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1.
J Cardiothorac Surg ; 16(1): 327, 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34749774

RESUMO

BACKGROUND: CircMMP11 is a characterized circRNA with oncogenic function in breast cancer. In this study, we explored the involvement of circMMP11 in non-small cell lung cancer (NSCLC). METHODS: Paired cancer and non-cancer tissues were collected from 66 NSCLC patients, and the expression of circMMP11 and miR-143 in these tissues were detected using RT-qPCRs. Overexpression levels of circMMP11 and miR-143 were performed by transfection, and their crosstalk was analyzed by RT-qPCRs. The effect of circMMP11 overexpression on miR-143 methylation was analyzed by methylation-specific PCR. CCK-8 assay was performed to analyze the roles of miR-143 and circMMP11 in regulating NSCLC cell proliferation. RESULTS: We found that circMMP11 was overexpressed in NSCLC and predicted patients' poor survival. Moreover, a close correlation between circMMP11 and miR143 was observed. In NSCLC cells, circMMP11 overexpression reduced miR-143 expression and increased miR-143 methylation. CCK-8 assay analysis showed that miR-143 reversed the enhancing effects of circMMP11 overexpression on cell proliferation. CONCLUSIONS: CircMMP11 is overexpressed in NSCLC and predicts poor survival. In addition, circMMP11 may downregulate miR-143 through methylation to suppress cell proliferation.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , RNA Complementar/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células , Humanos , Neoplasias Pulmonares/genética , Metilação , MicroRNAs/genética , MicroRNAs/metabolismo
2.
Bioorg Med Chem Lett ; 35: 127779, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33434643

RESUMO

To expand the variety of 2'-O-modified oligonucleotides, we synthesized 2'-O-carbamoylethyl-modified oligonucleotides bearing ethyl, n-propyl, n-butyl, n-pentyl, and n-octyl groups on their nitrogen atoms. The corresponding nucleosides were synthesized using 2'-O-benzyloxycarbonylethylthymidine, which was easily converted into the carboxylic acid through hydrogeneration; subsequent condensation with the appropriate amine gave the desired nucleoside. We evaluated the effect of the 2'-O-alkylcarbamoylethyl modifications on duplex stability by analyzing melting temperature, which revealed the formation of isostable duplexes. In addition, we also revealed that these modifications, especially octylcarbamoylethyl, endowed these oligonucleotides with resistance toward a 3'-exonuclease. These results highlight the usefulness of the 2'-O-alkylcarbamoylethyl modification for various biological applications.


Assuntos
Inibidores Enzimáticos/farmacologia , Exonucleases/antagonistas & inibidores , Oligonucleotídeos/farmacologia , RNA Complementar/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Exonucleases/metabolismo , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , RNA Complementar/metabolismo , Temperatura de Transição
3.
Int J Mol Sci ; 21(19)2020 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-32992595

RESUMO

Some plasma membrane intrinsic protein (PIP) aquaporins can facilitate ion transport. Here we report that one of the 12 barley PIPs (PIP1 and PIP2) tested, HvPIP2;8, facilitated cation transport when expressed in Xenopus laevis oocytes. HvPIP2;8-associated ion currents were detected with Na+ and K+, but not Cs+, Rb+, or Li+, and was inhibited by Ba2+, Ca2+, and Cd2+ and to a lesser extent Mg2+, which also interacted with Ca2+. Currents were reduced in the presence of K+, Cs+, Rb+, or Li+ relative to Na+ alone. Five HvPIP1 isoforms co-expressed with HvPIP2;8 inhibited the ion conductance relative to HvPIP2;8 alone but HvPIP1;3 and HvPIP1;4 with HvPIP2;8 maintained the ion conductance at a lower level. HvPIP2;8 water permeability was similar to that of a C-terminal phosphorylation mimic mutant HvPIP2;8 S285D, but HvPIP2;8 S285D showed a negative linear correlation between water permeability and ion conductance that was modified by a kinase inhibitor treatment. HvPIP2;8 transcript abundance increased in barley shoot tissues following salt treatments in a salt-tolerant cultivar Haruna-Nijo, but not in salt-sensitive I743. There is potential for HvPIP2;8 to be involved in barley salt-stress responses, and HvPIP2;8 could facilitate both water and Na+/K+ transport activity, depending on the phosphorylation status.


