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1.
Hum Pathol ; 116: 12-21, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34271067

RESUMEN

SMARCA4-deficient uterine sarcoma (SMARCA4-DUS) was recently proposed as a new entity of uterine sarcoma. Reported cases of SMARCA4-DUS showed the loss of SMARCA4 and SMARCA2 expression. However, the prevalence of their deficiency in uterine mesenchymal tumors remains unclear. This study immunohistochemically examined the expression of SMARCA4, SMARCA2, and SMARCB1 in 206 uterine mesenchymal tumors and detected a round cell tumor with the loss of SMARCA4 and SMARCA2 and a low-grade endometrial stromal sarcoma with SMARCA4 deficiency. The remaining 204 cases, including 170 smooth muscle tumors, 22 endometrial stomal nodule/sarcomas, seven undifferentiated uterine sarcomas, two adenosarcomas, one uterine tumor resembling ovarian sex cord tumor, and two perivascular epithelioid cell tumors, retained the expression of both SMARCA4 and SMARCA2. All tumors retained SMARCB1 expression. The round cell tumor with the loss of SMARCA4 and SMARCA2 was composed of diffuse small round cell growth with follicle-like spaces, which resembled small cell carcinoma of the ovary, hypercalcemic type. Immunohistochemically, the tumor showed the proficient expression of mismatch repair proteins and wild-type p53 expression, which favored SMARCA4-DUS; however, the tumor harbored the PIK3CA mutation, and thus, was reclassified as undifferentiated endometrial carcinoma. In conclusion, SMARCA4, SMARCA2, and SMARCB1 were rarely deficient in uterine mesenchymal tumors. SMARCA4 immunohistochemistry has potential in the diagnosis of SMARCA4-DUS with the exclusion of some tumors showing its deficiency, such as endometrial stromal sarcoma and undifferentiated carcinoma. Undifferentiated carcinoma may show an indistinguishable morphology and immunophenotype from SMARCA4-DUS, and thus, molecular analysis is required for their distinction in diagnostic practice.


Asunto(s)
ADN Helicasas/biosíntesis , Proteínas Nucleares/biosíntesis , Proteína SMARCB1/biosíntesis , Sarcoma/diagnóstico , Factores de Transcripción/biosíntesis , Neoplasias Uterinas/diagnóstico , Biomarcadores de Tumor/análisis , Carcinoma/diagnóstico , Diagnóstico Diferencial , Femenino , Humanos , Persona de Mediana Edad , Sarcoma/patología , Neoplasias Uterinas/patología
2.
Methods Mol Biol ; 2305: 153-174, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33950389

RESUMEN

The need to generate modified cell lines that express tagged proteins of interest has become increasingly important. Here, we describe a detailed protocol for facile CRISPR/Cas9-mediated gene tagging and isolation of modified cells. In this protocol, we combine two previously published strategies that promote CRISPR/Cas9-mediated gene tagging: using chemically modified single-stranded oligonucleotides as donor templates and a co-selection strategy targeting the ATP1A1 gene at the same time as the gene of interest. Altogether, the protocol proposed here is both easier and saves time compared to other approaches for generating cells that express tagged proteins of interest, which is crucial to purify native complex from human cells.


Asunto(s)
Biotecnología/métodos , Sistemas CRISPR-Cas , Edición Génica/métodos , Marcación de Gen/métodos , Línea Celular , ADN Helicasas/biosíntesis , ADN Helicasas/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Expresión Génica , Humanos , Células K562 , Oligonucleótidos/genética , ARN Guía de Kinetoplastida/metabolismo , Factor de Transcripción TFIIH/biosíntesis , Factor de Transcripción TFIIH/genética , Transfección
3.
Toxicology ; 456: 152772, 2021 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-33823233

RESUMEN

Exposure to environmental toxicants such as all-trans retinoic acid (atRA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may cause cleft palate (CP), which process is related to DNA damage. Rad54B, an important DNA damage repaired protein, has been proved to be associated with non-syndromic cleft lip with palate (NSCLP). In the present study, we sought to clarify the role of Rad54B in palatal development and environment-induced CP. atRA (100 mg/kg) and TCDD (40 µg/kg) were used to induce CP in mice (C57BL/6 J mice). In this study, mouse embryonic heads were collected on embryonic day (E) 13.5∼16.5. The expression level of DNA repair protein Rad54 homolog B (Rad54B) was significantly decreased while those of the DNA double-strand breaks (DSBs) marker γ-H2A.X, apoptosis marker caspase-3 and p53 were significantly increased in the palatal shelves upon exposure to atRA and TCDD relative to the control. Primary mouse embryonic palatal mesenchymal cells (MEPMs) were cultured and transfected with siRNA or adenovirus in vitro to knock down or increase the level of Rad54B. Rad54B knockdown resulted in increased cellular S-phase arrest and apoptosis as well as decreased cell proliferation. Rad54B overexpression also increased apoptosis and reduced cell proliferation. Western blotting was used to detect the level of γ-H2A.X in transfected cells stimulated with etoposide (ETO, a DSBs inducer), and after 5 µM ETO stimulation of transfected MEPMs, the expression of γ-H2A.X was increased in Rad54B-knockdown cells. The expression of Mdm2, Mdmx and p53 with changes in Rad54B was also detected and coimmunoprecipitation was performed to analyze the combination of Mdm2 and p53 when Rad54B was changed in MEPMs. Knockdown of Rad54B inhibited the expression of Mdm2 and Mdmx, while the level of p53 increased. The coimmunoprecipitation results showed a decreased combination of Mdm2 and p53 when Rad54B was knocked down. Therefore, Rad54B can regulate the cell cycle, proliferation, and apoptosis of MEPMs. The loss of Rad54B increased the sensitivity of MEPMs to DSBs inducers, promoted apoptosis, and suppressed the proliferation of MEPMs by inhibiting the degradation of p53. Taken together, these findings suggest that Rad54B may play a key regulatory role in environment-induced CP.


