RESUMEN
Atrial fibrillation (AF) is the most common form of sustained clinical arrhythmia. We previously mapped an AF locus to chromosome 5p13 in an AF family with sudden death in early childhood. Here we show that the specific AF gene underlying this linkage is NUP155, which encodes a member of the nucleoporins, the components of the nuclear pore complex (NPC). We have identified a homozygous mutation, R391H, in NUP155 that cosegregates with AF, affects nuclear localization of NUP155, and reduces nuclear envelope permeability. Homozygous NUP155(-/-) knockout mice die before E8.5, but heterozygous NUP155(+/-) mice show the AF phenotype. The R391H mutation and reduction of NUP155 are associated with inhibition of both export of Hsp70 mRNA and nuclear import of Hsp70 protein. These human and mouse studies indicate that loss of NUP155 function causes AF by altering mRNA and protein transport and link the NPC to cardiovascular disease.
Asunto(s)
Fibrilación Atrial/genética , Muerte Súbita Cardíaca , Proteínas de Complejo Poro Nuclear/genética , Secuencia de Aminoácidos , Animales , Femenino , Proteínas del Choque Térmico HSP72/genética , Proteínas del Choque Térmico HSP72/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Linaje , Alineación de SecuenciaRESUMEN
Cardiac sodium channel Nav1.5 is associated with cardiac arrhythmias and heart failure. Protein ubiquitination is catalyzed by an E1-E2-E3 cascade of enzymes. However, the E1 enzyme catalyzing Nav1.5 ubiquitination is unknown. Here, we show that UBE1 and UBA6 are two E1 enzymes regulating Nav1.5 ubiquitination and expression. Western blot analysis and patch-clamping recordings showed that overexpression of UBE1 or UBA6 increased the ubiquitination of Nav1.5 and significantly reduced Nav1.5 expression and sodium current density, and knockdown of UBE1 or UBA6 expression significantly increased Nav1.5 expression and sodium current density in HEK293/Nav1.5 cells. Similar results were obtained in neonatal cardiomyocytes. Bioinformatic analysis predicted two ubiquitination sites at K590 and K591. Mutations of K590 and K591 to K590A and K591A abolished the effects of overexpression or knockdown of UBE1 or UBA6 on Nav1.5 expression and sodium current density. Western blot analysis showed that the effects of UBE1 or UBA6 overexpression on the ubiquitination and expression of Nav1.5 were abolished by knockdown of UBC9, a putative E2 enzyme reported for Nav1.5 ubiquitination by us. Interestingly, real-time RT-PCR analysis showed that the expression level of UBE1, but not UBA6, was significantly up-regulated in ventricular tissues from heart failure patients. These data establish UBE1 and UBA6 as the E1 enzymes involved in Nav1.5 ubiquitination, and suggest that UBE1 and UBA6 regulate ubiquitination of Nav1.5 through UBC9. Our study is the first to reveal the regulatory role of the UBE1 or UBA6 E1 enzyme in the ubiquitination of an ion channel and links UBE1 up-regulation to heart failure.
Asunto(s)
Canales de Sodio/metabolismo , Enzimas Activadoras de Ubiquitina , Ubiquitinación/fisiología , Arritmias Cardíacas/metabolismo , Células HEK293 , Humanos , Mutación , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Sodio/metabolismo , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
Coronary artery disease (CAD) is the leading cause of death worldwide. Long noncoding RNAs (lncRNAs) are a class of noncoding transcripts of > 200 nucleotides and are increasingly recognized as playing functional roles in physiology and disease. ANRIL is an lncRNA gene mapped to the chromosome 9p21 genetic locus for CAD identified by the first series of genome-wide association studies (GWAS). However, ANRIL's role in CAD and the underlying molecular mechanism are unknown. Here, we show that the major ANRIL transcript in endothelial cells (ECs) is DQ485454 with a much higher expression level in ECs than in THP-1 monocytes. Of note, DQ485454 expression was down-regulated in CAD coronary arteries compared with non-CAD arteries. DQ485454 overexpression significantly reduced monocyte adhesion to ECs, transendothelial monocyte migration (TEM), and EC migration, which are critical cellular processes involved in CAD initiation, whereas siRNA-mediated ANRIL knockdown (KD) had the opposite effect. Microarray and follow-up quantitative RT-PCR analyses revealed that the ANRIL KD down-regulated expression of AHNAK2, CLIP1, CXCL11, ENC1, EZR, LYVE1, WASL, and TNFSF10 genes and up-regulated TMEM100 and TMEM106B genes. Mechanistic studies disclosed that overexpression of CLIP1, EZR, and LYVE1 reversed the effects of ANRIL KD on monocyte adhesion to ECs, TEM, and EC migration. These findings indicate that ANRIL regulates EC functions directly related to CAD, supporting the hypothesis that ANRIL is involved in CAD pathogenesis at the 9p21 genetic locus and identifying a molecular mechanism underlying lncRNA-mediated regulation of EC function and CAD development.