Assuntos
Aquaporinas/metabolismo , Cálcio/metabolismo , Hordeum/metabolismo , Transporte de Íons , Oócitos/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Animais , Aquaporinas/genética , Cátions/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Regulação da Expressão Gênica de Plantas , Hordeum/genética , Técnicas de Patch-Clamp , Fosforilação , Proteínas de Plantas/genética , Brotos de Planta/genética , RNA Complementar/administração & dosagem , Água/metabolismo , Xenopus laevis
4.
Reproduction ; 160(2): 319-330, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32585638

RESUMO

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.


Assuntos
Embrião de Mamíferos/fisiologia , Técnicas de Transferência Nuclear/estatística & dados numéricos , Oócitos/fisiologia , Fosfoinositídeo Fosfolipase C/metabolismo , RNA Complementar/administração & dosagem , Espermátides/fisiologia , Zigoto/fisiologia , Animais , Animais Recém-Nascidos , Embrião de Mamíferos/citologia , Feminino , Masculino , Camundongos Endogâmicos ICR , Oócitos/citologia , Fosfoinositídeo Fosfolipase C/administração & dosagem , Fosfoinositídeo Fosfolipase C/genética , Gravidez , Espermátides/citologia , Zigoto/citologia
5.
J Virol ; 94(13)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32295915

RESUMO

Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5' and 3' termini. Recent structural studies have identified a new surface binding site for the 3' vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription in vitro but is required to synthesize a full-length product. This is consistent with a model in which the 3' terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication.IMPORTANCE Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3' vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.


Assuntos
Vírus da Influenza A/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase Dependente de RNA/genética , Regiões 3' não Traduzidas/genética , Sítios de Ligação/genética , Genoma Viral/genética , Células HEK293 , Humanos , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Mutação/genética , Orthomyxoviridae/genética , Proteínas com Motivo de Reconhecimento de RNA , RNA Complementar/genética , RNA Complementar/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Genética/genética , Replicação Viral/genética
6.
FEBS Lett ; 594(10): 1608-1614, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32052435

RESUMO

The involvement of miRNAs in the pathogenesis of various diseases, including cancer, poses the need for developing miRNA inhibitors. Previously, using unmodified DNA, we designed LidNA, which inhibited miRNA function more potently than 2'-O-methylated RNA and locked nucleic acid. LidNA consists of a complementary sequence to miRNA flanked by two structured DNAs. Alterations in the connected sequences between the complementary region and structured region modestly affect miRNA inhibition activity. Surprisingly, variations in the mismatched insertion sequence in the center of the complementary sequence significantly affect activity. The central insertion sequence xxxA is required for the potent miRNA inhibitory effects of LidNA. This suggests that both the structure and insertion sequence of LidNA and other miRNA inhibitors should be considered for maximal miRNA inhibitory activity.


Assuntos
DNA/genética , MicroRNAs/genética , RNA Complementar/genética , Proteínas Argonauta/metabolismo , Sequência de Bases , Sítios de Ligação/genética , DNA/química , MicroRNAs/química , RNA Complementar/química
7.
Vet Microbiol ; 241: 108555, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31928702

RESUMO

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.


Assuntos
Escherichia coli Enteropatogênica/patogenicidade , Proteínas de Escherichia coli/análise , Macrófagos/microbiologia , Proteínas de Membrana/fisiologia , Animais , Galinhas , Biologia Computacional , Meios de Cultura/química , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/fisiologia , Deleção de Genes , Expressão Gênica , Concentração de Íons de Hidrogênio , Análise em Microsséries/veterinária , Mutação , Nitrogênio/deficiência , Doenças das Aves Domésticas/microbiologia , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação , RNA Complementar/química , RNA Complementar/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Organismos Livres de Patógenos Específicos , Virulência , beta-Galactosidase/metabolismo
8.
Bull Exp Biol Med ; 168(2): 270-274, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31784846

RESUMO

SmartFlare technology allows detection of mRNA in single living cells. We studied the possibility of using SmartFlare nanoprobes for detection of small subsets of CD4+ lymphocytes. It was found that SmartFlare allows detection of transcriptional master regulators of major CD4+T helper subsets in living human lymphocytes. Nanoprobes labeled with Cy5 fluorophore were better detected by flow cytometry than nanoprobes labeled with Cy3. Appropriate time of lymphocyte incubation with SmartFlare probes was 24 h.


Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes/química , RNA Complementar/genética , RNA Mensageiro/genética , Subpopulações de Linfócitos T/citologia , Linfócitos T Auxiliares-Indutores/citologia , Carbocianinas/química , Ouro/química , Humanos , Nanopartículas Metálicas/química
9.
BMC Bioinformatics ; 20(1): 558, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31703556

RESUMO

BACKGROUND: Vast amounts of next generation sequencing RNA data has been deposited in archives, accompanying very diverse original studies. The data is readily available also for other purposes such as genome annotation or transcriptome assembly. However, selecting a subset of available experiments, sequencing runs and reads for this purpose is a nontrivial task and complicated by the inhomogeneity of the data. RESULTS: This article presents the software VARUS that selects, downloads and aligns reads from NCBI's Sequence Read Archive, given only the species' binomial name and genome. VARUS automatically chooses runs from among all archived runs to randomly select subsets of reads. The objective of its online algorithm is to cover a large number of transcripts adequately when network bandwidth and computing resources are limited. For most tested species VARUS achieved both a higher sensitivity and specificity with a lower number of downloaded reads than when runs were manually selected. At the example of twelve eukaryotic genomes, we show that RNA-Seq that was sampled with VARUS is well-suited for fully-automatic genome annotation with BRAKER. CONCLUSIONS: With VARUS, genome annotation can be automatized to the extent that not even the selection and quality control of RNA-Seq has to be done manually. This introduces the possibility to have fully automatized genome annotation loops over potentially many species without incurring a loss of accuracy over a manually supervised annotation process.


Assuntos
Bases de Dados Genéticas , RNA Complementar/genética , Análise de Sequência de RNA/métodos , Software , Algoritmos , Animais , Drosophila melanogaster/genética , Eucariotos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Íntrons/genética , Anotação de Sequência Molecular , Transcriptoma/genética
10.
Acta Biochim Pol ; 66(3): 299-304, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442009

RESUMO

An electrochemical genosensor based on an epoxy-phenanthroline-Fe(III)-NH2-ssDNA layer for the detection of RNA derived from Avian Influenza is presented. The biosensor preparation consists of: (I) modification of gold electrodes with aminoethanethiol, (II) modification of the self-assembled monolayer of aminoethanethiol with 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline using "click" chemistry, (III) a first step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (IV) a second step of complexation of Fe(III) by 5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline, (V) immobilization of the single stranded amino-DNA probe via "click" chemistry between epoxy and amino groups. The interactions between the ssDNA probe and RNA targets were explored with Osteryoung Square Wave Voltammetry. The genosensor showed a remarkable detection limit of 3 copies/µL (5 aM) for RNA extracted from A/swan/Poland/305/06 (H5N1) containing a fully complementary sequence. A linear dynamic range for this sequence was observed from 3.0×103 to 3.0×105 [copies/µl]. RNA extracted from A/mallard/Poland/446/09 (H7N7), containing a non-complementary sequence, generated a much weaker response. Moreover, the developed genosensor allows to distinguish RNA present in biological samples having 2, 3 and 4 mismatches. This biosensing approach can become a potential alternative tool for detecting RNA samples in biomedical research and early clinical diagnosis of avian influenza viruses.


Assuntos
Sequência de Bases , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Vírus da Influenza A/genética , Influenza Aviária/virologia , Análise de Sequência de RNA/métodos , Animais , Técnicas Biossensoriais/instrumentação , Embrião de Galinha , DNA de Cadeia Simples/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Compostos de Epóxi/química , Compostos Férricos , Ouro , Vírus da Influenza A/isolamento & purificação , Polônia , Aves Domésticas/virologia , RNA Complementar , RNA Viral/química , Sensibilidade e Especificidade
11.
Nat Microbiol ; 4(10): 1750-1759, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31209309

RESUMO

The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the conserved viral genomic RNA (vRNA) or complementary RNA (cRNA) promoter. Here, we determined the apo and promoter-bound influenza D polymerase structures using cryo-electron microscopy and found the polymerase has an evolutionarily conserved stable core structure with inherently flexible peripheral domains. Strikingly, two conformations (mode A and B) of the vRNA promoter were observed where the 3'-vRNA end can bind at two different sites, whereas the cRNA promoter only binds in the mode B conformation. Functional studies confirmed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. This work advances our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for antiviral drug design.