Asunto(s)
Fisura del Paladar/inducido químicamente , Fisura del Paladar/metabolismo , Daño del ADN/efectos de los fármacos , ADN Helicasas/biosíntesis , Dibenzodioxinas Policloradas/toxicidad , Animales , Daño del ADN/fisiología , Susceptibilidad a Enfermedades , Femenino , Ratones , Ratones Endogámicos C57BL , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/metabolismo , Teratógenos/toxicidad
4.
Curr Eye Res ; 46(9): 1353-1362, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-33703976

RESUMEN

Purpose: DNA damage contributes to the pathogenesis of age-related cataract (ARC) and is repaired through the nucleotide excision repair (NER) pathway, which includes ERCC6. Evidence has demonstrated that defective autophagy leads to lens organelle degradation and cataract. This study aimed to investigate the effects of ERCC6 on autophagy and determine its mechanisms in ARC.Methods: The clinical case-control study comprised 30 patients with ARC and 30 age-matched controls who received transparent lens extraction. Transmission electron microscopy was used to assess the ultrastructure of autophagic vesicles in lens anterior capsule tissues and lens epithelial cell line (SRA01/04). Real-time polymerase chain reaction and western blot analyses were performed to measure relative gene expression levels. Gene expression levels and localization were assessed by immunofluorescence. A coimmunoprecipitation assay was used to investigate the relationship between CSB which encoded by ERCC6 and VCP. ERCC6-siRNA and let-7 c-5p mimic were used to alter the expression of ERCC6 and let-7 c-5p.Results: Autophagy induction occurred in lens anterior capsule tissues of patients with ARC and in UVB-induced SRA01/04 cells, where the number of LC3B puncta was increased. Consistent with this result, the expression of beclin1 (BECN1) and LC3B, in addition to that of p62, was increased. Additionally, ERCC6 expression decreased, and silencing ERCC6 induced increases in the expression of BECN1, LC3B and p62. Moreover, CSB interacted with VCP, and let-7 c-5p induced dysregulation of autophagy by targeting ERCC6.Conclusion: In ARC, Let-7 c-5p-mediated downregulation of ERCC6 might prevent the degradation of autophagic vacuoles. CSB binds to VCP, inducing autophagosomes to combine with lysosomes and be degraded.


Asunto(s)
Cápsula Anterior del Cristalino/metabolismo , Catarata/genética , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteína que Contiene Valosina/metabolismo , Anciano , Cápsula Anterior del Cristalino/ultraestructura , Autofagia , Western Blotting , Estudios de Casos y Controles , Catarata/metabolismo , Catarata/patología , Línea Celular , ADN Helicasas/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Células Epiteliales/ultraestructura , Femenino , Humanos , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Proteínas de Unión a Poli-ADP-Ribosa/biosíntesis
5.
Curr Eye Res ; 46(9): 1341-1352, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-33632032

RESUMEN

Purpose: To explore the regulatory role of ERCC6 in the circRNA-miRNA-mRNA network using a cellular ERCC6 overexpression model (OE-ERCC6) in lens epithelial cells.Methods: The expression profiles of circRNAs, miRNAs and mRNAs were determined by RNA-seq, and a regulatory circRNA-miRNA-mRNA network was constructed via bioinformatics. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used for the functional annotation of circRNA host genes, differentially expressed (DE) genes, and miRNA targets.Results: The DE molecules between the OE-ERCC6 and control groups included 269 circRNAs, 241 miRNAs and 3500 mRNAs. We validated 5 selected DE reads of circRNAs (hsa_circ_0001009, hsa_circ_0002024, hsa_circ_0004592, hsa_circ_0001900 and hsa_circ_0001017). Subsequent bioinformatics analysis revealed that the DE circRNAs are mainly involved in oxidative stress- and cell death-related signaling pathways. Finally, a circRNA-miRNA-mRNA network focusing on DNA damage and cell death, which involved 5 circRNAs, 13 miRNAs and 107 mRNAs, was constructed.Conclusion: We constructed a circRNA-miRNA-mRNA network that is regulated by ERCC6. DE circRNAs have the potential to become therapeutic targets related to the lens lesions observed in ARC. The establishment of related in vivo and in vitro models could be a future direction to confirm these hypotheses.


