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1.
Mol Med Rep ; 23(4)2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33655339

RESUMEN

Toll­like receptor (TLR) 2/4 serves an important regulatory role in nerve tissue injury. However, the downstream and potential mechanisms remain to be elucidated. The present study was designed to investigate the roles of the TLR2/4­major myeloid differentiation response gene 88 (MyD88)­NF­κB signaling pathway in the development of intracranial aneurysm. The expression of TLR2, TLR4 and MyD88 in the blood of normal controls and patients with intracranial aneurysm were detected by quantitative PCR and ELISA. Human brain vascular smooth muscle cells were treated by Angiotensin II (Ang II) to evaluate the involvement of TLR2/4­MyD88­NF­κB signaling pathway in the process. The in vitro experiment was divided into four groups: The control group, an Ang â…¡ group, an Ang â…¡ + small interfering (si)RNA control group and an Ang â…¡ + TLR2­group. Cell viability, migration, apoptosis and expression of TLR2, TLR4, MyD88, NF­κB and phosphorylated (p­)p65 expression were detected. The results demonstrated that the expression of TLR2, TLR4, MyD88 and NF­κB at mRNA and protein levels in patients with intracranial aneurysm was significantly higher compared with corresponding protein in normal controls (P<0.05). In vitro experiments demonstrated that Ang â…¡ treatment increased the cell proliferation and migration rate but reduced the apoptotic rate compared with the control (P<0.05). The expression of TLR2, TLR4, MyD88, NF­κB and p­p65 was significantly increased in the Ang II group (vs. control; P<0.05). By contrast, TLR2­short interfering RNA reduced the cell proliferation and migration rate, and reduced the expression of TLR2, TLR4, MyD88, NF­κB and p­p65 (vs. Ang â…¡ + short interfering RNA control; P<0.05). In conclusion, the data of the present study indicated that the TLR2/4­MyD88­NF­κB signaling pathway is involved in the pathogenesis of intracranial aneurysm.


Asunto(s)
Aneurisma Intracraneal/genética , Factor 88 de Diferenciación Mieloide/genética , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Angiotensina II/farmacología , Femenino , Regulación de la Expresión Génica/genética , Humanos , Aneurisma Intracraneal/patología , Aneurisma Intracraneal/terapia , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Factor 88 de Diferenciación Mieloide/antagonistas & inhibidores , FN-kappa B/genética , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 4/antagonistas & inhibidores , Factor de Transcripción ReIA/antagonistas & inhibidores , Factor de Transcripción ReIA/genética
2.
BMC Cancer ; 21(1): 207, 2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-33648461

RESUMEN

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.


Asunto(s)
Adenocarcinoma/genética , Adenoma/genética , Neoplasias Colorrectales/genética , Genes Relacionados con las Neoplasias , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-ets/fisiología , Adenocarcinoma/química , Adenocarcinoma/patología , Adenoma/química , Adenoma/patología , Anciano , Biomarcadores de Tumor/genética , Brasil , División Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , ADN de Neoplasias/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-ets/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ets/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Análisis de Matrices Tisulares , Transcriptoma , Ensayo de Tumor de Célula Madre
3.
Mol Med Rep ; 23(5)2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33760180