Assuntos
RNA Viral/biossíntese , RNA Viral/química , RNA Polimerase Dependente de RNA/metabolismo , Thogotovirus/enzimologia , Microscopia Crioeletrônica , Modelos Biológicos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , RNA Complementar/biossíntese , RNA Complementar/química , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Thogotovirus/ultraestrutura , Transcrição Genética , Replicação Viral
12.
PLoS One ; 14(4): e0214481, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31022205

RESUMO

The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com-RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.


Assuntos
Bacteriófago mu/genética , RNA Complementar/química , Proteínas Virais/química , Dedos de Zinco , Pareamento de Bases , Sítios de Ligação , DNA/metabolismo , Genes Virais , Haemophilus , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/genética , Técnica de Seleção de Aptâmeros , Solventes , Transcrição Genética
13.
Biochem Biophys Res Commun ; 508(2): 590-596, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30509492

RESUMO

The serotonin (5-hydroxytryptamine) type 3 (5-HT3) receptors are transmembrane ligand-gated ion channels. Although several 5-HT3 receptor agonists have been used as preclinical tools, SR 57227A is the most commonly used 5-HT3 receptor agonist with the ability to cross the blood brain barrier. However, the precise pharmacological profile of SR 57227A remains unclear. Therefore, we examined the pharmacological profile of SR 57227A at the 5-HT3A and 5-HT3AB receptors. We microinjected Xenopus laevis oocytes with human 5-HT3A complementary RNA (cRNA) or a combination of human 5-HT3A and human 5-HT3AB cRNA and performed two electrode voltage clamp recordings of 5-HT3A and 5-HT3AB receptor current in the presence of SR 57227A. Results showed that SR 57227A acts as partial agonist/partial antagonist at the 5-HT3 receptor. Interestingly, SR 57227A specifically reduced subsequent current amplitudes induced by 5-HT or SR 57227A. Based on its 5-HT3 receptor partial agonist/partial antagonist properties, we predict that SR 57227A functions as a serotonin stabilizer.


Assuntos
Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Piperidinas/farmacologia , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Animais , Barreira Hematoencefálica/metabolismo , Humanos , Oócitos , RNA Complementar , Agonistas do Receptor 5-HT3 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Xenopus laevis
14.
Eur Arch Otorhinolaryngol ; 275(11): 2773-2781, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30267217

RESUMO

PURPOSE: To identify potential molecular markers for induction chemotherapy of Laryngeal squamous cell carcinoma (LSCC). METHODS: Differently expressed genes between chemo-sensitive group (seven cases) and chemo-insensitive (five cases) group after induction chemotherapy by TPF were identified by microarrays. Bayes network and Random forest analyses were employed to identify core genes for induction chemotherapy. The diagnostic value of these core genes was also evaluated by ROC analysis. RESULTS: Six genes (SPP1, FOLR3, KYNU, LOC653219, ADH7 and XAGE1A) are highly expressed, while seven gene (CADM1, NDUFA4L2, CCND2, RARRES3, ERAP2, LYD6 and CNTNAP2) present significantly low expression. Among these genes, genes CADM1, FOLR3, KYNU, and CNTNAP2 are core candidates for LSCC chemo-sensitivity. And that the low expression of CADM1 may result in chemo-sensitivity, which leads to high expression of gene FOLR3 and KYNU, and low expression of gene CNTNAP2. Besides, ROC analysis shows that these four genes exhibit effective diagnostic value for induction chemo-sensitivity. CONCLUSIONS: CADM1 may be a potential molecular marker for LSCC induction chemotherapy, while CADM1, FOLR3, KYNU, and CNTNAP2 may provide essential guidance for LSCC diagnosis and follow-up treatment strategies.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Quimioterapia de Indução , Neoplasias Laríngeas/tratamento farmacológico , Neoplasias Laríngeas/genética , Idoso , Proteínas de Transporte/genética , Molécula 1 de Adesão Celular/genética , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Masculino , Proteínas de Membrana/genética , Análise em Microsséries , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , RNA Complementar/metabolismo
15.
Nucleic Acids Res ; 46(11): 5366-5380, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29790953

RESUMO

Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.


