RESUMO
Colorectal cancer (CRC) is the fourth leading cause of cancer-induced mortality. Histone deacetylase 2 (HDAC2) is involved in prognosis and therapy of CRC. This study aimed to explore novel therapeutic targets for CRC. The alteration of HDAC2 expression in CRC tissues was estimated by qRT-PCR. After lentivirus transfection, HDAC2 knockdown was confirmed by western blot analysis. The effect of HDAC2 knockdown on cell proliferation was then assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Screened by TargetScan, microRNA (miR)-455 was predicted to bind to 3'UTR of HDAC2 and the prediction was verified by luciferase assay. Finally, cells were transfected, respectively, with miR-455 mimics or miR-455 negative control (miR-NC) and the expression of HDAC2, cell proliferation and apoptosis of transfected cells were respectively evaluated by western blot analysis, MTT assay and flow cytometry. Results showed that the HDAC2 expression was up-regulated in CRC tissues (P<0.05). HDAC2 knockdown significantly decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after infection. Then, miR-455 was verified to directly target HDAC2, resulting in a significant difference in luciferase activity (P<0.01). Moreover, miR-455 decreased the expression of HDAC2 (P<0.01). miR-455 remarkably decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after transfection while inducing cell apoptosis (P<0.001). In conclusion, miR-455 inhibited cell proliferation while inducing cell apoptosis by targeting HDAC2 in CRC cells.
Assuntos
Neoplasias Colorretais/enzimologia , Histona Desacetilase 2/metabolismo , MicroRNAs/metabolismo , Idoso , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Neoplasias Colorretais/genética , Regulação para Baixo , Feminino , Células HCT116 , Histona Desacetilase 2/genética , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Transfecção , Regulação para CimaRESUMO
Colorectal cancer (CRC) is the fourth leading cause of cancer-induced mortality. Histone deacetylase 2 (HDAC2) is involved in prognosis and therapy of CRC. This study aimed to explore novel therapeutic targets for CRC. The alteration of HDAC2 expression in CRC tissues was estimated by qRT-PCR. After lentivirus transfection, HDAC2 knockdown was confirmed by western blot analysis. The effect of HDAC2 knockdown on cell proliferation was then assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Screened by TargetScan, microRNA (miR)-455 was predicted to bind to 3′UTR of HDAC2 and the prediction was verified by luciferase assay. Finally, cells were transfected, respectively, with miR-455 mimics or miR-455 negative control (miR-NC) and the expression of HDAC2, cell proliferation and apoptosis of transfected cells were respectively evaluated by western blot analysis, MTT assay and flow cytometry. Results showed that the HDAC2 expression was up-regulated in CRC tissues (P<0.05). HDAC2 knockdown significantly decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after infection. Then, miR-455 was verified to directly target HDAC2, resulting in a significant difference in luciferase activity (P<0.01). Moreover, miR-455 decreased the expression of HDAC2 (P<0.01). miR-455 remarkably decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after transfection while inducing cell apoptosis (P<0.001). In conclusion, miR-455 inhibited cell proliferation while inducing cell apoptosis by targeting HDAC2 in CRC cells.
Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Neoplasias Colorretais/enzimologia , Histona Desacetilase 2/metabolismo , MicroRNAs/metabolismo , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Neoplasias Colorretais/genética , Regulação para Baixo , Células HCT116 , Histona Desacetilase 2/genética , MicroRNAs/genética , Transfecção , Regulação para CimaRESUMO
Maternal post-traumatic stress disorder (PTSD) increases the risk of adverse neurodevelopmental outcomes in the child. Epigenetic alternations may play an essential role in the negative effects of PTSD. This study was aimed to investigate the possible epigenetic alterations of maternal PTSD, which underpins the developmental and behavioral impact. 24 pregnant Sprague-Dawley (SD) rats were randomly grouped into PTSD and control groups. Open-field tests (OFTs), elevated pull maze (EPM) assays, gene expression profile chip tests, and methylated DNA immunoprecipitation sequencing (MeDIP-Seq) were performed on the offsprings 30 days after birth. The results showed that PTSD offsprings had lower body weights and OFT scores than control offsprings. Enzyme-linked immunosorbent assays showed that serotonin receptor (5-HT) and dopamine levels were significantly lower in PTSD offsprings than in control offsprings. In contrast, corticosterone levels were higher in the PTSD group than in the control group. In a comparison of the PTSD group versus the control group, 4,160 significantly differentially methylated loci containing 30,657 CpGs were identified; 2,487 genes, including 13 dysmethylated genes, were validated by gene expression profiling, showing a negative correlation between methylation and gene expression (R = -0.617, P = 0.043). In conclusion, maternal PTSD could delay the physical and behavioral development of offsprings, and the underlying mechanism could contribute to changes in neurotransmitters and gene expression, owing to dysregulation of whole-genome methylation. These findings could support further clinical research on appropriate interventions for maternal PTSD to prevent methylation dysregulation and developmental retardation.
