclonais/farmacologia , Pontos de Checagem do Ciclo Celular , Técnicas de Silenciamento de Genes , Genes jun , Genes myc , Humanos , Células MCF-7 , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Mutação , Taurina , Vacinas de Subunidades
5.
Arch Virol ; 164(11): 2659-2669, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31385116

RESUMO

Interferon gamma (IFN-γ) is best known for its ability to regulate host immune responses; however, its direct antiviral activity is less well studied. Transmissible gastroenteritis virus (TGEV) is an economically important swine enteric coronavirus and causes acute diarrhea in piglets. At present, little is known about the function of IFN-γ in the control of TGEV infection. In this study, we demonstrated that IFN-γ inhibited TGEV infection directly in ST cells and intestine epithelial IPEC-J2 cells and that the anti-TGEV activity of IFN-γ was independent of IFN-α/ß. Moreover, IFN-γ suppressed TGEV infection in ST cells more efficiently than did IFN-α, and the combination of IFN-γ and IFN-α displayed a synergistic effect against TGEV. Mechanistically, using overexpression and functional knockdown experiments, we demonstrated that porcine interferon regulatory factor 1 (poIRF1) elicited by IFN-γ primarily mediated IFN-γ signaling cascades and the inhibition of TGEV infection by IFN-γ. Importantly, we found that TGEV elevated the expression of poIRF1 and IFN-γ in infected small intestines and peripheral blood mononuclear cells. Thus, IFN-γ plays a crucial role in curtailing enteric coronavirus infection and may serve as an effective prophylactic and/or therapeutic agent against TGEV infection.


Assuntos
Gastroenterite Suína Transmissível/imunologia , Fator Regulador 1 de Interferon/metabolismo , Interferon gama/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Animais , Linhagem Celular , Cercopithecus aethiops , Fator Regulador 1 de Interferon/genética , Interferon-alfa/imunologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/imunologia , Suínos , Células Vero
6.
Eur J Histochem ; 63(3)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31455073

RESUMO

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.


Assuntos
Técnicas de Transferência de Genes , Lipídeos/química , Polímeros/química , RNA Interferente Pequeno/genética , Transfecção/métodos , Sequência de Bases , Carbocianinas/química , Carbocianinas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Inativação Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Metabolismo dos Lipídeos , Lipídeos/toxicidade , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/toxicidade , Neoplasias Hepáticas/genética , Polímeros/metabolismo , Polímeros/toxicidade , Interferência de RNA
7.
Cell Physiol Biochem ; 53(1): 215-228, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31299143

RESUMO

BACKGROUND/AIMS: With the prevalence of asthma being greater in women, detrimental effects of female sex steroids have been explored, but potential protective effects of androgens are not established. Airway smooth muscle (ASM) is a key cell type in contractility and remodelling of asthma. There are no data on expression and functionality of androgen receptor (AR) in human ASM cells. METHODS: We used primary human ASM cells from non-asthmatics vs. asthmatics to determine AR expression at baseline and with inflammation measured using Western blotting/qRT-PCR, and the role of AR in regulating intracellular Ca2+ ([Ca2+]i) measured using Fluo-3 loaded real time [Ca2+]i imaging. RESULTS: We found that compared to females, baseline AR is greater in male ASM and increases with inflammation/asthma. Androgens, via AR, blunted TNFα or IL-13-induced enhancement of ASM [Ca2+]i in both males and females, with retained efficacy in asthmatics. AR effects involve reduced Ca2+ influx via L-type channels and store-operated Ca2+ entry, the latter by downregulating STIM1 and Orai1 and increasing TMEM66. CONCLUSION: Our data show AR expression is increased in female ASM with asthma, but has retained functionality that could be used to reduce [Ca2+]i towards alleviating airway hyperresponsiveness.