Assuntos
Terapia Genética/métodos , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA Complementar/metabolismo , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Animais , Apolipoproteínas B/genética , Sítios de Ligação/genética , Células Cultivadas , Feminino , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/genética
16.
PLoS Negl Trop Dis ; 12(5): e0006535, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29813122

RESUMO

BACKGROUND: Blood flukes of the genus Schistosoma cause schistosomiasis-a neglected tropical disease (NTD) that affects more than 200 million people worldwide. Studies of schistosome genomes have improved our understanding of the molecular biology of flatworms, but most of them have focused largely on protein-coding genes. Small non-coding RNAs (sncRNAs) have been explored in selected schistosome species and are suggested to play essential roles in the post-transcriptional regulation of genes, and in modulating flatworm-host interactions. However, genome-wide small RNA data are currently lacking for key schistosomes including Schistosoma haematobium-the causative agent of urogenital schistosomiasis of humans. METHODOLOGY: MicroRNAs (miRNAs) and other sncRNAs of male and female adults of S. haematobium and small RNA transcription levels were explored by deep sequencing, genome mapping and detailed bioinformatic analyses. PRINCIPAL FINDINGS: In total, 89 transcribed miRNAs were identified in S. haematobium-a similar complement to those reported for the congeners S. mansoni and S. japonicum. Of these miRNAs, 34 were novel, with no homologs in other schistosomes. Most miRNAs (n = 64) exhibited sex-biased transcription, suggestive of roles in sexual differentiation, pairing of adult worms and reproductive processes. Of the sncRNAs that were not miRNAs, some related to the spliceosome (n = 21), biogenesis of other RNAs (n = 3) or ribozyme functions (n = 16), whereas most others (n = 3798) were novel ('orphans') with unknown functions. CONCLUSIONS: This study provides the first genome-wide sncRNA resource for S. haematobium, extending earlier studies of schistosomes. The present work should facilitate the future curation and experimental validation of sncRNA functions in schistosomes to enhance our understanding of post-transcriptional gene regulation and of the roles that sncRNAs play in schistosome reproduction, development and parasite-host cross-talk.


Assuntos
RNA Complementar/genética , Pequeno RNA não Traduzido/genética , Schistosoma haematobium/genética , Esquistossomose Urinária/parasitologia , Animais , Biologia Computacional , Cricetinae , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , RNA Complementar/metabolismo , Pequeno RNA não Traduzido/metabolismo , Schistosoma haematobium/metabolismo , Análise de Sequência de RNA
17.
Am J Hum Genet ; 102(4): 649-657, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29606300

RESUMO

Fertilization is a fundamental process of development and is a prerequisite for successful human reproduction. In mice, although several receptor proteins have been shown to play important roles in the process of fertilization, only three genes have been shown to cause fertilization failure and infertility when deleted in vivo. In clinical practice, some infertility case subjects suffer from recurrent failure of in vitro fertilization and intracytoplasmic sperm injection attempts due to fertilization failure, but the genetic basis of fertilization failure in humans remains largely unknown. Wee2 is a key oocyte-specific kinase involved in the control of meiotic arrest in mice, but WEE2 has not been associated with any diseases in humans. In this study, we identified homozygous mutations in WEE2 that are responsible for fertilization failure in humans. All four independent affected individuals had homozygous loss-of-function missense mutations or homozygous frameshift protein-truncating mutations, and the phenotype of fertilization failure was shown to follow a Mendelian recessive inheritance pattern. All four mutations significantly decreased the amount of WEE2 protein in vitro and in affected individuals' oocytes in vivo, and they all led to abnormal serine phosphorylation of WEE2 and reduced tyrosine 15 phosphorylation of Cdc2 in vitro. In addition, injection of WEE2 cRNA into affected individuals' oocytes rescued the fertilization failure phenotype and led to the formation of blastocysts in vitro. This work presents a novel gene responsible for human fertilization failure and has implications for future therapeutic treatments for infertility cases.