Assuntos
Deficiências do Desenvolvimento/metabolismo , Epigênese Genética , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Transtornos de Estresse Pós-Traumáticos/complicações , Animais , Peso Corporal , Corticosterona/sangue , Metilação de DNA , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/psicologia , Feminino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/psicologia , Ratos Sprague-Dawley , Transtornos de Estresse Pós-Traumáticos/genética , Transtornos de Estresse Pós-Traumáticos/metabolismo , Transcriptoma , Aumento de PesoRESUMO
PURPOSE: Human epithelial growth factor receptor 2 (HER2) is over-expressed in several malignancies and represents an important therapeutic target. Aptamers are oligonucleotides that may potentially serve as tumor-homing ligand with excellent affinity and specificity for targeted cancer therapy. However, aptamers need to have nuclease resistance in order to function in vivo. The aim of this study was to generate a novel HER2 thioaptamer with enhanced nuclease resistance. METHODS: The HER2 thioaptamer is selected in an evolutionary process called systematic evolution of ligands by exponential enrichment. RESULTS: The thioaptamer could bind to the extracellular domain of HER2 with a K d of 172 nM and had minimal cross reactivity to trypsin or IgG. Moreover, the thioaptamer was found capable of binding with the HER2-positive breast cancer cells SK-BR-3 and MDA-MB-453, but not the HER2-negative cells MDA-MB-231. Notably, the thioaptamer HY6 largely maintained its structural integrity facing the nucleases in serum, while regular DNA aptamers were mostly digested. Additionally, the thioaptamer retained the capability of binding with the HER2-positive cells in the presence of serum, whereas non-thionated HER2 aptamer lost the binding function. CONCLUSION: The results indicated that the selected thioaptamer was more resistant to nuclease than regular DNA aptamers and might potentially function as a HER2-targeting ligand in complicated environment.
Assuntos
Antineoplásicos/administração & dosagem , Aptâmeros de Nucleotídeos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Receptor ErbB-2/antagonistas & inibidores , Neoplasias da Mama/patologia , Feminino , Citometria de Fluxo , Humanos , Técnica de Seleção de Aptâmeros , Células Tumorais CultivadasRESUMO
We investigated the polymorphisms of PRLR and FOLR1 genes in Xinong Saanen, Guanzhong, and Boer goat breeds by DNA sequencing and PCR-RFLP. Two novel SNPs were identified: KC109741: g.62130C>T in the 3ê-UTR of goat gene PRLR, and KC136296: g.7884A>C in exon 3 of goat gene FOLR1. In the three goat breeds, the polymorphism information content was 0.20-0.27 at the g.62130C>T locus. At the g.7884A>C locus, it was 0.36 in Boer goats. The three goat breeds were in Hardy-Weinberg disequilibrium at the g.62130C>T locus. The g.62130C>T SNP was found to be significantly associated with milk production traits in Xinong Saanen and Guanzhong breeds. These results are consistent with the regulatory function of PRLR in mammary gland development, milk secretion, and expression of milk protein genes; they extend the spectrum of genetic variation of the goat PRLR gene, which could be useful for breeding programs.
Assuntos
Receptor 1 de Folato/genética , Estudos de Associação Genética , Leite , Receptores da Prolactina/genética , Regiões 3' não Traduzidas , Animais , Cruzamento , Genótipo , Cabras/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides in length, which play important roles in regulating gene expression post-transcriptionally. Several computational methods and algorithms have been developed to predict miRNA targets. In this study, we described a method that can be used to integrate miRNA target prediction data from multiple sources and gene expression data to predict target genes of particular miRNAs. We used hsa-miR-375 as an example to test the feasibility of our method. A total of 5645 target genes of hsa-miR-375 were identified from five prediction programs, and among them, 2440 target genes were shared by at least 2 of these 5 programs. By using our method, the number was further reduced to 149 and 5 of the 149 target genes had been validated by previous study. This is a simple yet highly effective approach.