Assuntos
Cálcio/metabolismo , Receptores Androgênicos/metabolismo , Asma/metabolismo , Asma/patologia , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Interleucina-13/farmacologia , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Androgênicos/química , Receptores Androgênicos/genética , Fatores Sexuais , Molécula 1 de Interação Estromal/metabolismo , Testosterona/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
8.
J Agric Food Chem ; 67(32): 8875-8883, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31347830

RESUMO

Glucan synthase (GLS) gene is known to be involved in the fungal biosynthesis of cell wall, differentiation, and growth. In the present study, a glucan synthase gene (GFGLS) in the edible mushroom Grifola frondosa with a full sequence of 5927 bp encoding a total of 1781 amino acids was cloned and characterized for the first time. GFGLSp is a membrane protein containing two large transmembrane domains connected with a hydrophilic cytoplasmic domain. With a constructed dual promoter RNA silencing vector pAN7-gfgls-dual, a GFGLS-silencing transformant iGFGLS-3 had the lowest GFGLS transcriptional expression level (26.1%) with a shorter length and thinner appearance of the mycelia, as well as decreased mycelial biomass and exo-polysaccharide production of 5.02 and 0.38 g/L, respectively. Further analysis indicated that GFGLS silence influenced slightly the monosaccharide compositions and ratios of mycelial and exo-polysaccharide. These findings suggest that GFGLS could affect mycelial growth and polysaccharide production by downregulating the glucan synthesis.


Assuntos
Polissacarídeos Fúngicos/biossíntese , Proteínas Fúngicas/metabolismo , Glucosiltransferases/metabolismo , Grifola/enzimologia , Micélio/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Glucosiltransferases/genética , Grifola/genética , Grifola/crescimento & desenvolvimento , Grifola/metabolismo , Micélio/enzimologia , Micélio/genética , Micélio/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
9.
Parasite ; 26: 39, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31294687

RESUMO

Glycogen synthase kinase 3 (GSK-3), which belongs to the serine/threonine kinase family, regulates glycogen metabolism, Wnt signaling, hormonal regulation, and embryonic development in many eukaryotes. Here, we cloned a complete open reading frame (ORF) of glycogen synthase kinase 3ß (GSK-3ß) from Haemaphysalis longicornis and characterized its transcriptional and functional status. The ORF of GSK-3ß possesses 1242 nucleotides encoding a mature protein of 413 amino acid residues. GSK-3ß nucleotide and protein sequences are highly conserved among different vertebrate and invertebrate animals, with identity between 47.8-100% and 63.2-88.7%, respectively. Sequence comparison showed one signature domain between the residues of 51 and 335 amino acids, which was identified as a protein kinase (serine/threonine). RT-PCR showed GSK-3ß mRNA present in all developmental stages of H. longicornis. Interestingly, a higher transcript level was observed in nymph and 7-day-old eggs compared with others by real-time PCR, indicating a role of GSK-3ß in the early stages of life. The functional status of GSK-3ß was characterized by RNA interference (RNAi) and caused significant (p < 0.05) reduction in feeding and reproduction, as well as an abnormality in eggs and hatching. Taken together, our results suggest that GSK-3ß may be an important candidate for a multiple antigen vaccine for controlling the tick population.


Assuntos
Glicogênio Sintase Quinase 3 beta/genética , Ixodidae/enzimologia , Ixodidae/genética , Animais , Clonagem Molecular , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Estágios do Ciclo de Vida , Masculino , Ninfa/genética , Oócitos , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Interferência de RNA , RNA Mensageiro , Transdução de Sinais
10.
Yi Chuan ; 41(7): 653-661, 2019 Jul 20.
Artigo em Chinês | MEDLINE | ID: mdl-31307974

RESUMO

RNA interference is a gene silencing phenomenon mediated by short double-stranded RNAs, which has become a widely used research technology for reverse genetics. In order to make students understand the technology better, the students were required to select target genes, to design small interfering RNAs (siRNAs) and primers, and then to test the effect of gene silencing mediated by siRNAs. Taking the fifth group in 2018 as an example, Mus musculus acyl-CoA synthetase long-chain family member 1 (Acsl1) was selected as the target gene, two pairs of siRNAs targeting Acsl1 mRNA were designed and transfected into 3T3-L1 by electroporation, then the total RNAs were extracted and synthesized to cDNA, and the expression levels of mRNAs were finally tested by relative quantitative PCR. The results showed that both pairs of siRNAs had more than 60% silencing effects. In the past three years, about 83% of the students completed all the experiments successfully and screened out at least a pair of effective siRNA. This teaching practice for undergraduates enhances students' understanding of RNA interference principle and technology, and exercises students' lab experience and scientific research ability.