Asunto(s)
Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Proteínas del Citoesqueleto/metabolismo , Células Endoteliales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , ARN Largo no Codificante/metabolismo , Regulación hacia Arriba , Proteínas de Transporte Vesicular/metabolismo , Movimiento Celular , Células Cultivadas , Proteínas del Citoesqueleto/genética , Humanos , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Proteínas de Neoplasias/genética , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Transporte Vesicular/genéticaRESUMEN
Each gene typically has multiple alternatively spliced transcripts. Different transcripts are assumed to play a similar biological role; however, some transcripts may simply lose their function due to loss of important functional domains. Here, we show that two different transcripts of lncRNA gene ANRIL associated with coronary artery disease (CAD) play antagonizing roles against each other. We previously reported that DQ485454, the short transcript, is downregulated in coronary arteries from CAD patients, and reduces monocyte adhesion to endothelial cells (ECs) and transendothelial monocyte migration (TEM). Interestingly, the longest transcript NR_003529 is significantly upregulated in coronary arteries from CAD patients. Overexpression of ANRIL transcript NR_003529 increases monocyte adhesion to ECs and TEM, whereas knockdown of NR_003529 expression reduces monocyte adhesion to ECs and TEM. Much more dramatic effects were observed for the combination of overexpression of NR_003529 and knockdown of DQ485454 or the combination of knockdown of NR_003529 and overexpression of DQ485454. The antagonizing effects of ANRIL transcripts NR_003529 and DQ485454 were associated with their opposite effects on expression of downstream target genes EZR, CXCL11 or TMEM106B. Our results demonstrate that different transcripts of lncRNA can exert antagonizing effects on biological functions, thereby providing important insights into the biology of lncRNA. The data further support the hypothesis that ANRIL is the causative gene at the 9p21 CAD susceptibility locus.
Asunto(s)
Empalme Alternativo , Enfermedad de la Arteria Coronaria/genética , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , ARN Largo no Codificante/genética , Biomarcadores , Adhesión Celular/genética , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/metabolismo , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Susceptibilidad a Enfermedades , Técnicas de Silenciamiento del Gen , Humanos , Monocitos/metabolismo , Monocitos/patología , Isoformas de ARN , Migración Transendotelial y Transepitelial/genéticaRESUMEN
Nav1.5 is the α-subunit of the cardiac sodium channel complex. Abnormal expression of Nav1.5 on the cell surface because of mutations that disrupt Nav1.5 trafficking causes Brugada syndrome (BrS), sick sinus syndrome (SSS), cardiac conduction disease, dilated cardiomyopathy, and sudden infant death syndrome. We and others previously reported that Ran-binding protein MOG1 (MOG1), a small protein that interacts with Nav1.5, promotes Nav1.5 intracellular trafficking to plasma membranes and that a substitution in MOG1, E83D, causes BrS. However, the molecular basis for the MOG1/Nav1.5 interaction and how the E83D substitution causes BrS remains unknown. Here, we assessed the effects of defined MOG1 deletions and alanine-scanning substitutions on MOG1's interaction with Nav1.5. Large deletion analysis mapped the MOG1 domain required for the interaction with Nav1.5 to the region spanning amino acids 146-174, and a refined deletion analysis further narrowed this domain to amino acids 146-155. Site-directed mutagenesis further revealed that Asp-148, Arg-150, and Ser-151 cluster in a peptide loop essential for binding to Nav1.5. GST pulldown and electrophysiological analyses disclosed that the substitutions E83D, D148Q, R150Q, and S151Q disrupt MOG1's interaction with Nav1.5 and significantly reduce its trafficking to the cell surface. Examination of MOG1's 3D structure revealed that Glu-83 and the loop containing Asp-148, Arg-150, and Ser-151 are spatially proximal, suggesting that these residues form a critical binding site for Nav1.5. In conclusion, our findings identify the structural elements in MOG1 that are crucial for its interaction with Nav1.5 and improve our understanding of how the E83D substitution causes BrS.