RESUMEN

Nasopharyngeal carcinoma (NPC) is an epithelial carcinoma originating from the nasopharyngeal mucosal tissue and is highly prevalent in southeast Asia. Galectin­3 (gal­3) serves crucial roles in many cancers but its role in NPC remains to be elucidated. The aim of the present study was to investigate the role of gal­3 in NPC. Immunohistochemistry and ELISA were used to determine the expression level of gal­3 in patients with NPC or chronic rhinitis (CR). Gal­3 short hairpin (sh)RNA was established to knockdown gal­3 in 5­8F and 6­10B cells, allowing for the evaluation of the roles of gal­3 in proliferation, migration and apoptosis in NPC cell lines. Immunohistochemistry staining of IL­6 and IL­8 was applied to access the inflammatory state of tumor tissues, and the correlation between the inflammatory state and gal­3 was analyzed. The results demonstrated that gal­3 was upregulated in patients with NPC compared with patients with CR. Knockdown of gal­3 inhibited proliferation and migration in 5­8F and 6­10B cells, as well as promoted apoptosis in these cells. The expression levels of MMP­9 and IL­8 were also decreased in 5­8F and 6­10B cells after transfection with gal­3 shRNA. A positive correlation was identified between the expression level of gal­3 and the inflammatory state of NPC. The phosphorylation levels of ERK1/2 and Akt were downregulated after knockdown of gal­3 in 5­8F and 6­10B cells. In conclusion, the expression level of gal­3 was upregulated in patients with NPC and was positively correlated with the inflammatory state of NPC. The results suggested that gal­3 promoted the proliferation and migration of 5­8F and 6­10B cells, while inhibiting the apoptosis of these cells. Moreover, activation of ERK1/2 and Akt may be the underlying mechanism of the effects of gal­3 on NPC.


Asunto(s)
Galectina 3/genética , Inflamación/genética , Interleucina-8/genética , Metaloproteinasa 9 de la Matriz/genética , Carcinoma Nasofaríngeo/genética , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Galectina 3/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inflamación/patología , Sistema de Señalización de MAP Quinasas/genética , Masculino , Persona de Mediana Edad , Carcinoma Nasofaríngeo/patología , Carcinoma Nasofaríngeo/terapia , Proteína Oncogénica v-akt/genética , ARN Interferente Pequeño/farmacología
4.
Methods Mol Biol ; 2265: 621-634, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704743

RESUMEN

RNA interference (RNAi) is a posttranscriptional regulatory mechanism that employs siRNA. It typically results in the degradation of a target mRNA that encodes a particular protein. Treatment with siRNA therapeutics requires the use of an effective drug delivery system to assist in delivering these therapeutics into the cytoplasm of the transfected cells. Here we describe the transfection of melanoma cancer cells with siRNA using cationic niosome nanoparticles as a delivery system. The method of niosome preparation is first introduced and is followed by complex formation with siRNA and the transfection method.


Asunto(s)
Melanoma , Nanopartículas , ARN Interferente Pequeño , Transfección , Humanos , Liposomas , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Nanopartículas/química , Nanopartículas/uso terapéutico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacología
5.
Int J Nanomedicine ; 16: 1961-1976, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33727809

RESUMEN

Introduction: Metastatic breast cancer seriously harms women's health and is currently the tumour type with the highest mortality rate in women. Recently, the combinatorial therapeutic approaches that integrate anti-cancer drugs and genetic agents is an attractive and promising strategy for the treatment of metastatic breast cancer. Moreover, such a combination strategy requires better drug carriers that can effectively deliver the cargo to the breast cancer cells and achieve controlled release in the cells to achieve better therapeutic effects. Methods: The tumour-targeted and redox-responsive mesoporous silica nanoparticles (MSNs) functionalised with DNA aptamers (AS1411) as a co-delivery system was developed and investigated for the potential against metastatic breast cancer. Doxorubicin (Dox) was loaded onto the MSNs, while AS1411 and a small interfering RNA (siTIE2) were employed as gatekeepers via attachment to the MSNs with redox-sensitive disulfide bonds. Results: The controlled release of Dox and siTIE2 was associated with intracellular glutathione. AS1411 mediated the targeted delivery of Dox by increasing its cellular uptake in metastatic breast cancer, ultimately resulting in a lower IC50 in MDA-MB-231 cells (human breast cancer cell line with high metastatic potency), improved biodistribution in tumour-bearing mice, and enhanced in vivo anti-tumour effects. The in vitro cell migration/invasion assay and in vivo anti-metastatic study revealed synergism in the co-delivery system that suppresses cancer cell metastasis. Conclusion: The tumour-targeted and redox-responsive MSN prepared in this study are promising for the effective delivery and controlled release of Dox and siTIE2 for improved treatment of metastatic breast cancer.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/uso terapéutico , Nanopartículas/química , ARN Interferente Pequeño/administración & dosificación , Dióxido de Silicio/química , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada/farmacología , Preparaciones de Acción Retardada/uso terapéutico , Doxorrubicina/farmacología , Portadores de Fármacos/química , Endocitosis/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/ultraestructura , Invasividad Neoplásica , Metástasis de la Neoplasia , Oxidación-Reducción , Porosidad , ARN Interferente Pequeño/farmacología , Distribución Tisular/efectos de los fármacos
6.
Anticancer Res ; 41(2): 783-794, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33517283

