RESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of death in the world. NSCLC diagnosed at an early stage can be highly curable with a positive prognosis, but biomarker limitations make it difficult to diagnose lung cancer at an early stage. To identify biomarkers for lung cancer development, we previously focused on the oncogenic roles of transcription factor TFAP2C in lung cancers and revealed the molecular mechanism of several oncogenes in lung tumorigenesis based on TFAP2C-related microarray analysis. RESULTS: In this study, we analyzed microarray data to identify tumor suppressor genes and nine genes downregulated by TFAP2C were screened. Among the nine genes, we focused on growth arrest and DNA-damage-inducible beta (GADD45B) and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) as representative TFAP2C-regulated tumor suppressor genes. It was observed that overexpressed TFAP2C resulted in inhibition of GADD45B and PMAIP1 expressions at both the mRNA and protein levels in NSCLC cells. In addition, downregulation of GADD45B and PMAIP1 by TFAP2C promoted cell proliferation and cell motility, which are closely associated with NSCLC tumorigenesis. CONCLUSION: This study indicates that GADD45B and PMAIP1 could be promising tumor suppressors for NSCLC and might be useful as prognostic markers for use in NSCLC therapy.
Assuntos
Antígenos de Diferenciação/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Neoplasias Pulmonares/genética , Fator de Transcrição AP-2/genética , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Humanos , RNA Mensageiro/análise , RNA Interferente Pequeno/análiseRESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of death in the world. NSCLC diagnosed at an early stage can be highly curable with a positive prognosis, but biomarker limitations make it difficult to diagnose lung cancer at an early stage. To identify biomarkers for lung cancer development, we previously focused on the oncogenic roles of transcription factor TFAP2C in lung cancers and revealed the molecular mechanism of several oncogenes in lung tumorigenesis based on TFAP2C-related microarray analysis. RESULTS: In this study, we analyzed microarray data to identify tumor suppressor genes and nine genes downregulated by TFAP2C were screened. Among the nine genes, we focused on growth arrest and DNA-damage-inducible beta (GADD45B) and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) as representative TFAP2C-regulated tumor suppressor genes. It was observed that overexpressed TFAP2C resulted in inhibition of GADD45B and PMAIP1 expressions at both the mRNA and protein levels in NSCLC cells. In addition, downregulation of GADD45B and PMAIP1 by TFAP2C promoted cell proliferation and cell motility, which are closely associated with NSCLC tumorigenesis. CONCLUSION: This study indicates that GADD45B and PMAIP1 could be promising tumor suppressors for NSCLC and might be useful as prognostic markers for use in NSCLC therapy.
Assuntos
Humanos , Antígenos de Diferenciação/genética , Regulação para Baixo/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/genética , Fator de Transcrição AP-2/genética , Neoplasias Pulmonares/genética , RNA Mensageiro/análise , Biomarcadores Tumorais/análise , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , RNA Interferente Pequeno/análise , Linhagem Celular TumoralRESUMO
Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs. Distinguishing piRNAs from other non-coding RNAs is important because of their important role in the physiological regulation of spermatogenesis, genome protection from transposons, and regulation of mRNAs and long non-coding RNAs. Few computational studies have addressed piRNAs detection, and both effectiveness and efficiency of piRNA detection tools require improvement. In this study, a piRNA detection method based on sequence features and a support vector machine was developed. Four types of features are proposed: weighted k-mer, weighted k-mer with wildcards, position-specific base, and piRNA length. The piRNA sequences from human, mouse, rat, and drosophila were respectively used in this experiment. Compared to existing algorithms, the proposed method provides a better balance between precision and sensitivity (both are approximately 90%), and although these values were slightly slower than those obtained using the piRNA annotation approach, the proposed method was four-fold faster than piRPred and 229-fold faster than piRNA predictor.