Asunto(s)
Síndrome de Brugada/metabolismo , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Proteína de Unión al GTP ran/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Síndrome de Brugada/genética , Eliminación de Gen , Humanos , Mutación Missense , Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.5/genética , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Proteína de Unión al GTP ran/química , Proteína de Unión al GTP ran/genéticaRESUMEN
Brugada syndrome (BrS) is a fatal arrhythmia that causes an estimated 4% of all sudden death in high-incidence areas. SCN5A encodes cardiac sodium channel NaV1.5 and causes 25 to 30% of BrS cases. Here, we report generation of a knock-in (KI) mouse model of BrS (Scn5aG1746R/+). Heterozygous KI mice recapitulated some of the clinical features of BrS, including an ST segment abnormality (a prominent J wave) on electrocardiograms and development of spontaneous ventricular tachyarrhythmias (VTs), seizures, and sudden death. VTs were caused by shortened cardiac action potential duration and late phase 3 early afterdepolarizations associated with reduced sodium current density (INa) and increased Kcnd3 and Cacna1c expression. We developed a gene therapy using adeno-associated virus serotype 9 (AAV9) vector-mediated MOG1 delivery for up-regulation of MOG1, a chaperone that binds to NaV1.5 and traffics it to the cell surface. MOG1 was chosen for gene therapy because the large size of the SCN5A coding sequence (6048 base pairs) exceeds the packaging capacity of AAV vectors. AAV9-MOG1 gene therapy increased cell surface expression of NaV1.5 and ventricular INa, reversed up-regulation of Kcnd3 and Cacna1c expression, normalized cardiac action potential abnormalities, abolished J waves, and blocked VT in Scn5aG1746R/+ mice. Gene therapy also rescued the phenotypes of cardiac arrhythmias and contractile dysfunction in heterozygous humanized KI mice with SCN5A mutation p.D1275N. Using a small chaperone protein may have broad implications for targeting disease-causing genes exceeding the size capacity of AAV vectors.
Asunto(s)
Síndrome de Brugada , Cardiomiopatías , Animales , Arritmias Cardíacas/terapia , Síndrome de Brugada/genética , Síndrome de Brugada/metabolismo , Síndrome de Brugada/terapia , Cardiomiopatías/genética , Cardiomiopatías/terapia , Muerte Súbita , Modelos Animales de Enfermedad , Terapia Genética , Ratones , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Transporte de ProteínasRESUMEN
Electronic states of the PbSi molecule up to 4 eV have been studied by carrying out ab initio based MRDCI calculations which include relativistic effective core potentials (RECPs) of both the atoms. The use of semicore RECPs of Pb produces better dissociation limits than the full-core one. However, the (3)P(0)-(3)P(1) splitting due to Pb is underestimated by about 4000 cm(-1). At least 25 bound electronic states of the Λ-S symmetry are predicted for PbSi. The computed zero-field-splitting in the ground state is about 544 cm(-1). A strong spin-orbit mixing changes the nature of the potential energy curves of many Ω states. The overall splitting among the spin components of A(3)Π is computed to be 4067 cm(-1). However, the largest spin-orbit splitting is reported for the (3)Δ state. A number of spin-allowed and spin-forbidden transitions are predicted. The partial radiative lifetime for the A(3)Π-X(3)Σ(-) transition is of the order of milliseconds. The computed bond energy in the ground state is 1.68 eV, considering the spin-orbit coupling. The vertical ionization energy for the ionization to the X(4)Σ(-) ground state of PbSi(+) is about 6.93 eV computed at the same level of calculations.
RESUMEN
Electronic structure and spectroscopic properties of the ground and low-lying excited states of SnSi within 4 eV have been investigated by using a multireference singles and doubles configuration interaction (MRDCI) method that includes relativistic effective core potentials. Potential energy curves of a number of Lambda-S states of singlet, triplet, and quintet spin multiplicities are constructed. Spectroscopic parameters (T(e), r(e), omega(e), D(e), and mu(e)) of 27 bound Lambda-S states are reported. The ground state of SnSi belongs to the X(3)Sigma(-) symmetry with an estimated dissociation energy (D(e)) of 2.49 eV. However, with the inclusion of the spin-orbit coupling, D(e) reduces to 2.11 eV. Spectroscopic properties of at least 36 Omega states are determined. Transition probabilities of several singlet-singlet and triplet-triplet transitions are calculated. Partial radiative lifetimes of some of these transitions are estimated. A number of weak Omega-Omega transitions with partial radiative lifetimes of the order of milliseconds or more is also predicted here.