Assuntos
MicroRNAs/genética , Modelos Genéticos , Algoritmos , Simulação por Computador , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNARESUMO
Kisspeptins, the product of the KISS1 gene, play an essential role in the regulation of reproductive functions, acting primarily at the hypothalamic level of the gonadotropic axis. We detected polymorphisms of the goat KISS1 gene in 723 individuals from three goat breeds (Xinong Saanen, Guanzhong, and Boer) by DNA pooling, PCR-RFLP, and DNA sequencing methods. We cloned the promoter sequence of this gene and found it to share high similarity with that of the bovine KISS1 promoter. Six TATA boxes were found in the goat KISS1 promoter region. Two novel SNPs (g.2124T>A and g.2270C>T) were identified in the intron 1 of the KISS1 gene of all three goat breeds. The three goat breeds were in Hardy-Weinberg disequilibrium at g.2124T>A and g.2270C>T loci. The g.2124T>A and g.2270C>T loci were closely linked in the three goat breeds (r2 > 0.33). The g.2124T>A and g.2270C>T SNPs were significantly associated with litter size, and the C1 female goats had a larger litter size than did those with the other genotypes. These results extend the spectrum of genetic variation of the goat KISS1 gene, which contributes to our knowledge of goat genetic resources for breeding programs.
Assuntos
Cabras/genética , Kisspeptinas/genética , Tamanho da Ninhada de Vivíparos/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Animais , Clonagem Molecular , Feminino , Frequência do Gene , Estudos de Associação Genética , Loci Gênicos , Genótipo , Desequilíbrio de Ligação , Mutação Puntual , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNARESUMO
Ovarian-specific promoter 1 (OSP-1) is a retrovirus-like element isolated from the complementary DNA library of rat that has been thought to be specifically expressed in ovary. To exploit this promoter in dairy goat ovary granulosa cells (GCs), OSP-1 from rat was used to construct the reporter vector pOSP-1-EGFP, in which egfp coding for enhanced green fluorescent protein (EGFP) was used as a reporter to examine the activity of OSP-1 in GCs. EGFP was successfully expressed in dairy goat GCs transfected with pOSP-1-EGFP. Reverse transcriptase-polymerase chain reaction analysis confirmed the tissue-specific transcription of EGFP messenger RNA in dairy goat GCs transfected with pOSP-1-EGFP. We concluded that OSP-1 promoter from rat can specifically drive foreign gene expression in dairy goat GCs. Thus, we obtained a tissue-specific regulation element and provided a potential tool for the research of regulation and development of the ovary in dairy goats.
Assuntos
Claudinas/genética , Cabras/genética , Células da Granulosa/fisiologia , Ovário/metabolismo , Animais , Células Cultivadas , DNA Complementar/genética , Feminino , Expressão Gênica/genética , Genes Reporter/genética , Vetores Genéticos/genética , Cabras/metabolismo , Células da Granulosa/metabolismo , Proteínas de Fluorescência Verde/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Ratos , Retroelementos , Transcrição Gênica , Transfecção/métodosRESUMO
Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.
Assuntos
Clonagem Molecular/métodos , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Genoma Humano/genética , Fator Inibidor de Leucemia/genética , Mucosa Bucal/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Éxons/genética , Humanos , Fator Inibidor de Leucemia/química , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Splicing de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In the field of anxiety research, animal models are used as screening tools in the search for compounds with therapeutic potential and as simulations for research on mechanism underlying emotional behaviour. However, a solely pharmacological approach to the validation of such tests has resulted in distinct problems with their applicability to systems other than those involving the benzodiazepine/GABAA receptor complex. In this context, recent developments in our understanding of mammalian defensive behaviour have not only prompted the development of new models but also attempts to refine existing ones. The present review focuses on the application of ethological techniques to one of the most widely used animal models of anxiety, the elevated plus-maze paradigm. This fresh approach to an established test has revealed a hitherto unrecognized multidimensionality to plus-maze behaviour and, as it yields comprehensive behavioural profiles, has many advantages over conventional methodology. This assertion is supported by reference to recent work on the effects of diverse manipulations including psychosocial stress, benzodiazepines, GABA receptor ligands, neurosteroids, 5-HT1A receptor ligands, and panicolytic/panicogenic agents. On the basis of this review, it is suggested that other models of anxiety may well benefit from greater attention to behavioural detail.