Asunto(s)
ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Cápsula del Cristalino/citología , MicroARNs/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Circular/genética , ARN Mensajero/genética , Western Blotting , Catarata/genética , Catarata/metabolismo , Catarata/patología , Células Cultivadas , ADN Helicasas/biosíntesis , Reparación del ADN , Enzimas Reparadoras del ADN/biosíntesis , Células Epiteliales/patología , Perfilación de la Expresión Génica/métodos , Humanos , Cápsula del Cristalino/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/biosíntesis
6.
Acta Pharmacol Sin ; 42(11): 1900-1912, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-33536604

RESUMEN

Ras-GTPase activating SH3 domain-binding protein 1 (G3BP1) is a multifunctional binding protein involved in the development of a variety of human cancers. However, the role of G3BP1 in breast cancer progression remains largely unknown. In this study, we report that G3BP1 is upregulated and correlated with poor prognosis in breast cancer. Overexpression of G3BP1 promotes breast cancer cell proliferation by stimulating ß-catenin signaling, which upregulates a number of proliferation-related genes. We further show that G3BP1 improves the stability of ß-catenin by inhibiting its ubiquitin-proteasome degradation rather than affecting the transcription of ß-catenin. Mechanistically, elevated G3BP1 interacts with and inactivates GSK-3ß to suppress ß-catenin phosphorylation and degradation. Disturbing the G3BP1-GSK-3ß interaction accelerates the degradation of ß-catenin, impairing the proliferative capacity of breast cancer cells. Our study demonstrates that the regulatory mechanism of the G3BP1/GSK-3ß/ß-catenin axis may be a potential therapeutic target for breast cancer.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proliferación Celular/fisiología , ADN Helicasas/biosíntesis , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/biosíntesis , ARN Helicasas/biosíntesis , Proteínas con Motivos de Reconocimiento de ARN/biosíntesis , beta Catenina/metabolismo , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , beta Catenina/antagonistas & inhibidores
7.
Development ; 147(15)2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32816902

RESUMEN

The evolutionarily conserved lethal-7 (let-7) microRNAs (miRNAs) are well-known activators of proliferative quiescence and terminal differentiation. However, in the murine auditory organ, let-7g overexpression delays the differentiation of mechano-sensory hair cells (HCs). To address whether the role of let-7 in auditory-sensory differentiation is conserved among vertebrates, we manipulated let-7 levels within the chicken auditory organ: the basilar papilla. Using a let-7 sponge construct to sequester let-7 miRNAs, we found that endogenous let-7 miRNAs are essential for limiting the self-renewal of HC progenitor cells. Furthermore, let-7b overexpression experiments revealed that, similar to mice, higher than normal let-7 levels slow/delay HC differentiation. Finally, we identify CHD7, a chromatin remodeler, as a candidate for mediating the repressive function of let-7 in HC differentiation and inner ear morphogenesis. Our analysis uncovered an evolutionarily conserved let-7-5p-binding site within the chicken Chd7 gene and its human and murine homologs, and we show that let-7g overexpression in mice limits CHD7 expression in the developing inner ear, retina and brain. Haploinsufficiency of CHD7 in humans causes CHARGE syndrome and attenuation of let-7 function may be an effective method for treating CHD7 deficiency.


Asunto(s)
Proteínas Aviares/biosíntesis , Pollos/metabolismo , ADN Helicasas/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Células Ciliadas Auditivas/metabolismo , MicroARNs/metabolismo , Células Madre/metabolismo , Animales , Proteínas Aviares/genética , Diferenciación Celular , Embrión de Pollo , Pollos/genética , ADN Helicasas/genética , Células Ciliadas Auditivas/citología , Humanos , Ratones , MicroARNs/genética , Células Madre/citología
8.
Biochemistry ; 59(26): 2401-2409, 2020 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-32478505

RESUMEN

G-Quadruplexes are secondary structures that can form in guanine-rich DNA and RNA that have been implicated in regulating multiple biological processes, including transcription. G-Quadruplex-forming sequences are prevalent in promoter regions of proto-oncogenes and DNA repair proteins. HELB is a human helicase involved in DNA replication and repair with 12 runs of three to four guanines in the proximal promoter. This sequence has the potential to form three canonical three-tetrad G-quadruplexes. Our results show that although all three G-quadruplexes can form, a structure containing two noncanonical G-quadruplexes with longer loops containing runs of three to four guanines is the most prevalent. These HELB G-quadruplexes are stable under physiological conditions. In cells, stabilization of the G-quadruplexes results in a decrease in the level of HELB expression, suggesting that the G-quadruplexes in the HELB promoter serve as transcriptional repressors.