Assuntos
Genética/educação , Interferência de RNA , Estudantes , Ensino , Animais , Coenzima A Ligases , Expressão Gênica , Humanos , Camundongos , RNA Interferente Pequeno
11.
Plant Sci ; 286: 17-27, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300138

RESUMO

The plant-specific gibberellic acid (GA)-stimulated transcript gene family is critical for plant growth and development. There are 10 family members in rice (Oryza sativa), known as OsGASRs. However, few have been functionally characterized. Here, we investigated the function of OsGASR9 in rice. OsGASR9 transcripts were detected in various tissues, with the lowest and highest levels in leaves and panicles, respectively. Greater mRNA levels accumulated in young, compared with in old, panicles and spikelets. OsGASR9 localized to the plasma membrane, cytoplasm and nucleus. Transgenic RNA interference-derived lines in the Zhonghua 11 background exhibited reduced plant height, grain size and yield compared with the wild-type. The two osgasr9 mutants in the Nipponbare background showed similar phenotypes. Conversely, the overexpression of OsGASR9 in the two backgrounds increased plant height and grain size. A significantly increased grain yield per plant was also observed in the overexpression lines having a Nipponbare background. Furthermore, by measuring the GA-induced lengths of the second leaf sheaths and α-amylase activity levels of seeds, we concluded that OsGASR9 is a positive regulator of responses to GA in rice. Thus, OsGASR9 may regulate plant height, grain size and yield through the GA pathway and could have an application value in breeding.


Assuntos
Giberelinas/metabolismo , Oryza/genética , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Família Multigênica , Oryza/crescimento & desenvolvimento , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Interferência de RNA
12.
Gene ; 712: 143958, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278963

RESUMO

The Wnt signaling pathway has been identified for its function in carcinogenesis and embryonic development. It is known to play a vital role in the initiation and development of colorectal cancer (CRC). Therefore, it is of great importance for CRC research to illuminate the mechanisms which regulate Wnt pathway activity. Here, we intended to examine the effect of hsa-miR-942 (miR-942) on the Wnt signaling activity, cell cycle progression, and its expression in CRC tissues. RT-qPCR results indicated that miR-942 is significantly upregulated in colorectal cancer. Then, overexpression of miR-942 promoted, whereas its inhibition decreased the Wnt signaling activity, detected by RT-qPCR and Top/Fop flash assay. Inhibition of Wnt signaling by using PNU-74654 or IWP-2 small molecules indicated that miR-942 applies its effect to the ß-catenin degradation complex level. Then, RT-qPCR and dual luciferase assay showed that miR-942 upregulated Wnt signaling through direct targeting of APC, which is a tumor suppressor in Wnt signaling pathway. Furthermore, the western blotting analysis indicated that ß.catenin, as a main member of Wnt signaling pathway is upregulated following the overexpression of miR-942. Finally, miR-942 overexpression resulted in cell cycle progression in SW480 cells. Taken together, our findings established an oncogenic role for miR-942 in CRC and indicated that this miRNA might be a crucial target for CRC therapy.


Assuntos
Neoplasias Colorretais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Via de Sinalização Wnt , Regiões 3' não Traduzidas , Carcinogênese/genética , Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Biologia Computacional , Ciclina D1/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , Masculino , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Longo não Codificante/genética , Transdução de Sinais , Regulação para Cima , beta Catenina/metabolismo
13.
Gene ; 712: 143963, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31279706

RESUMO

BACKGROUND: The aim of this study was to identify the expression of LIM and calponin-homology domains 1 (LIMCH1) in lung cancer and normal tissues, to determine the interaction between LIMCH1 and HUWE1 in regulating p53 stability. METHODS: The expression of LIMCH1 was detected by the Oncomine and Cancer Genome Atlas databases. Expression of LIMCH1 mRNA was identified using qRT-PCR. In transfected human lung cancer cells, co-immunoprecipitation experiments were performed. The mechanism that HUWE1 sustained lung cancer malignancy was verified by western blotting. The proliferation of tranfected cells was assessed by CCK-8 assay and colony formation. RESULTS: Bioinformatic data and e TCGA database suggested LIMCH1 mRNA levels in tumor tissues were down-regulated compared to tumor adjacent tissues. We found low expression of LIMCH1 mRNA in tumor sites and tumor cell line. Exogenous expression of LIMCH1 interacts with HUWE1 promotes expression of p53. Use of siRNA or shRNA against LIMCH1 resulted in decreased p53 protein levels. LIMCH1 deletion lead to enhance of p53 ubiquitination and protein expression of p53 and substrate p21, puma. Growth curve showed that LIMCH1 deletion significantly promoted the proliferation of A549 cells. CONCLUSIONS: LIMCH1 was a negative regulator and indicated a new molecular mechanism for the pathogenesis of lung cancer via modulating HUWE1 and p53.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas com Domínio LIM/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células A549 , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Biologia Computacional , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Proteínas com Domínio LIM/genética , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
14.
Microbiol Res ; 226: 1-9, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284938