Assuntos
Proteínas de Ciclo Celular/genética , Fertilização/genética , Infertilidade Feminina/genética , Mutação/genética , Proteínas Tirosina Quinases/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/química , Família , Feminino , Células HeLa , Homozigoto , Humanos , Masculino , Proteínas Mutantes/metabolismo , Oócitos/metabolismo , Linhagem , Fenótipo , Fosforilação , Proteínas Tirosina Quinases/química , RNA Complementar/administração & dosagem , Injeções de Esperma Intracitoplásmicas , Zigoto/metabolismo
18.
Sci Rep ; 8(1): 4377, 2018 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-29531265

RESUMO

The blood-brain barrier (BBB) is increasingly regarded as a dynamic interface that adapts to the needs of the brain, responds to physiological changes, and gets affected by and can even promote diseases. Modulation of BBB function at the molecular level in vivo is beneficial for a variety of basic and clinical studies. Here we show that our heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide and its complementary RNA, conjugated to α-tocopherol as a delivery ligand, efficiently reduced the expression of organic anion transporter 3 (OAT3) gene in brain microvascular endothelial cells in mice. This proof-of-concept study demonstrates that intravenous administration of chemically synthesized HDO can remarkably silence OAT3 at the mRNA and protein levels. We also demonstrated modulation of the efflux transport function of OAT3 at the BBB in vivo. HDO will serve as a novel platform technology to advance the biology and pathophysiology of the BBB in vivo, and will also open a new therapeutic field of gene silencing at the BBB for the treatment of various intractable neurological disorders.


Assuntos
Barreira Hematoencefálica/metabolismo , Oligonucleotídeos/metabolismo , Animais , Barreira Hematoencefálica/fisiologia , Células Endoteliais/metabolismo , Inativação Gênica , Camundongos , Oligonucleotídeos Antissenso/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , RNA Complementar/metabolismo
19.
J Neurochem ; 144(1): 50-57, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29023772

RESUMO

l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAA ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAA ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAA ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAA ρ1 receptors.


Assuntos
Cisteína/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Animais , Ligação Competitiva , Cloretos/metabolismo , Cistina/farmacologia , Relação Dose-Resposta a Droga , Etilmaleimida/farmacologia , Homocisteína/farmacologia , Humanos , Transporte de Íons/efeitos dos fármacos , Oócitos , Técnicas de Patch-Clamp , RNA Complementar/genética , Receptores de GABA-A/fisiologia , Proteínas Recombinantes/metabolismo , Xenopus laevis , Ácido gama-Aminobutírico/farmacologia
20.
Gut Liver ; 12(3): 306-315, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29271183

RESUMO

Background/Aims: The failure to correctly differentiate between intrahepatic cholangiocarcinoma (CC) and hepatocellular carcinoma (HCC) is a significant clinical problem, particularly in terms of the different treatment goals for both cancers. In this study a specific gene expression profile to discriminate these two subgroups of liver cancer was established and potential diagnostic markers for clinical use were analyzed. Methods: To evaluate the gene expression profiles of HCC and intrahepatic CC, Oligonucleotide arrays (AffymetrixU133A) were used. Overexpressed genes were checked for their potential use as new markers for discrimination and their expression values were validated by reverse transcription polymerase chain reaction and immunohistochemistry analyses. Results: 695 genes/expressed sequence tags (ESTs) in HCC (245 up-/450 down-regulated) and 552 genes/ESTs in CC (221 up-/331 down-regulated) were significantly dysregulated (p<0.05, fold change >2, ≥70%). Using a supervised learning method, and one-way analysis of variance a specific 270-gene expression profile that enabled rapid, reproducible differentiation between both tumors and nonmalignant liver tissues was established. A panel of 12 genes (e.g., HSP90ß, ERG1, GPC3, TKT, ACLY, and NME1 for HCC; SPT2, T4S3, CNX43, TTD1, HBD01 for CC) were detected and partly described for the first time as potential discrimination markers. Conclusions: A specific gene expression profile for discrimination of primary liver cancer was identified and potential marker genes with feasible clinical impact were described.


Assuntos
Neoplasias dos Ductos Biliares/diagnóstico , Carcinoma Hepatocelular/diagnóstico , Colangiocarcinoma/diagnóstico , Perfilação da Expressão Gênica/métodos , Neoplasias Hepáticas/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Idoso , Neoplasias dos Ductos Biliares/classificação , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/classificação , Carcinoma Hepatocelular/genética , Colangiocarcinoma/classificação , Colangiocarcinoma/genética , Feminino , Humanos , Neoplasias Hepáticas/classificação , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , RNA Complementar/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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