Asunto(s)
ADN Helicasas/biosíntesis , G-Cuádruplex , Regulación Enzimológica de la Expresión Génica , Regiones Promotoras Genéticas , ADN Helicasas/genética , Células HEK293 , Humanos
9.
Br J Cancer ; 122(4): 564-568, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31844183

RESUMEN

BACKGROUND: Ovarian small cell carcinoma, hypercalcaemic type (SCCOHT) is a rare and lethal disease affecting young women. As histological diagnosis is challenging and urgent, there is a clear need for a robust diagnostic test. While mutations in the chromatin-remodelling gene, SMARCA4, appear to be typical, it may not be feasible routinely to be clinically relevant. METHODS: Previous studies have described the value of SMARCA4 IHC to differentiate SCCOHT from ovarian neoplasms (ON), with similar histologic appearances. We aimed to evaluate its clinical utility among a cohort of 44 SCCOHT and 94 rare ON frequently misdiagnosed as SCCOHT. RESULTS: Forty-three percent (16/36) of SCCOHT had been classified locally as non-SCCOHT confirming the diagnosis challenge. Sensitivity and specificity of SMARCA4 IHC were excellent at 88% and 94%, respectively. In a community setting with a much lower prevalence of the disease, estimated PPV is 40% while NPV remained high at 99%. Finally, among the 16 SCCOHT misclassified locally, SMARCA4 IHC testing would have resulted in corrected diagnosis in 88% of cases. CONCLUSIONS: SMARCA4 IHC is a highly sensitive, and specific test for the diagnosis of SCCOHT and is of huge clinical utility in providing a timely and accurate diagnosis of this challenging disease.


Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Pequeñas/diagnóstico , ADN Helicasas/biosíntesis , Proteínas Nucleares/biosíntesis , Neoplasias Ováricas/diagnóstico , Factores de Transcripción/biosíntesis , Adulto , Carcinoma de Células Pequeñas/metabolismo , ADN Helicasas/análisis , Femenino , Humanos , Hipercalcemia , Inmunohistoquímica , Proteínas Nucleares/análisis , Neoplasias Ováricas/metabolismo , Sensibilidad y Especificidad , Factores de Transcripción/análisis
10.
Cell Prolif ; 53(1): e12700, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31667976

RESUMEN

OBJECTIVES: Long non-coding RNA (lncRNA) MATN1-AS1 is a newfound lncRNA that has been rarely explored in cancers. Herein, we would like to investigate its role in glioma. MATERIALS AND METHODS: qRT-PCR was conducted to examine gene expression in glioma. Then, MTT assay, colony formation assay and flow cytometry analysis were applied to evaluate the function of MATN1-AS1 on glioma cells. Western blot was performed to measure the protein levels of genes. Besides, the luciferase reporter assay, RNA pull-down assay, RIP assay and Spearman's correlation analysis were also performed as needed. RESULTS: Firstly, a data from TCGA showed that MATN1-AS1 might be largely implicated in glioma. Meanwhile, MATN1-AS1 upregulation confirmed in glioma predicted poor clinical outcomes. Functionally, MATN1-AS1 knockdown restrained cell proliferation but stimulated apoptosis in vitro and repressed tumour growth in vivo. Mechanistic investigations validated that MATN1-AS1 functioned as a ceRNA for miR-200b/c/429 to upregulate CHD1 which was also verified to exert a growth-promoting role in glioma cells here. Importantly, both CHD1 overexpression and miR-200b/c/429 inhibition could rescue the obstructive role of MATN1-AS1 silence in glioma cells. CONCLUSIONS: MATN1-AS1 promotes glioma progression through regulating miR-200b/c/429-CHD1 axis, suggesting MATN1-AS1 as a probable target for glioma treatment.


Asunto(s)
ADN Helicasas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , MicroARNs/metabolismo , Proteínas de Neoplasias/biosíntesis , ARN Largo no Codificante/metabolismo , ARN Neoplásico/metabolismo , Anciano , Línea Celular Tumoral , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Femenino , Glioma/genética , Glioma/patología , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Proteínas de Neoplasias/genética , ARN Largo no Codificante/genética , ARN Neoplásico/genética
11.
J Clin Invest ; 128(8): 3475-3489, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-30010625

RESUMEN

Chromatin remodeler Brahma related gene 1 (BRG1) is silenced in approximately 10% of human pancreatic ductal adenocarcinomas (PDAs). We previously showed that BRG1 inhibits the formation of intraductal pancreatic mucinous neoplasm (IPMN) and that IPMN-derived PDA originated from ductal cells. However, the role of BRG1 in pancreatic intraepithelial neoplasia-derived (PanIN-derived) PDA that originated from acinar cells remains elusive. Here, we found that exclusive elimination of Brg1 in acinar cells of Ptf1a-CreER; KrasG12D; Brg1fl/fl mice impaired the formation of acinar-to-ductal metaplasia (ADM) and PanIN independently of p53 mutation, while PDA formation was inhibited in the presence of p53 mutation. BRG1 bound to regions of the Sox9 promoter to regulate its expression and was critical for recruitment of upstream regulators, including PDX1, to the Sox9 promoter and enhancer in acinar cells. SOX9 expression was downregulated in BRG1-depleted ADMs/PanINs. Notably, Sox9 overexpression canceled this PanIN-attenuated phenotype in KBC mice. Furthermore, Brg1 deletion in established PanIN by using a dual recombinase system resulted in regression of the lesions in mice. Finally, BRG1 expression correlated with SOX9 expression in human PDAs. In summary, BRG1 is critical for PanIN initiation and progression through positive regulation of SOX9. Thus, the BRG1/SOX9 axis is a potential target for PanIN-derived PDA.


Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Transformación Celular Neoplásica/metabolismo , ADN Helicasas/biosíntesis , Proteínas Nucleares/biosíntesis , Neoplasias Pancreáticas/metabolismo , Factor de Transcripción SOX9/metabolismo , Transducción de Señal , Factores de Transcripción/biosíntesis , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , ADN Helicasas/genética , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Elementos de Respuesta , Factor de Transcripción SOX9/genética , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias Pancreáticas
12.
Biomed Res Int ; 2018: 6135016, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29862278

RESUMEN

Hilar cholangiocarcinoma (HC) is a devastating malignancy that carries a poor overall prognosis. As a member of the AAA+ superfamily, Pontin becomes highly expressed in several malignant tumors, which contributes to tumor progression and influences tumor prognosis. In our research, Pontin expression in tumor specimens resected from 86 HC patients was detected by immunohistochemistry. Interestingly, high expression of Pontin was significantly associated with lymph node metastasis (p = 0.011) and tumor node metastasis (TNM) stage (p = 0.005). The Kaplan-Meier overall survival rate and multivariate analyses were performed to evaluate the prognosis of patients with HC. Patients with high Pontin expression had significantly poorer overall survival outcomes. Multivariate analyses found that Pontin was an independent prognostic factor (p = 0.001). Moreover, bioinformatics analysis confirmed the increase in Pontin mRNA expression levels in cholangiocarcinoma tissues. In addition, in vitro experiments showed that Pontin expression was inhibited at the mRNA as well as protein levels after transfection with Pontin siRNA in human cholangiocarcinoma cell lines. Moreover, significant suppression of cell invasion was observed after the downregulation of Pontin. Taken together, the present study suggested that Pontin could act as a potential prognostic predictor, which might be a new valuable molecular candidate for the prevention and treatment of HC.


Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/biosíntesis , Neoplasias de los Conductos Biliares , Biomarcadores de Tumor/biosíntesis , Proteínas Portadoras/biosíntesis , ADN Helicasas/biosíntesis , Tumor de Klatskin , Proteínas de Neoplasias/biosíntesis , Adulto , Anciano , Neoplasias de los Conductos Biliares/enzimología , Neoplasias de los Conductos Biliares/mortalidad , Neoplasias de los Conductos Biliares/patología , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Tumor de Klatskin/enzimología , Tumor de Klatskin/mortalidad , Tumor de Klatskin/patología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Valor Predictivo de las Pruebas , Tasa de Supervivencia
13.
Gynecol Oncol ; 149(2): 341-349, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29572031

RESUMEN

OBJECTIVE: To investigate the role of HELQ in chemo-resistance of epithelial ovarian carcinoma (EOC), which is a critical factor of patients' prognosis. METHODS: Immunohistochemistry, survival analysis of our 87 EOC patients and bioinformatics analysis of The Cancer Genome Atlas (TCGA) datasets (Nature, 2011) disclosed the clinical importance of HELQ expression. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western Blot analyses of EOC tissue were used to confirm it. Ectopic overexpression and RNA interference knockdown of HELQ were carried out in OVCAR3 and A2780 cell lines, respectively, to determine the effect of altered HELQ expression on cellular response to cisplatin by CCK8 assay. The DNA repair capacity of these cells was evaluated by using host-cell reactivation assay. Western Blot analyses were carried out to determine the effect of HLEQ on the DNA repair genes by using cells with altered HELQ expression. RESULTS: HELQ expression associates with response of EOC patients to platinum-based chemotherapy and their overall survival (OS), disease free survival (DFS). HELQ overexpression or knockdown, respectively, increased and decreased the cellular resistance to cisplatin, DNA repair activity, and expression of DNA repair proteins of Nucleotide excision repair (NER) pathway. CONCLUSIONS: HELQ plays an important role in regulating the expression of DNA repair proteins NER pathway which, in turn, contributes to cellular response to cisplatin and patients' response to platinum-based chemotherapy. Our results demonstrated that HELQ could serve as a novel indicator for chemo-resistance of EOC, which can predict the prognosis of the disease.