RESUMEN

BACKGROUND/AIM: The inflammatory cytokine IL-8 and its receptor CXCR2 are key signalling pathway molecules in cancer development. We hypothesized that IL-8/CXCR2 signalling promotes tumour progression in oesophageal squamous cell carcinoma (ESCC) patients. MATERIALS AND METHODS: We examined the relationship between IL-8/CXCR2 expression and clinicopathological factors by immunohistochemistry in samples from 63 patients with resectable ESCC. The effects of IL-8/CXCR2 signalling on cell proliferation and gene expression were examined in vitro and in vivo using ESCC cell lines. RESULTS: Increased IL-8/CXCR2 signalling was associated with shorter overall survival (p<0.05) and recurrence-free survival (p<0.05) in ESCC patients. Multivariate analysis identified IL-8/CXCR2 expression as a prognostic factor for surgically treated ESCC (p<0.05). In vitro, IL-8 exposure or over-expression significantly enhanced ESCC cell proliferation. SB225002, a CXCR2-specific antagonist, and IL-8 siRNA significantly suppressed cell proliferation. CONCLUSION: IL-8/CXCR2 expression is an independent prognostic factor for surgically treated ESCC, and IL-8/CXCR2 signalling contributes to ESCC cell proliferation.


Asunto(s)
Neoplasias Esofágicas/cirugía , Carcinoma de Células Escamosas de Esófago/cirugía , Interleucina-8/metabolismo , Receptores de Interleucina-8B/metabolismo , Regulación hacia Arriba , Anciano , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-8/antagonistas & inhibidores , Interleucina-8/genética , Masculino , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Compuestos de Fenilurea/farmacología , Pronóstico , ARN Interferente Pequeño/farmacología , Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/genética , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia
7.
Nucleic Acids Res ; 49(2): 928-953, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33406258

RESUMEN

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP's function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP's carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.


Asunto(s)
Reparación del ADN por Unión de Extremidades/fisiología , Replicación del ADN , Endodesoxirribonucleasas/fisiología , Procesamiento Proteico-Postraduccional , Sumoilación , Sustitución de Aminoácidos , Arginina/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular , Quinasas Ciclina-Dependientes/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Genes Reporteros , Inestabilidad Genómica , Humanos , Lisina/química , Proteínas de Unión a Poli-ADP-Ribosa/fisiología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Inhibidoras de STAT Activados/fisiología , Mapeo de Interacción de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Reparación del ADN por Recombinación/genética , Reparación del ADN por Recombinación/fisiología
8.
Nucleic Acids Res ; 49(2): 805-817, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33410907

RESUMEN

Pin1 is a peptidyl-prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II (Pol II) through interaction with the C-terminal domain (CTD) of Rpb1, the largest subunit of Pol II. We demonstrated that this function is important for cellular response to oxidative stress in the fission yeast Schizosaccharomyces pombe. In response to oxidative stress, the Atf1 transcription factor targets Sty1, the mitogen-activated protein kinase (MAPK), to specific stress-responsive promoters. Anchored Sty1 recruits Pol II through direct association with Rpb1-CTD and phosphorylates the reiterated heptad sequence at Serine 5. Pin1 binds phosphorylated CTD to promote dissociation of Sty1 from it, and directly recruits Ssu72 phosphatase to facilitate dephosphorylation of CTD for transcription elongation. In the absence of Pin1, the association of Sty1-Atf1 with Rpb1 persists on stress-responsive promoters failed to generate transcripts of the corresponding genes effectively. The identified characteristic features of the fission yeast Pin1 are conserved in humans. We demonstrated that elevated Pin1 level in cancer cells might help to sustain survival under oxidative stress generated from their altered metabolic pathways. Together, these results suggest a conserved function of Pin1 in cellular response to oxidative stress among eukaryotic cells that might have clinical implication.


Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/fisiología , Estrés Oxidativo/genética , Fosfoproteínas Fosfatasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Inmunoprecipitación de Cromatina , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Especies Reactivas de Oxígeno , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/genética , Transcripción Genética
9.
Arch Biochem Biophys ; 699: 108754, 2021 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-33450239

RESUMEN

Drug resistance is one of the major challenges for treatment of hepatocellular carcinoma (HCC) with sorafenib. Our present study found that sorafenib resistant (SR) HCC cells showed epithelial-mesenchymal transition (EMT) characteristics with the downregulation of epithelial marker and upregulation of mesenchymal makers. The expression of Snail, a core factor of EMT, was increased in HCC/SR cells, while knockdown of Snail can restore sorafenib sensitivity and EMT potential of HCC/SR cells. Further, the upregulation of protein stability was responsible for the upregulation of Snail in HCC/SR cells. ATM and CSN2, which can stabilize Snail protein, were increased in HCC/SR cells. Knockdown of ATM and CSN2 can suppress the expression of Snail and increase sorafenib sensitivity of HCC/SR cells. It indicated that targeted inhibition of Snail might be helpful to overcome sorafenib resistance of HCC patients.


Asunto(s)
Carcinoma Hepatocelular/fisiopatología , Resistencia a Antineoplásicos/fisiología , Neoplasias Hepáticas/fisiopatología , Factores de Transcripción de la Familia Snail/metabolismo , Sorafenib/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Complejo del Señalosoma COP9/genética , Complejo del Señalosoma COP9/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/fisiología , Silenciador del Gen , Humanos , Estabilidad Proteica , ARN Interferente Pequeño/farmacología , Factores de Transcripción de la Familia Snail/química , Factores de Transcripción de la Familia Snail/genética , Regulación hacia Arriba/efectos de los fármacos
10.
ACS Appl Mater Interfaces ; 13(5): 6109-6118, 2021 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-33497198

RESUMEN

siRNA is found to effectively knock down the target gene in cells, which is considered a promising strategy for gene therapy. However, the application of siRNA is limited due to its low efficiency of the cellular uptake. Tetrahedral framework nucleic acids (tFNAs) are synthesized by four single-stranded DNAs and show multiple biological functions in recent studies, especially suitable for drug delivery. More than 60% of malignant melanomas are associated with Braf gene mutation, an attractive therapeutic target for RNA interference. In this study, we modified anti-Braf siRNA (siBraf) with tFNAs to downregulate the target gene. Meanwhile, we directly incorporated AS1411 (a DNA aptamer) to our nanostructure, which assists tFNAs to improve the cellular uptake efficacy of siBraf significantly. The results indicated that tFNAs-AS1411-siBraf exhibited more potent activity to cleave Braf mRNA than free siBraf. This study may provide a new idea for the combination therapy of siRNA and aptamers via DNA nanomaterials to achieve gene silencing.


Asunto(s)
Aptámeros de Nucleótidos/química , ADN/química , Sistemas de Liberación de Medicamentos , Melanoma/tratamiento farmacológico , ARN Interferente Pequeño/farmacología , ADN/síntesis química , Silenciador del Gen/efectos de los fármacos , Humanos , Melanoma/genética , Melanoma/metabolismo , Tamaño de la Partícula , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , Propiedades de Superficie , Células Tumorales Cultivadas
11.
ACS Appl Mater Interfaces ; 13(5): 5999-6010, 2021 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-33506682

