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1.
PLoS One ; 8(7): e68488, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840894

RESUMO

Rho family GTPases act as molecular switches to regulate a range of physiological functions, including the regulation of the actin-based cytoskeleton, membrane trafficking, cell morphology, nuclear gene expression, and cell growth. Rho function is regulated by its ability to bind GTP and by its localization. We previously demonstrated functional and physical interactions between Rho3 and the clathrin-associated adaptor protein-1 (AP-1) complex, which revealed a role of Rho3 in regulating Golgi/endosomal trafficking in fission yeast. Sip1, a conserved AP-1 accessory protein, recruits the AP-1 complex to the Golgi/endosomes through physical interaction. In this study, we showed that Sip1 is required for Rho3 localization. First, overexpression of rho3⁺ suppressed defective membrane trafficking associated with sip1-i4 mutant cells, including defects in vacuolar fusion, Golgi/endosomal trafficking and secretion. Notably, Sip1 interacted with Rho3, and GFP-Rho3, similar to Apm1-GFP, did not properly localize to the Golgi/endosomes in sip1-i4 mutant cells at 27°C. Interestingly, the C-terminal region of Sip1 is required for its localization to the Golgi/endosomes, because Sip1-i4-GFP protein failed to properly localize to Golgi/endosomes, whereas the fluorescence of Sip1ΔN mutant protein co-localized with that of FM4-64. Consistently, in the sip1-i4 mutant cells, which lack the C-terminal region of Sip1, binding between Apm1 and Rho3 was greatly impaired, presumably due to mislocalization of these proteins in the sip1-i4 mutant cells. Furthermore, the interaction between Apm1 and Rho3 as well as Rho3 localization to the Golgi/endosomes were significantly rescued in sip1-i4 mutant cells by the expression of Sip1ΔN. Taken together, these results suggest that Sip1 recruits Rho3 to the Golgi/endosomes through physical interaction and enhances the formation of the Golgi/endosome AP-1/Rho3 complex, thereby promoting crosstalk between AP-1 and Rho3 in the regulation of Golgi/endosomal trafficking in fission yeast.


Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Complexo 1 de Proteínas Adaptadoras/análise , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/genética , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Mutação , Mapas de Interação de Proteínas , Transporte Proteico , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/análise , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais
2.
J Hum Genet ; 56(4): 270-6, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21289630

RESUMO

MicroRNAs (miRNAs) are endogenous non-coding RNAs that function as negative regulators of gene expression. Alterations in miRNA expression have been shown to affect tumor growth and response to chemotherapy. In this study, we explored the possible role of miRNAs in cisplatin resistance in esophageal squamous cell carcinoma (ESCC). First we assessed the sensitivity of nine human ESCC cell lines (KYSE series) to cisplatin using an in vitro cell viability assay, and then we compared the miRNA profiles of the cisplatin-sensitive and -resistant cell lines by miRNA microarray analysis. The two groups showed markedly different miRNA expression profiles, and 10 miRNAs were found to be regulated differentially between the two groups. When miR-141, which was the most highly expressed miRNA in the cisplatin-resistant cell lines, was expressed ectopically in the cisplatin-sensitive cell lines, cell viability after cisplatin treatment was increased significantly. Furthermore, we found that miR-141 directly targeted the 3'-untranslated region of YAP1, which is known to have a crucial role in apoptosis induced by DNA-damaging agents, and thus downregulated YAP1 expression. Our study highlights an important regulatory role for miR-141 in the development of cisplatin resistance in ESCC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma de Células Escamosas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Esofágicas/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/metabolismo , Fosfoproteínas/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular , Cisplatino/metabolismo , Cisplatino/farmacologia , Primers do DNA/genética , Humanos , MicroRNAs/genética , Análise em Microsséries , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição , Azul Tripano , Proteínas de Sinalização YAP
3.
BMC Genomics ; 11: 137, 2010 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-20181289

RESUMO

BACKGROUND: Cancer cells undergo massive alterations to their DNA methylation patterns that result in aberrant gene expression and malignant phenotypes. However, the mechanisms that underlie methylome changes are not well understood nor is the genomic distribution of DNA methylation changes well characterized. RESULTS: Here, we performed methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) to obtain whole-genome DNA methylation profiles for eight human breast cancer cell (BCC) lines and for normal human mammary epithelial cells (HMEC). The MeDIP-seq analysis generated non-biased DNA methylation maps by covering almost the entire genome with sufficient depth and resolution. The most prominent feature of the BCC lines compared to HMEC was a massively reduced methylation level particularly in CpG-poor regions. While hypomethylation did not appear to be associated with particular genomic features, hypermethylation preferentially occurred at CpG-rich gene-related regions independently of the distance from transcription start sites. We also investigated methylome alterations during epithelial-to-mesenchymal transition (EMT) in MCF7 cells. EMT induction was associated with specific alterations to the methylation patterns of gene-related CpG-rich regions, although overall methylation levels were not significantly altered. Moreover, approximately 40% of the epithelial cell-specific methylation patterns in gene-related regions were altered to those typical of mesenchymal cells, suggesting a cell-type specific regulation of DNA methylation. CONCLUSIONS: This study provides the most comprehensive analysis to date of the methylome of human mammary cell lines and has produced novel insights into the mechanisms of methylome alteration during tumorigenesis and the interdependence between DNA methylome alterations and morphological changes.


Assuntos
Neoplasias da Mama/genética , Metilação de DNA , DNA de Neoplasias/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ilhas de CpG , Feminino , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Imunoprecipitação , Análise de Sequência de DNA
4.
Nucleic Acids Res ; 37(11): 3821-7, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19386621

RESUMO

MicroRNAs are small noncoding RNA species, some of which are playing important roles in cell differentiation. However, the level of participations of microRNAs in epithelial cell differentiation is largely unknown. Here, utilizing an epithelial differentiation model with T84 cells, we demonstrate that miR-338-3p and miR-451 contribute to the formation of epithelial basolateral polarity by facilitating translocalization of beta1 integrin to the basolateral membrane. Among 250 microRNAs screened in this study, the expression levels of four microRNAs (miR-33a, 210, 338-3p and 451) were significantly elevated in the differentiated stage of T84 cells, when epithelial cell polarity was established. To investigate the involvement of these microRNAs in terms of epithelial cell polarity, we executed loss-of- and gain-of-function analyses of these microRNAs. The blockade of endogenous miR-338-3p or miR-451 via each microRNA-specific antisense oligonucleotides inhibited the translocalization of beta1 integrin to the basolateral membrane, whereas inhibition of miR-210 or miR-33a had no effect on it. On the other hand, simultaneous transfection of synthetic miR-338-3p and miR-451 accelerated the translocalization of beta1 integrin to the basolateral membrane, although the introduction of individual synthetic microRNAs exhibited no effect. Therefore, we concluded that both miR-338-3p and miR-451 are necessary for the development of epithelial cell polarity.


Assuntos
Polaridade Celular/genética , Células Epiteliais/metabolismo , MicroRNAs/fisiologia , Diferenciação Celular , Linhagem Celular Tumoral , Células Epiteliais/citologia , Regulação da Expressão Gênica , Humanos , MicroRNAs/antagonistas & inibidores
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