Asunto(s)
Cisplatino/farmacología , ADN Helicasas/metabolismo , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/enzimología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Antineoplásicos/farmacología , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , ADN Helicasas/biosíntesis , ADN Helicasas/deficiencia , ADN Helicasas/genética , Reparación del ADN , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Tasa de Supervivencia
14.
Connect Tissue Res ; 59(2): 120-128, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-28362515

RESUMEN

PURPOSE: FBP1, one of the far-upstream element binding proteins(FBPs), is a distal upstream binding protein of c-myc, which is highly expressed in tumor tissues. This study aimed to investigate FBP1 expression in human hypertrophic scars and to determine the effects of FBP1 on fibroblasts. MATERIALS AND METHODS: Human normal skin and scar specimens were collected during clinical surgery. One portion of each tissue specimen was embedded in paraffin and sliced to observe differences in histological features and FBP1 expression by immunohistochemistry and western blotting. The other portion of each tissue specimen was cultured to obtain fibroblasts. Fibroblasts from the second to the sixth passage were used for the experiments, which were divided into the following two groups: an experimental group, whose cells were transfected with an siRNA targeting FBP1, and a control group, whose cells where not transfected. MTT and TUNEL assays were performed, respectively, to assess fibroblast proliferation and apoptosis, and western blotting was performed to assess protein expression. RESULTS: We obtained fibroblasts by primary tissue culture and found that FBP1 was highly expressed in hypertrophic scars. MTT assay showed that an siRNA targeting FBP1 significantly reduced fibroblast proliferation in siRNA-treated cells compared to control cells. TUNEL assay showed that there was no difference in apoptosis between the two groups; however, western blotting showed that collagen I, collagen III, c-myc, caspase-3, and caspase-9 expression levels were all decreased in the experimental group. CONCLUSION: FBP1 is highly expressed in human hypertrophic scars and increases fibroblast proliferation, apoptosis and collagen expression.


Asunto(s)
Apoptosis , Proliferación Celular , Cicatriz Hipertrófica/metabolismo , Colágeno/biosíntesis , ADN Helicasas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Cicatriz Hipertrófica/genética , Cicatriz Hipertrófica/patología , Colágeno/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Femenino , Fibroblastos/patología , Humanos , Masculino , Proteínas de Unión al ARN
15.
Hum Pathol ; 73: 66-73, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29288038

RESUMEN

Brahma-related gene 1 (BRG1), a component of the chromatin-remodeling complex, regulates transcription by remodeling the chromatin structure. The present study aimed to elucidate the clinicopathological significance and prognostic role of BRG1 in colorectal cancer (CRC). We investigated the correlation between BRG1 expression and clinicopathological parameters, including prognosis, using immunohistochemistry on 266 archival paraffin-embedded CRC tissues. In addition, to confirm the prognostic role of BRG1 in malignant tumors, we performed a meta-analysis of 9 eligible studies and the current study. BRG1 was highly expressed in 67.7% of the 266 CRCs analyzed. High BRG1 expression significantly correlated with poor overall and recurrence-free survival (P < .001 and P < .001, respectively). The high expression of BRG1 also significantly correlated with high expression of SNAI (P < .001) but not E-cadherin (P = .432). However, there was no significant correlation between BRG1 expression and other clinicopathological parameters. The meta-analysis also demonstrated that high BRG1 expression positively correlated with poor overall and recurrence-free survival (hazard ratio 1.572, 95% confidence interval 1.106-2.235 and hazard ratio 2.050, 95% confidence interval 1.610-2.610, respectively). However, subgroup analysis based on tumor type showed that the correlation between BRG1 expression and poor prognosis was only prevalent in CRC and breast cancer. Taken together, the results of this study suggest that high BRG1 expression was associated with high SNAI expression and was significantly correlated with poor prognosis.


Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/patología , ADN Helicasas/biosíntesis , Proteínas Nucleares/biosíntesis , Factores de Transcripción/biosíntesis , Neoplasias Colorrectales/mortalidad , ADN Helicasas/análisis , Supervivencia sin Enfermedad , Humanos , Estimación de Kaplan-Meier , Proteínas Nucleares/análisis , Pronóstico , Factores de Transcripción/análisis
16.
Biochem Biophys Res Commun ; 491(4): 1047-1054, 2017 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-28780352

RESUMEN

The far upstream element (FUSE)-binding protein 1 (FUBP1), a well-known transcriptional regulator of the proto-oncogene c-Myc, has been demonstrated by previous work to be aberrantly expressed in a variety of tumors and plays a critical role in tumor progression; however, its expression and function in relatively rare and aggressive chordomas remains unclear. In this retrospective study, we reviewed clinicopathologic characteristics of 40 patients diagnosed with sacral chordoma, and analyzed 40 tumor and 20 distant normal tissues obtained from patients during the primary surgical tumor excision. Using immunohistochemistry, we observed an up-regulation in the expression of FUBP1 and c-Myc in sacral chordomas compared with the normal tissues (P = 0.001 for both). Additionally, positive correlations of FUBP1 expression with c-Myc (γ = 0.651, P < 0.001) and the cell proliferation index Ki-67 expression (γ = 0.447, P = 0.004) were indicated using Spearman's rank correlation coefficient. Increased expression of FUBP1 was significantly associated with tumor invasion into the surrounding muscles (P = 0.002). Kaplan-Meier curves demonstrated the association between FUBP1 levels and the patients' local recurrence-free survival (LRFS) (P < 0.001) but not with the overall survival (OS) (P = 0.070). The independent prognostic significance of FUBP1 levels for the LRFS was indicated by multivariate analysis (HR = 4.272; 95% CI, 1.133-16.112; P = 0.032). Our findings demonstrate an association between FUBP1 levels and chordoma progression and prognosis, suggesting that FUBP1 can be used as a biomarker and a potential therapeutic target.