RESUMEN

Cellular FLIP (cFLIP) is a crucial player of apoptosis-regulated pathways that is frequently overexpressed in solid cancers. To inhibit c-FLIP, pre- and post-transcriptionally, a multifunctional nanoparticle (NP) was created to deliver cFLIP-specific small interfering RNA (siRNA) into cancer cells. Specifically, Vorinostat (Vor)-loaded mesoporous silica nanoparticles (MSN) were conjugated with polyethylenimine-biotin (PB), followed by electrostatically binding with cFLIP siRNA (Vor/siR@MSN-PB). To stabilize and prolong the circulation time of nanoparticles, a bialdehyde-modified poly(ethylene glycol) (PEG) was cross-linked onto the polyethylenimine (PEI) backbone via the formation of the imine linkage (Schiff base) (Vor/siR@MSN-PB-PEG). The Schiff base is highly stable at physiological pH 7.4 but labile under slightly acidic pH conditions. In the acidic tumor microenvironment (TME), the PEG outer layer could be rapidly cleaved, resulting in the switching of the nanoparticle surface charge to positive, which specifically enhances internalization of the NPs to the biotin-positive tumor cells. Our results demonstrated the successful preparation of Vor/siR@MSN-PB-PEG NPs, in which the siRNA was effectively protected in serum and regulated the expression of cFlip, post-transcriptionally. The presence of the PEG layer resulted in high tumor accumulation and high efficacy in tumor inhibition, which was a result of the efficient cFLIP suppression. Furthermore, in the low-dose regimen of Vorinostat-the pre-transcriptional cFLIP suppressor, treatment with Vor/siR@MSN-PB-PEG NPs was found to be safe with the treated mice, indicating a promising combination regimen for cancer therapy.


Asunto(s)
Antineoplásicos/farmacología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/antagonistas & inhibidores , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Nanopartículas/química , ARN Interferente Pequeño/farmacología , Vorinostat/farmacología , Animales , Antineoplásicos/química , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Tamaño de la Partícula , Polietilenglicoles/química , ARN Interferente Pequeño/química , Propiedades de Superficie , Vorinostat/química
12.
Am J Hematol ; 96(4): 480-492, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33476437

RESUMEN

Efficient erythropoiesis relies on the expression of the transferrin receptor type 2 (TFR2). In erythroid precursors, TFR2 facilitates the export of the erythropoietin receptor (EPOR) to cell surface, which ensures the survival and proliferation of erythroblasts. Although TFR2 has a crucial role in erythropoiesis regulation, its mechanism of action remains to be clarified. To understand its role better, we aimed at identifying its protein partners by mass-spectrometry after immunoprecipitation in erythroid cells. Here we report the kinase MRCKα (myotonic dystrophy kinase-related CDC42-binding kinase α) as a new partner of both TFR2 and EPOR in erythroblasts. We show that MRCKα is co-expressed with TFR2, and TFR1 during terminal differentiation and regulates the internalization of the two types of transferrin receptors. The knockdown of MRCKα by shRNA in human primary erythroblasts leads to a decreased cell surface expression of both TFR1 and TFR2, an increased cell-surface expression of EPOR, and a delayed differentiation. Additionally, knockout of Mrckα in the murine MEDEP cells also leads to a striking delay in erythropoiesis, showcasing the importance of this kinase in both species. Our data highlight the importance of MRCKα in the regulation of erythropoiesis.


Asunto(s)
Eritropoyesis/fisiología , Proteína Quinasa de Distrofia Miotónica/fisiología , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Endocitosis , Eritroblastos/citología , Eritroblastos/metabolismo , Técnicas de Inactivación de Genes , Humanos , Hierro/metabolismo , Ratones , Proteína Quinasa de Distrofia Miotónica/aislamiento & purificación , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Receptores de Eritropoyetina/metabolismo , Receptores de Transferrina/metabolismo , Proteína de Unión al GTP cdc42/metabolismo
13.
Genomics ; 113(1 Pt 1): 331-343, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33321203