RESUMEN
Electronic structure and spectroscopic properties of the ground and low-lying excited states of GeSi+ have been studied by using multireference singles and doubles configuration interaction (MRDCI) method that includes relativistic effective core potentials of Ge and Si atoms. At least 17 Lambda-Sigma bound states of GeSi+ are reported within 5 eV. Potential energy curves of 24 Lambda-Sigma states which correlate with the lowest two dissociation limits, Ge+(2Pu)+Si(3Pg) and Ge(3Pg)+Si+ (2Pu) are constructed. The ground-state dissociation energy of GeSi+ is estimated to be 3.116 eV. The spin-orbit coupling among all states correlating the two dissociation limits is also included in the calculations. Spectroscopic constants of 27 Omega states of the ion are reported. Effects of the spin-orbit coupling on the spectroscopic properties are also studied. Potential energy curves of Omega states show several avoided crossings. Transition probabilities of many electric dipole allowed and spin-forbidden transitions are computed. Transitions such as 24Sigma(-)-X4Sigma- and 44Sigma(-)-X4Sigma- are found to be highly probable. Partial lifetimes for some weak spin-forbidden transitions to the ground-state components, X4Sigma(1/2)- and X4Sigma(3/2)- are also computed.
RESUMEN
Dysfunctional inflammatory pathways are associated with an increased risk of cancer, including colorectal cancer. We have previously identified and enriched for a self-renewing, colon cancer stem cell (CCSC) subpopulation in primary sporadic colorectal cancers (CRC) and a related subpopulation in ulcerative colitis (UC) patients defined by the stem cell marker, aldehyde dehydrogenase (ALDH). Subsequent work demonstrated that CCSC-initiated tumors are dependent on the inflammatory chemokine, CXCL8, a known inducer of tumor proliferation, angiogenesis and invasion. Here, we use RNA interference to target CXCL8 and its receptor, CXCR1, to establish the existence of a functional signaling pathway promoting tumor growth initiated by sporadic and colitis CCSCs. Knocking down either CXCL8 or CXCR1 had a dramatic effect on inhibiting both in vitro proliferation and angiogenesis. Likewise, tumorigenicity was significantly inhibited due to reduced levels of proliferation and angiogenesis. Decreased expression of cycle cell regulators cyclins D1 and B1 along with increased p21 levels suggested that the reduction in tumor growth is due to dysregulation of cell cycle progression. Therapeutically targeting the CXCL8-CXCR1 signaling pathway has the potential to block sustained tumorigenesis by inhibiting both CCSC- and pCCSC-induced proliferation and angiogenesis.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/etiología , Neoplasias del Colon/metabolismo , Inflamación/metabolismo , Interleucina-8/metabolismo , Células Madre Neoplásicas/metabolismo , Receptores de Interleucina-8A/metabolismo , Transducción de Señal , Animales , Biomarcadores , Línea Celular Tumoral , Proliferación Celular , Colitis/complicaciones , Colitis/genética , Colitis/metabolismo , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Dosificación de Gen , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Inmunofenotipificación , Inflamación/complicaciones , Inflamación/genética , Interleucina-8/genética , Ratones , Modelos Biológicos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Receptores de Interleucina-8A/genéticaRESUMEN
Inflammatory bowel disease (IBD) affects one million people in the US. Ulcerative colitis (UC) is a subtype of IBD that can lead to colitis-associated cancer (CAC). In UC, the rate of CAC is 3-5-fold greater than the rate of sporadic colorectal cancer (CRC). The pathogenesis of UC and CAC are due to aberrant interactions between host immune system and microenvironment, but precise mechanisms are still unknown. In colitis and CAC, microenvironmental fibroblasts exhibit an activated, inflammatory phenotype that contributes to tumorigenesis accompanied by excessive secretion of the chemokine CXCL8. However, mechanisms regulating CXCL8 secretion are unclear. Since it is known that miRNAs regulate chemokines such as CXCL8, we queried a microRNA library for mimics affecting CXCL8 secretion. Among the identified microRNAs, miR-20a/b was further investigated as its stromal expression levels inversely correlated with the amounts of CXCL8 secreted and predicted fibroblast tumor-promoting activity. Indeed, miR-20a directly bound to the 3'UTR of CXCL8 mRNA and regulated its expression by translational repression. In vivo co-inoculation studies with CRC stem cells demonstrated that fibroblasts characterized by high miR-20a expression had reduced tumor-promoting activities. These studies reveal that in stromal fibroblasts, miR-20a modulates CXCL8 function, therefore influencing tumor latency.