Asunto(s)
Biomarcadores de Tumor/biosíntesis , Cordoma/metabolismo , ADN Helicasas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Sacro/metabolismo , Adulto , Anciano , Cordoma/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proto-Oncogenes Mas , Proteínas de Unión al ARN , Sacro/patología
17.
Am J Surg Pathol ; 41(9): 1238-1246, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28719461

RESUMEN

Differential diagnosis based on morphology and immunohistochemistry between a clinically nonfunctioning pituitary neuroendocrine tumor (NET)/pituitary adenoma and a primary or secondary NET of nonpituitary origin in the sellar region may be difficult. Serotonin, a frequently expressed marker in the NETs, has not been systematically evaluated in pituitary NETs. Although mutations in ATRX or DAXX have been reported in a significant proportion of pancreatic NETs, the mutational status of ATRX and DAXX and their possible pathogenetic role in pituitary NETs are unknown. Facing a difficult diagnostic case of an invasive serotonin and adrenocorticotroph hormone immunoreactive NET in the sellar region, we explored the immunohistochemical expression of serotonin, ATRX, and DAXX in a large series of pituitary endocrine tumors of different types from 246 patients and in 2 corticotroph carcinomas. None of the pituitary tumors expressed serotonin, suggesting that serotonin immunoreactive sellar tumors represent primary or secondary NETs of nonpituitary origin. Normal expression of ATRX and DAXX in pituitary tumors suggests that ATRX and DAXX do not play a role in the pathogenesis of pituitary endocrine tumors that remain localized to the sellar and perisellar region. A lack of ATRX or DAXX in a sellar NET suggests a nonpituitary NET, probably of pancreatic origin. One of the 2 examined corticotroph carcinomas, however, demonstrated negative ATRX immunolabeling due to an ATRX gene mutation. Further studies on a larger cohort of pituitary carcinomas are needed to clarify whether ATRX mutations may contribute to the metastatic potential in a subset of pituitary NETs.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Adenoma/metabolismo , Biomarcadores de Tumor/biosíntesis , ADN Helicasas/biosíntesis , Tumores Neuroendocrinos/diagnóstico , Proteínas Nucleares/biosíntesis , Neoplasias Hipofisarias/metabolismo , Silla Turca , Serotonina/biosíntesis , Neoplasias Craneales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Adenoma/diagnóstico , Adulto , Anciano , Biomarcadores de Tumor/análisis , Proteínas Co-Represoras , ADN Helicasas/análisis , Diagnóstico Diferencial , Femenino , Humanos , Inmunohistoquímica , Masculino , Chaperonas Moleculares , Proteínas Nucleares/análisis , Neoplasias Hipofisarias/diagnóstico , Serotonina/análisis , Neoplasias Craneales/diagnóstico , Proteína Nuclear Ligada al Cromosoma X
18.
Oncotarget ; 8(26): 43140-43152, 2017 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-28562347

RESUMEN

Excision repair cross-complementing group 6 and 8 (ERCC6 and ERCC8) are two indispensable genes for the initiation of transcription-coupled nucleotide excision repair pathway. This study aimed to evaluate the interactions between single nucleotide polymorphisms of ERCC6 (rs1917799) and ERCC8 (rs158572 and rs158916) in gastric cancer and its precancerous diseases. Besides, protein level analysis were performed to compare ERCC6 and ERCC8 expression in different stages of gastric diseases, and to correlate SNPs jointly with gene expression. Sequenom MassARRAY platform method was used to detect polymorphisms of ERCC6 and ERCC8 in 1916 subjects. In situ ERCC6 and ERCC8 protein expression were detected by immunohistochemistry in 109 chronic superficial gastritis, 109 chronic atrophic gastritis and 109 gastric cancer cases. Our results demonstrated pairwise epistatic interactions between ERCC6 and ERCC8 SNPs that ERCC6 rs1917799-ERCC8 rs158572 combination was associated with decreased risk of chronic atrophic gastritis and increased risk of gastric cancer. ERCC6 rs1917799 also showed a significant interaction with ERCC8 rs158916 to reduce gastric cancer risk. The expressions of ERCC6, ERCC8 and ERCC6-ERCC8 combination have similarities that higher positivity was observed in chronic superficial gastritis compared with chronic atrophic gastritis and gastric cancer. As for the effects of ERCC6 and ERCC8 SNPs on the protein expression, single SNP had no correlation with corresponding gene expression, whereas the ERCC6 rs1917799-ERCC8 rs158572 pair had significant influence on ERCC6 and ERCC6-ERCC8 expression. In conclusion, ERCC6 rs1917799, ERCC8 rs158572 and rs158916 demonstrated pairwise epistatic interactions to associate with chronic atrophic gastritis and gastric cancer risk. The ERCC6 rs1917799-ERCC8 rs158572 pair significantly influence ERCC6 and ERCC6-ERCC8 expression.