RESUMEN

An outbreak, caused by an RNA virus, SARS-CoV-2 named COVID-19 has become pandemic with a magnitude which is daunting to all public health institutions in the absence of specific antiviral treatment. Surface glycoprotein and nucleocapsid phosphoprotein are two important proteins of this virus facilitating its entry into host cell and genome replication. Small interfering RNA (siRNA) is a prospective tool of the RNA interference (RNAi) pathway for the control of human viral infections by suppressing viral gene expression through hybridization and neutralization of target complementary mRNA. So, in this study, the power of RNA interference technology was harnessed to develop siRNA molecules against specific target genes namely, nucleocapsid phosphoprotein gene and surface glycoprotein gene. Conserved sequence from 139 SARS-CoV-2 strains from around the globe was collected to construct 78 siRNA that can inactivate nucleocapsid phosphoprotein and surface glycoprotein genes. Finally, based on GC content, free energy of folding, free energy of binding, melting temperature, efficacy prediction and molecular docking analysis, 8 siRNA molecules were selected which are proposed to exert the best action. These predicted siRNAs should effectively silence the genes of SARS-CoV-2 during siRNA mediated treatment assisting in the response against SARS-CoV-2.


Asunto(s)
/terapia , Química Computacional , Diseño de Fármacos , Terapia Genética/métodos , Simulación del Acoplamiento Molecular , Interferencia de ARN , ARN Mensajero/antagonistas & inhibidores , ARN Interferente Pequeño/química , ARN Viral/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas Argonauta/química , Proteínas Argonauta/genética , Composición de Base , /virología , Evolución Molecular , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Pandemias , Fosfoproteínas/genética , Filogenia , Pliegue del ARN , ARN Guia/química , ARN Guia/genética , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , ARN Viral/genética , Alineación de Secuencia , Termodinámica
14.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118856, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32931817

RESUMEN

NIR, a novel INHAT, negatively regulates the transcription activity of tumor repressor p53. However, if NIR functions in the tumorigenesis dependent on the regulation of p53 remains unknown. Here, we report that NIR promotes progression of colorectal cancer (CRC) through regulating RB function. Firstly, we found that NIR expression is upregulated in the human CRC tissues and significantly associated with the poor outcome of the patients. Sequence alignment shows that NIR contains an RB-binding motif LxCxE in its INHAT-2 domain. We demonstrate that NIR interacts with RB via INHAT-2 in CRC cells and promotes RB degradation through proteasome-mediated pathway. Further, either full-length GFP-NIR or GFP-NIR-INHAT2 facilitates poly-ubiquitination of RB. In addition, NIR inhibits RB acetylation by INHAT-2, suggesting NIR might promote RB degradation through inhibiting RB acetylation. Importantly, endogenous NIR is downregulated upon DNA damage, which is consistent with the upregulation of total level and acetylation of RB. We further show that Flag-NIR inhibits DNA damage-induced RB acetylation. Thus, downregulation of NIR might contribute to maintain the cellular homeostasis under DNA damage. Consequently, depletion of NIR inhibits cell proliferation and tumor growth in mouse xenografts. Taken together, we demonstrate that NIR promotes CRC progression partially through inhibiting RB acetylation and promoting RB degradation. Targeting NIR may provide a potential therapeutic strategy for NIR-upregulated CRC patients.


Asunto(s)
Carcinogénesis/genética , Neoplasias Colorrectales/genética , Proteínas Represoras/genética , Proteína de Retinoblastoma/genética , Proteína p53 Supresora de Tumor/genética , Acetilación/efectos de los fármacos , Anciano , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/patología , Daño del ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteolisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Ubiquitinación
15.
Carbohydr Polym ; 251: 117008, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-33142574