RESUMEN
BACKGROUND: The cardiac sodium channel Nav1.5 is essential for the physiological function of the heart and causes cardiac arrhythmias and sudden death when mutated. Many disease-causing mutations in Nav1.5 cause defects in protein trafficking, a cellular process critical to the targeting of Nav1.5 to cell surface. However, the molecular mechanisms underlying the trafficking of Nav1.5, in particular, the exit from the endoplasmic reticulum (ER) for cell surface trafficking, remain poorly understood. METHODS AND RESULTS: Here we investigated the role of the SAR1 GTPases in trafficking of Nav1.5. Overexpression of dominant-negative mutant SAR1A (T39N or H79G) or SAR1B (T39N or H79G) significantly reduces the expression level of Nav1.5 on cell surface, and decreases the peak sodium current density (INa) in HEK/Nav1.5 cells and neonatal rat cardiomyocytes. Simultaneous knockdown of SAR1A and SAR1B expression by siRNAs significantly reduces the INa density, whereas single knockdown of either SAR1A or SAR1B has minimal effect. Computer modeling showed that the three-dimensional structure of SAR1 is similar to RAN. RAN was reported to interact with MOG1, a small protein involved in regulation of the ER exit of Nav1.5. Co-immunoprecipitation showed that SAR1A or SAR1B interacted with MOG1. Interestingly, knockdown of SAR1A and SAR1B expression abolished the MOG1-mediated increases in both cell surface trafficking of Nav1.5 and the density of INa. CONCLUSIONS: These data suggest that SAR1A and SAR1B are the critical regulators of trafficking of Nav1.5. Moreover, SAR1A and SAR1B interact with MOG1, and are required for MOG1-mediated cell surface expression and function of Nav1.5.
Asunto(s)
Proteínas de Unión al GTP Monoméricas/metabolismo , Miocitos Cardíacos/fisiología , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Proteína de Unión al GTP ran/metabolismo , Animales , Animales Recién Nacidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Mutagénesis , Miocitos Cardíacos/citología , Canal de Sodio Activado por Voltaje NAV1.5/economía , Técnicas de Placa-Clamp , Cultivo Primario de Células , Transporte de Proteínas/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína de Unión al GTP ran/químicaRESUMEN
Thiourea (TU), a commercially available laboratory chemical, has been discovered to introduce metallogelation when reacted with copper(II) chloride in aqueous medium. The chemistry involves the reduction of Cu(II) to Cu(I) with concomitant oxidation of thiourea to dithiobisformamidinium dichloride. The gel formation is triggered through metal-ligand complexation, i.e., Cu(I)-TU coordination and extensive hydrogen bonding interactions involving thiourea, the disulfide product, water, and chloride ions. Entangled network morphology of the gel selectively develops in water, maybe for its superior hydrogen-bonding ability, as accounted from Kamlet-Taft solvent parameters. Complete and systematic chemical analyses demonstrate the importance of both Cu(I) and chloride ions as the key ingredients in the metal-organic coordination gel framework. The gel is highly fluorescent. Again, exclusive presence of Cu(I) metal centers in the gel structure makes the gel redox-responsive and therefore it shows reversible gel-sol phase transition. However, the reversibility does not cause any morphological change in the gel phase. The gel practically exhibits its multiresponsive nature and therefore the influences of different probable interfering parameters (pH, selective metal ions and anions, selective complexing agents, etc.) have been studied mechanistically and the results might be promising for different applications. Finally, the gel material shows a highly selective visual response to a commonly used nitroexplosive, picric acid among a set of 19 congeners and the preferred selectivity has been mechanistically interpreted with density functional theory-based calculations.