Asunto(s)
ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Gastritis Atrófica/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Neoplasias Gástricas/genética , Factores de Transcripción/genética , Estudios de Casos y Controles , ADN Helicasas/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Femenino , Gastritis Atrófica/enzimología , Gastritis Atrófica/patología , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas de Unión a Poli-ADP-Ribosa/biosíntesis , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/patología , Factores de Transcripción/biosíntesis
19.
Mod Pathol ; 30(10): 1422-1432, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28643792

RESUMEN

A distinct subset of thoracic sarcomas with undifferentiated rhabdoid morphology and SMARCA4 inactivation has recently been described, and potential targeted therapy for SMARC-deficient tumors is emerging. We sought to validate the clinicopathological features of SMARCA4-deficient thoracic sarcomas. Clinicopathological information was gathered for 40 undifferentiated thoracic tumors with rhabdoid morphology (mediastinum (n=18), lung (n=14), pleura (n=8)). Thymic carcinomas (n=11) were used as a comparison group. Immunohistochemistry included BRG1 (SMARCA4), BRM (SMARCA2), INI-1 (SMARCB1), pan-cytokeratin, desmin, NUT, S-100 protein, TTF1, CD34, and SOX2. BRG1 loss was present in 12 of 40 rhabdoid thoracic tumors (30%): 7 of 18 in mediastinum (39%), 2 of 8 in pleura (25%), and 3 of 14 in lung (21%). All BRG1-deficient tumors tested for BRM (n=8) showed concomitant loss. All thymic carcinomas showed retained BRG1 and INI-1. Morphologically, tumors with BRG1 loss showed sheets of monotonous ovoid cells with indistinct cell borders, abundant eosinophilic cytoplasm, and prominent nucleoli. Scattered areas with rhabdoid morphology (ie, eccentric nuclei, dense eosinophilic cytoplasm, discohesion) were present in all the cases. SMARCA4/BRG1-deficient sarcomas showed rare cells positive for cytokeratin in 10 cases (83%). One showed rare TTF1-positive cells. All were negative for desmin, NUT, and S-100 protein. CD34 was positive in three of five (60%) BRG1-deficient tumors tested. SOX2 was positive in all four BRG1-deficient tumors tested, and negative in all seven tested cases with retained BRG1. SMARCA4/BRG1-deficient sarcomas occurred at median age of 59 years (range 44-76) with male predominance (9:3) and had worse 2-year survival compared with BRG1-retained tumors (12.5% vs 64.4%, P=0.02). SMARCA4-deficient thoracic sarcomas can be identified based on their distinctive high-grade rhabdoid morphology, and the diagnosis can be confirmed by immunohistochemistry. Identification of these tumors is clinically relevant due to their aggressive behavior, poor prognosis, and potential targeted therapy.


Asunto(s)
ADN Helicasas/genética , Proteínas Nucleares/genética , Sarcoma/genética , Sarcoma/patología , Neoplasias Torácicas/genética , Neoplasias Torácicas/patología , Factores de Transcripción/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , ADN Helicasas/análisis , ADN Helicasas/biosíntesis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Nucleares/análisis , Proteínas Nucleares/biosíntesis , Sarcoma/diagnóstico , Neoplasias Torácicas/diagnóstico , Factores de Transcripción/análisis , Factores de Transcripción/biosíntesis , Adulto Joven
20.
Dig Dis Sci ; 62(9): 2376-2385, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28646284

RESUMEN

BACKGROUND: The chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) plays a key role in controlling various cellular phenomena, including immune-mediated inflammation, transformation, apoptosis, cell cycle progression, and proliferation. METHODS: This study investigated the function and clinical significance of CHD1L protein expression in pancreatic cancer (PC). We analyzed CHD1L expression in surgical specimens from 112 PC patients. The correlation between the clinical characteristics and prognosis was also determined. Futhermore, cell proliferation were measured using EDU, and a molecular mechanism of Wnt/ß-catenin pathway regulation by CHD1L was explored. RESULT: CHD1L protein expression was significantly higher in PC patients with regard to the tumor grade, stage, size, differentiation and lymph node status. Increased CHD1L protein expression was significantly associated with poor overall survival. Multivariate analyses revealed that high CHD1L expression was an independent predictive marker for the recurrence and poor prognosis of pancreatic cancer. Furthermore, silencing of CHD1L expression by RNAi effectively abolished the proliferative abilities of CHD1L in vivo and in vitro. We found that the Wnt/ß-catenin pathway contributed to the effect of CHD1L-mediated pancreatic cancer proliferation. CONCLUSION: Taken together, our data provide a novel evidence for the biological and clinical significance of CHD1L as a potential biomarker, and we demonstrate that CHD1L-Wnt/ß-catenin might be a novel pathway involved in pancreatic cancer progression.


Asunto(s)
Biomarcadores de Tumor/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Anciano , Biomarcadores de Tumor/biosíntesis , Línea Celular Tumoral , ADN Helicasas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismo , Valor Predictivo de las Pruebas , Pronóstico , Tasa de Supervivencia/tendencias
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