RESUMO

Citrus crops have great economic importance worldwide. However, citrus production faces many diseases caused by different pathogens, such as bacteria, oomycetes, fungi and viruses. To overcome important plant diseases in general, new technologies have been developed and applied to crop protection, including RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. RNAi has been demonstrated to be a powerful tool for application in plant defence mechanisms against different pathogens as well as their respective vectors, and CRISPR/Cas system has become widely used in gene editing or reprogramming or knocking out any chosen DNA/RNA sequence. In this article, we provide an overview of the use of RNAi and CRISPR/Cas technologies in management strategies to control several plants diseases, and we discuss how these strategies can be potentially used against citrus pathogens.


Assuntos
Citrus/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controle , Interferência de RNA , Bactérias/genética , Bactérias/patogenicidade , Sistemas CRISPR-Cas , Citrus/microbiologia , Produtos Agrícolas , Fungos/genética , Fungos/patogenicidade , Edição de Genes/métodos , Engenharia Genética , Vírus/genética , Vírus/patogenicidade
15.
Chem Commun (Camb) ; 55(62): 9112-9115, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31298670

RESUMO

We designed novel 4'-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in ≈4.9-fold more efficient knockdown than unmodified siRNA.


Assuntos
Guanidina/química , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Guanidina/análogos & derivados , Modelos Moleculares , RNA Interferente Pequeno/genética , Ribonucleases/sangue , Ribonucleases/metabolismo , Termodinâmica
17.
Artigo em Chinês | MEDLINE | ID: mdl-31327201

RESUMO

Objective:The aim of this study is to screen the targeting chemokine receptor 3-RNA interference (CCR3-RNAi) lentiviral expression vector, infect mouse mast cells,observe the expression of this gene in mast cells and the interference efficiency of the virus vector.The pathogenesis of allergic rhinitis lays the foundation.Method:Three pairs of CCR3-shRNA sequences were constructed,and three pairs of double-stranded shRNA oligo were inserted into shRNA lentiviral vectors to construct three shRNA lentiviral recombinant plasmids.The recombinant vector and virus-packed auxiliary plasmids were co-transfected into 293T cells to obtain lentiviral plasmids.The lentiviral plasmids were then transfected into mouse bone marrow-derived mast cells in vitro and purified. The expression level of CCR3 mRNA in mast cells was verified by qRT-PCR,and the expression level of CCR3 protein in mast cells was detected by Western Blot.Result: It was confirmed by sequencing that the lentiviral vector of CCR3 shRNA was successfully constructed, transfected into 293T cells and packaged with virus. Finally the high purity PDSO19-PL-CCR3 lentiviral plasmid was obtained with a virus titer of 3.7×108TU/ml.The lentiviral plasmid was used to infect mouse mast cells.RT-PCR and Western Blot detection assay showed that CCR3shRNA reduced the expression of CCR3 gene in mouse mast cells at the level of mRNA and protein.Conclusion: The CCR3 gene RNAi lentivirus expression vector was successfully constructed.It was found that it downregulated the expression level of CCR3 gene mRNA and protein in mouse mast cells,which laid the foundation for further research on its role in the pathogenesis of allergic rhinitis.


Assuntos
Vetores Genéticos , Mastócitos/citologia , Interferência de RNA , Receptores CCR3/genética , Animais , Lentivirus , Camundongos , RNA Interferente Pequeno , Rinite Alérgica/patologia , Transfecção
18.
Oncology ; 97(3): 180-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31330520