Assuntos
RNA Interferente Pequeno/análise , RNA Interferente Pequeno/genética , Análise de Sequência de RNA/métodos , Algoritmos , Animais , Drosophila/genética , Genoma , Humanos , RNA Interferente Pequeno/isolamento & purificação , RNA Interferente Pequeno/metabolismo , Software , Máquina de Vetores de SuporteRESUMO
BACKGROUND: MiRNAs and phasiRNAs are negative regulators of gene expression. These small RNAs have been extensively studied in plant model species but only 10 mature microRNAs are present in miRBase version 21, the most used miRNA database, and no phasiRNAs have been identified for the model legume Phaseolus vulgaris. Thanks to the recent availability of the first version of the common bean genome, degradome data and small RNA libraries, we are able to present here a catalog of the microRNAs and phasiRNAs for this organism and, particularly, we suggest new protagonists in the symbiotic nodulation events. RESULTS: We identified a set of 185 mature miRNAs, including 121 previously unpublished sequences, encoded by 307 precursors and distributed in 98 families. Degradome data allowed us to identify a total of 181 targets for these miRNAs. We reveal two regulatory networks involving conserved miRNAs: those known to play crucial roles in the establishment of nodules, and novel miRNAs present only in common bean, suggesting a specific role for these sequences. In addition, we identified 125 loci that potentially produce phased small RNAs, with 47 of them having all the characteristics of being triggered by a total of 31 miRNAs, including 14 new miRNAs identified in this study. CONCLUSIONS: We provide here a set of new small RNAs that contribute to the broader knowledge of the sRNAome of Phaseolus vulgaris. Thanks to the identification of the miRNA targets from degradome analysis and the construction of regulatory networks between the mature microRNAs, we present here the probable functional regulation associated with the sRNAome and, particularly, in N2-fixing symbiotic nodules.
Assuntos
Phaseolus/genética , Proteínas de Plantas/genética , RNA de Plantas/análise , Análise de Sequência de RNA/métodos , Sequência Conservada , Bases de Dados Genéticas , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , MicroRNAs/análise , MicroRNAs/metabolismo , Phaseolus/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , RNA de Plantas/metabolismo , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/metabolismoRESUMO
Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.
Assuntos
Inativação Gênica , Interações Hospedeiro-Patógeno , Nicotiana/virologia , Doenças das Plantas/virologia , Vírus Eruptivo da Ameixa/crescimento & desenvolvimento , Interferência de RNA , Resistência à Doença , Perfilação da Expressão Gênica , Plantas Geneticamente Modificadas , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/genéticaRESUMO
Hyperglycemia alters the tight control of intracellular calcium dynamics in retinal cells and may lead to the development of diabetic retinopathy. The potassium channel interacting protein 3 (KChIP3) also known as DREAM (Downstream Regulatory Element Antagonist Modulator) or calsenilin (KChIP3/DREAM/calsenilin), a member of the neuronal calcium sensor protein family, is expressed in Müller glial cells and upregulated under high glucose experimental culture conditions. Here, we analyzed the expression and function of KChIP3 in the retina of streptozotocin induced diabetic Long Evans rats by immunofluorescence confocal microscopy, western blot, co-immunoprecipitation, whole cell patch clamp recording on isolated cells and KChIP3 gene silencing by RNA interference. Three weeks after streptozotocin application, KChIP3 was increased throughout the different retinal layers and this process was not linked to augmented apoptosis. KChIP3 co-immunoprecipitated with voltage gated K(+) channels of the K(V)4.2-4.3 subtype in retinal extracts from control and hyperglycemic rats. Electrophysiological analysis showed that control cells did not express A type (K(V)4-mediated) K(+) currents but most of the cells from streptozotocin treated retinas displayed macroscopic currents with an inactivating component sensitive to 4-AP, suggesting the persistence of the A type currents at early times after treatment. siRNA analysis in Müller cells cultures grown under high glucose experimental conditions corroborated that, when the expression of KChIP3 is 50% reduced, the number of cells expressing A type currents decreases significantly. Together these data suggest an altered expression and function of KChIP3 after streptozotocin induced hyperglycemia that might help explain some pathological alterations in early diabetic retinopathy.