RESUMEN

A novel folic acid mediated chitosan oligosaccharide-grafted disulfide-containing polyethylenimine copolymer-based silica nanohybrids were fabricated for co-delivering paclitaxel and P-shRNA. These nanoparticles could efficiently protect P-shRNA against degradation, and exhibited well redox-responsive P-shRNA release and pH-responsive drug release behaviors. Folic acid as the targeting head, could improve cellular uptake of nanoparticles by multidrug-resistant breast cancer cells. Moreover, these nanoparticles showed excellent delivery P-shRNA into cells and displayed high gene silencing efficiency at the targeted mRNAs to downregulate the expression of P-gp which induced up to 63% decrease. Finally, nanoparticles could completely reverse the resistance of breast cancer cells to paclitaxel and the resistance reversion index was 50.59. These results suggested that our nanoparticles could efficiently co-deliver paclitaxel and P-shRNA into cancer cells to exert its synergistic antitumor effect, and opened up a new avenue for overcoming multidrug resistance.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Portadores de Fármacos/química , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/farmacología , Quitosano/análogos & derivados , Quitosano/química , Liberación de Fármacos , Ácido Fólico/química , Humanos , Concentración de Iones de Hidrógeno , Células MCF-7 , Nanopartículas/química , Oxidación-Reducción , Paclitaxel/farmacología , Polietileneimina/análogos & derivados , Polietileneimina/química , ARN Interferente Pequeño/farmacología
16.
Methods Mol Biol ; 2174: 263-275, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32813256

RESUMEN

In recent decades, zebrafish (Danio rerio) has become a major in vivo model for the evaluation of drug efficacies and toxicities. In the field of drug delivery research, zebrafish larvae are a suitable model for the use of fluorescent-labeled chemicals, nanoparticle, liposome, or micelle-mediated delivery systems because of their transparent body wall. In the current chapter, we describe the method to perform micelle-based siRNA delivery using cancer cells implanted into the circulation of zebrafish.


Asunto(s)
Técnicas de Transferencia de Gen , ARN Interferente Pequeño/farmacología , Pez Cebra , Animales , Animales Modificados Genéticamente , Carbocianinas/química , Línea Celular Tumoral , Trasplante de Células , Sistemas de Liberación de Medicamentos/métodos , Colorantes Fluorescentes/química , Humanos , Larva/genética , Proteínas Luminiscentes/genética , Melanoma/patología , Micelas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Trasplantes , Pez Cebra/genética
17.
Phytomedicine ; 80: 153369, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33070082

RESUMEN

BACKGROUND: Impairment of mitochondrial biogenesis is associated with the pathological progression of Parkinson's disease (PD). Parkin-interacting substrate (PARIS) can be ubiquitinated by parkin and prevents the repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α). PURPOSE: This study investigated whether the neuroprotective mechanism of carnosic acid (CA) from rosemary is mediated via the regulation of PARIS and PGC-1α by parkin. METHODS: The Western blotting and RT-PCR were used to determine protein and mRNA, respectively. To investigate the protein-protein interaction of between PARIS and ubiquitin, the immunoprecipitation assay (IP assay) was utilized. Silencing of endogenous parkin or PGC-1α was performed by using transient transfection of small interfering RNA (siRNA). RESULTS: SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA) increased PARIS protein, decreased PGC-1α protein, and reduced protein and mRNA of mitochondrial biogenesis-related genes. CA pretreatment reversed the effects of 6-OHDA. By IP assay, the interaction of PARIS with ubiquitin protein caused by CA was stronger than that caused by 6-OHDA. Moreover, knockdown of parkin attenuated the ability of CA to reverse the 6-OHDA-induced increase in PARIS and decrease in PGC-1α expression. PGC-1α siRNA was used to investigate how CA influenced the effect of 6-OHDA on the modulation of mitochondrial biogenesis and apoptosis. In the presence of PGC-1α siRNA, CA could no longer significantly reverse the reduction of mitochondrial biogenesis or the induction of cleavage of apoptotic-related proteins by 6-OHDA. CONCLUSION: The cytoprotective of CA is related to the enhancement of mitochondrial biogenesis by inhibiting PARIS and inducing PGC-1α by parkin. The activation of PGC-1α-mediated mitochondrial biogenesis by CA prevents the degeneration of dopaminergic neurons, CA may have therapeutic application in PD.