RESUMEN
BACKGROUND: Loss-of-function mutations in Na(v)1.5 cause sodium channelopathies, including Brugada syndrome, dilated cardiomyopathy, and sick sinus syndrome; however, no effective therapy exists. MOG1 increases plasma membrane (PM) expression of Na(v)1.5 and sodium current (I(Na)) density, thus we hypothesize that MOG1 can serve as a therapeutic target for sodium channelopathies. METHODS AND RESULTS: Knockdown of MOG1 expression using small interfering RNAs reduced Na(v)1.5 PM expression, decreased I(Na) densities by 2-fold in HEK/Na(v)1.5 cells and nearly abolished I(Na) in mouse cardiomyocytes. MOG1 did not affect Na(v)1.5 PM turnover. MOG1 small interfering RNAs caused retention of Na(v)1.5 in endoplasmic reticulum, disrupted the distribution of Na(v)1.5 into caveolin-3-enriched microdomains, and led to redistribution of Na(v)1.5 to noncaveolin-rich domains. MOG1 fully rescued the reduced PM expression and I(Na) densities by Na(v)1.5 trafficking-defective mutation D1275N associated with sick sinus syndrome/dilated cardiomyopathy/atrial arrhythmias. For Brugada syndrome mutation G1743R, MOG1 restored the impaired PM expression of the mutant protein and restored I(Na) in a heterozygous state (mixture of wild type and mutant Na(v)1.5) to a full level of a homozygous wild-type state. CONCLUSIONS: Use of MOG1 to enhance Na(v)1.5 trafficking to PM may be a potential personalized therapeutic approach for some patients with Brugada syndrome, dilated cardiomyopathy, and sick sinus syndrome in the future.
Asunto(s)
Síndrome de Brugada/genética , Mutación , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , ARN/genética , Síndrome del Seno Enfermo/genética , Animales , Síndrome de Brugada/metabolismo , Síndrome de Brugada/patología , Células Cultivadas , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Miocitos Cardíacos/patología , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Síndrome del Seno Enfermo/metabolismo , Síndrome del Seno Enfermo/patologíaRESUMEN
BACKGROUND: A subset of head and neck squamous cell carcinoma (HNSCC) exhibits a microsatellite instability (MIN) phenotype. The authors correlated alterations in the mismatch-repair genes human mutL homolog 1 (hMLH1) and human mutS homolog 2 (hMSH2) in primary head and neck squamous cell carcinoma (HNSCC) tumors and in samples of leukoplakia with the MIN phenotype. METHODS: One hundred twenty-three paired HNSCC normal and tumor tissues and 27 leukoplakia samples were examined for hypermethylation of hMLH1 and hMSH2 promoters. The hypermethylation status of the tissues was confirmed by expression studies. Sixty-three of 123 randomly selected tumors and all 27 leukplakia samples were genotyped with 8 microsatellite markers to determine MIN. RESULTS: Fifty percent of HNSCC tumors and 63% of leukoplakia samples harbored hypermethylation at either or both hMLH1 and hMSH2 promoters. Normal tissues adjacent to methylation-positive tumors also demonstrated hypermethylation of both promoters at a high frequency (25%). A positive correlation between tobacco habit and promoter hypermethylation was observed (P = .001). A correlation was observed between MIN and the frequency of promoter hypermethylation in the leukoplakia samples, but no such trend was observed in the HNSCC tumors. It is noteworthy that patients who had a high frequency of MIN-positive tumors exhibited hypermethylation in both the affected tissues and the adjacent normal tissues (P = .007). Patients with a tobacco habit who had promoter hypermethylation at both the affected tissues and the adjacent normal tissues had tumors that mostly were MIN positive (P = .047). CONCLUSIONS: The current results suggested that tobacco-addicted individuals are more susceptible to promoter hypermethylation of hMLH1 and hMSH2 and that, if such hypermethylation occurs in the normal squamous epithelium of the head and neck region, then those tissues are likely to develop into tumors that involve the MIN pathway.
Asunto(s)
Carcinoma de Células Escamosas/genética , Proteínas Portadoras/genética , Metilación de ADN , Neoplasias de Cabeza y Cuello/genética , Leucoplasia/genética , Repeticiones de Microsatélite/genética , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Reparación de la Incompatibilidad de ADN , ADN de Neoplasias/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
Intrinsic genomic instability of an incipient tumor cell drives the development of cancer. In colorectal cancer, an inverse relationship between microsatellite instability (MIN) and chromosomal instability (CIN) has been reported. The relationship between MIN and CIN in head and neck squamous cell carcinoma (HNSCC) is uncertain. In the present study, we examined these two types of instabilities in HNSCC using microsatellite markers and arbitrary primed PCR (APPCR) technique. HNSCC tumors showed high frequency of MIN and loss of heterozygosity (LOH). We observed that, in contrast to colorectal tumors, the frequency of LOH increased with the increase in MIN. There was no significant difference between MIN- and MIN+ groups of HNSCC tumors in the extent of overall genomic alterations; rather higher MIN+ tumors exhibited higher incidence of deletion. Thus, in sporadic HNSCC, both MIN and CIN pathways operate simultaneously to drive tumor formation.