Assuntos
Cálcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Hiperglicemia/metabolismo , Proteínas Interatuantes com Canais de Kv/biossíntese , Proteínas Repressoras/biossíntese , Retina/metabolismo , Animais , Células Cultivadas , Hiperglicemia/induzido quimicamente , Proteínas Interatuantes com Canais de Kv/genética , Neuroglia/química , Neuroglia/metabolismo , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos LEC , Proteínas Repressoras/genética , Regulação para CimaRESUMO
Background: Advances in the analyses of human and other higher eukaryotic genomes have disclosed a large fraction of the genetic material (ca 98%) which does not code for proteins. Major portion of this non-coding genome is in fact transcribed into an enormous repertoire of functional RNA molecules (ncRNAs) rather than encoding any proteins. Broadly ncRNAs fall into three size classes namely, ~20 nucleotides for the large family of microRNAs, to 25-200 nucleotides for other different families of small RNAs and finally to over thousands of nucleotides for macro ncRNAs involved in eukaryotic gene regulation. Among the ncRNAs that have been revolutionized our understanding of eukaryotic gene expression, microRNAs (miRNAs) have recently been emphasized extensively with enormous potential for playing their pivotal roles in diseases, fertility and development. The miRNAs are estimated to comprise 15% of animal genes or a given genome could encode nearly thousands of miRNAs. Moreover, a typical miRNA regulates hundreds of target genes and altogether they could target a large proportion of genes up to 30% of the genome. Review: It was reviewed the involvement of miRNAs for reproductive biology in mammals known so far. Several studies expanding from identification and expression profiling to functional involvement of miRNAs in the ovary have been carried out in different animal species. Several studies highlighted the expression and regulation of some individual miRNAs in different ovarian cells especially in oocyte and granulosa cells. Furthermore the impact of miRNAs for embryonic development was considered. The well-orchestrated expression of genes that are derived from the maternal and/or embryonic genome is required for the onset and maintenance of distinct morphological changes during the embryonic development. Optimum regulation of genes or critical gene regulatory event in favor of early embryonic development have been shown directly (individual miRNAs study) or indirectly (disrupting miRNAs biogenesis) under the control of miRNAs. Finally, miRNA effects on DNA methylation pattern are reported by the review. Reversible DNA methylation and histone modifications are known to have profound effects on controlling gene expression. Correct DNA methylation patterns are paramount for the generation of functional gametes with pluripotency states, embryo development, placental function and the maintenance of genome architecture and expression in somatic cells. Aberrancies in both the epigenetic and in the miRNA regulation of genes have been documented to be important in diseases and early development. Interestingly, it has been evident that there is an effect of miRNAs on epigenetic machinery. On the other hand miRNA expression also found to be controlled by epigenetic mechanisms. Conclusion: Significant advancements have been made in recent years on understanding the involvement of miRNA's in ovarian function, gene regulation well as early embryonic development. Since this area of research is rapidly moving forward it is expected that a lot of information regarding miRNA-mediated posttranscriptional gene regulation and their epigenetic regulation in ruminant reproduction biology will be known within the next several years. Studies to identify the specific miRNAs, their target genes and post transcriptional regulatory network will further shed light on the importance of specific miRNA both for the development and function of reproductive tissues as well as disease condition. Once relevant miRNAs and functional targets are identified, possible clinical use for these molecules will represent the next front line and may lead to novel strategies for better enhancing or manipulating reproductive efficiency.
Assuntos
Animais , MicroRNAs/análise , MicroRNAs/fisiologia , RNA Interferente Pequeno/análise , Interferência de RNA , Técnicas de Reprodução Assistida/veterináriaRESUMO
We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.