Asunto(s)
Abietanos/farmacología , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Proteínas Represoras/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Biogénesis de Organelos , Oxidopamina/toxicidad , Enfermedad de Parkinson/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , ARN Interferente Pequeño/farmacología , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
18.
Gene ; 772: 145358, 2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33340561

RESUMEN

FAM135B (family with sequence similarity 135, member B) is related to the progression of esophageal squamous cell carcinoma (ESCC). However, the role played by the gene in radiosensitivity remains unknown. Herein, we examined the relationship between FAM135B and radiosensitivity. According to the results, FAM135B is highly expressed in ESCC cells, and ESCC cells with high levels of FAM135B are resistant to irradiation. Silencing FAM135B inhibits colony formation capability and cell cycle protein expression (pP53, CDK1), promotes cell cycle arrest at the G2/M phase following irradiation. Moreover, transcriptome sequencing analysis demonstrates that FAM135B regulates downstream PI3K/Akt/mTOR signaling pathway, and western blot verifies the result. One of the mechanisms of increasing radiosensitivity by silencing FAM135B expression in ESCC cells may be achieved by regulating the PI3K/Akt/mTOR signaling pathway. Silencing FAM135B shows synergy with PI3K/Akt/mTOR pathway inhibitor (rapamycin) in increasing radiosensitivity, regulating the expression of cell cycle protein and inducing apoptosis of ESCC cells. The results indicate that FAM135B could be a potential treatment target for ESCC in management of radiosensitivity.


Asunto(s)
Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Péptidos y Proteínas de Señalización Intracelular/genética , ARN Interferente Pequeño/farmacología , Regulación hacia Arriba/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas de Esófago/terapia , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Silenciador del Gen , Humanos , Tolerancia a Radiación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Sirolimus/farmacología
19.
Sci Rep ; 10(1): 22343, 2020 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-33339841

RESUMEN

Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40-70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60-90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.


Asunto(s)
Silenciador del Gen/efectos de los fármacos , Terapia Genética , ARN Interferente Pequeño/farmacología , Receptores de N-Metil-D-Aspartato/genética , Enfermedades de la Retina/terapia , Animales , Genes Esenciales/genética , Humanos , Inyecciones Intravítreas , ARN Bicatenario/genética , ARN Bicatenario/farmacología , Ratas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Retina/patología , Enfermedades de la Retina/genética , Enfermedades de la Retina/patología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Cuerpo Vítreo/efectos de los fármacos
20.
Inflamm Res ; 69(12): 1215-1234, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33044562

RESUMEN

OBJECTIVE AND DESIGN: Macrophages exhibit strong phenotypic plasticity and can mediate renal inflammation by polarizing into an M1 phenotype. They play a pivotal role in diabetic nephropathy (DN). Here, we have investigated the regulatory role of transforming growth factor ß-activated kinase 1-binding protein 1 (TAB1) in glycolysis and activation of macrophages during DN. METHODS: TAB1 was inhibited using siRNA in high glucose (HG)-stimulated bone marrow-derived macrophages (BMMs) and lentiviral vector-mediated TAB1 knockdown was used in streptozotocin (STZ)-induced diabetic mice. Western blotting, flow cytometry, qRT-PCR, ELISA, PAS staining and immunohistochemical staining were used for assessment of TAB1/nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α), iNOS, glycolysis, inflammation and the clinical and pathological manifestations of diabetic nephropathy. RESULTS: We found that TAB1/NF-κB/HIF-1α, iNOS and glycolysis were up-regulated in BMMs under HG conditions, leading to release of further inflammatory factors, Downregulation of TAB1 could inhibit glycolysis/polarization of macrophages and inflammation in vivo and in vitro. Furthermore, albuminuria, the tubulointerstitial damage index and glomerular mesangial expansion index of STZ-induced diabetic nephropathy mice were decreased by TAB1 knockdown. CONCLUSIONS: Our results suggest that the TAB1/NF-κB/HIF-1α signaling pathway regulates glycolysis and activation of macrophages in DN.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neuropatías Diabéticas/genética , Neuropatías Diabéticas/metabolismo , Glucólisis/genética , Activación de Macrófagos/genética , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Albuminuria/tratamiento farmacológico , Albuminuria/patología , Animales , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Técnicas de Silenciamiento del Gen , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Células Mesangiales/patología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/efectos de los fármacos , Nefritis Intersticial/patología , Óxido Nítrico Sintasa de Tipo II/efectos de los fármacos , ARN Interferente Pequeño/farmacología
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