RESUMO

BACKGROUND: Hormone receptor-positive breast cancer accounts for nearly two-thirds of breast cancer cases; it ultimately acquires resistance during endocrine treatment and becomes more aggressive. This study evaluated the role of developmental endothelial locus (Del)-1 in tamoxifen-resistant (TAM-R) breast cancer. METHODS: Del-1 expression in recurrent TAM-R breast cancer tissue was evaluated and compared to that in the original tumor tissue from the same patients. Del-1 expression was also evaluated in TAM-R cells by quantitative real-time PCR, western blotting, and enzyme-linked immunosorbent assay. The effects of Del-1 knockdown on the proliferation, migration, and invasion of TAM-R cells was assessed with wound-healing and Matrigel transwell assays. RESULTS: Del-1 was more highly expressed in recurrent breast cancer as compared to the original tumor tissues before initiation of endocrine treatment. Del-1 mRNA was upregulated in TAM-R and small interfering RNA-mediated knockdown of Del-1 suppressed the migration and proliferation of TAM-R cells while partly restoring TAM sensitivity. And the TAM resistance was recovered by knockdown of Del-1. CONCLUSIONS: TAM-R breast cancer is characterized by Del-1 overexpression and tumor progression can be inhibited by Del-1 depletion, which restores TAM sensitivity. Thus, therapeutic strategies that target Del-1 may be effective for the treatment of hormone-resistant breast cancer.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Tamoxifeno/farmacologia , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Técnicas de Silenciamento de Genes , Humanos , Interferência de RNA , RNA Interferente Pequeno
19.
Chem Biol Interact ; 311: 108773, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31351048

RESUMO

Hemangioma (HA) is tumor formed by hyper-proliferation of vascular endothelial cells. However, the potential effects of mono-(2-ethylhexyl) phthalate (MEHP) on the progression of HA are not well illustrated. Our present study revealed that MEHP exposure can significantly increase the in vitro proliferation of hemangioma-derived endothelial cells (HemECs). MEHP treatment can activate yes-associated protein (YAP), a key effector of Hippo pathway, by inhibiting its phosphorylation. The dephosphorylation of YAP induced by MEHP can promote the nuclear accumulation of YAP. Knockdown of YAP or its inhibitor can block MEHP triggered cell proliferation. MEHP can increase the levels of precursor and mature mRNA of YAP in HemECs. As well, MEHP extended the half-life of YAP protein. Mechanistically, MEHP can decrease the phosphorylation of YAP via suppressing the activity of large tumor suppressor kinase 1/2 (LATS1/2) to inhibit it induced degradation of YAP. Further, MEHP increased the expression of interferon regulatory factor 1 (IRF1), which can bind to the promoter of YAP to initiate its transcription. Collectively, we revealed that Hippo-YAP signal is involved in MEHP-induced proliferation of HA cells.


Assuntos
Proliferação de Células/efeitos dos fármacos , Dietilexilftalato/análogos & derivados , Hemangioma/patologia , Proteínas Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Dietilexilftalato/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Hemangioma/metabolismo , Humanos , Fatores Reguladores de Interferon/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Estabilidade Proteica/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Genética/efeitos dos fármacos
20.
Chem Biol Interact ; 311: 108772, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31351049

RESUMO

Atherosclerosis is a common type of cardiovascular disease (CVD), remaining one of the leading causes of global death. Tripartite motif-containing 28 (TRIM28) is a member of TRIM family that has been found to be involved in atherosclerosis. However, the role of TRIM28 in atherosclerosis remains unknown. This study aimed to investigate the effects of TRIM28 on the phenotypic switching of human aortic smooth muscle cells (HASMCs), which is considered as a fundamental event during the development of atherosclerosis. The results showed that TRIM28 was highly expressed in human atherosclerotic tissues, as well in cultured HASMCs stimulated by platelet-derived growth factor subunit B homodimer (PDGF-BB). Knockdown of TRIM28 by transfection with siRNA targeting TRIM28 (si-TRIM28) significantly suppressed the PDGF-BB-induced cell proliferation and migration of HASMCs. Besides, knockdown of TRIM28 inhibited the expressions of matrix metalloproteinase (MMP)-2 and MMP-9. The VSMC markers including α-smooth muscle actin (α-SMA), calponin and SM22α were upregulated in TRIM28 knocked down HASMCs. Furthermore, knockdown of TRIM28 blocked PDGF-BB-induced NF-κB activation in HASMCs. Collectively, knockdown of TRIM28 prevented PDGF-BB-induced phenotypic switching of HASMCs, which might be mediated by the regulation of NF-κB signaling pathway.


Assuntos
Becaplermina/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteína 28 com Motivo Tripartido/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Proteínas dos Microfilamentos/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição RelA/metabolismo , Proteína 28 com Motivo Tripartido/antagonistas & inibidores , Proteína 28 com Motivo Tripartido/genética , Regulação para Cima/efeitos dos fármacos
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