ATP2B1-AS1 exacerbates sepsis-induced cell apoptosis and inflammation by regulating miR-23a-3p/TLR4 axis

Background: Sepsis is a life-threatening disease with dominant mortality. Its early diagnosis and treatment can improve prognosis and reduce mortality. Long noncoding RNAs (lncRNAs) ATPase plasma membrane Ca2+ transporting 1 antisense RNA 1 (ATP2B1-AS1) is dysregulated and is involved in the progression of various diseases. Nevertheless, the role of ATP2B1-AS1 in sepsis remains unclear. Methods: A human monocytic cell line, THP-1 cells, was stimulated to induce a model of sepsis in vitro. The levels of ATP2B1-AS1, miR-23a-3p, and TLR4 were assessed by real-time quantitative polymerase chain reaction. The role of ATP2B1-AS1 in cell apoptosis and inflammation was explored by flow cytometry, Western blot analysis and enzyme-linked immunosorbent serologic assay. The binding sites between ATP2B1-AS1 and miR-23a-3p, and between miR-23a-3p and TLR4 were predicted by BiBiServ and the Encyclopedia of RNA Interactomes (ENCORI) online sites, respectively, and confirmed by the luciferase assay. Results: The level of ATP2B1-AS1 was increased in lipopolysaccharide (LPS)-treated THP-1 cells. LPS increased apoptosis ratio, relative protein expressions of pro-apoptotic factors, and relative messenger RNA (mRNA) level and concentrations of pro-inflammatory cytokines, but decreased the relative expression of anti-apoptosis protein and relative mRNA level and concentrations of anti-inflammatory factor. All these alterations were reversed with transfection of shATP2B1-AS1 into THP-1 cells. Moreover, ATP2B1-AS1 directly bound miR-23a-3p and negatively modulated the level of miR-23a-3p. Meanwhile, TLR4 was directly targeted by miR-23a-3p, and negatively and positively modulated by miR-23a-3p and ATP2B1-AS1, respectively. Conclusion: ATP2B1-AS1 aggravated apoptosis and inflammation by modulating miR-23a-3p/TLR4 axis in LPS-treated THP-1 cells (AU)

CHAC1 exacerbates LPS-induced ferroptosis and apoptosis in HK-2 cells by promoting oxidative stress

Background: Sepsis-induced acute kidney injury (AKI) is a singularly grievous and life-threatening syndrome. Its pathogenesis is closely related to inflammatory response, apoptosis, oxidative stress, and ferroptosis. Cation transport regulator-like protein 1 (CHAC1), as a proapoptic factor, may be involved in apoptosis, oxidative stress, and ferroptosis. This study aimed to explore the role of CHAC1 in the lipopolysaccharide (LPS)-induced the human renal proximal tubular epithelial (HK-2) cells. Methods: HK-2 cells were challenged with LPS to construct a model of sepsis-induced AKI in vitro. The role of CHAC1 in the LPS-induced HK-2 cells was explored using Western blot assay, cell counting kit-8 (CCK-8), flow cytometry, and colorimetric assays. Additionally, N-acetyl cysteine (NAC) was incubated with HK-2 cells to define deeply the relation between oxidative stress and apoptosis or ferroptosis. Results: The expression of CHAC1 was enhanced in the kidney tissues of mice with sepsis--induced multiple organ dysfunction syndrome (MODS), through the Gene Expression Omnibus database (GSE60088 microarray dataset), and in the LPS-induced HK-2 cells. The cell viability was significantly reduced by LPS treatment, which was at least partly restored by the transfection of siCHAC1#1 and siCHAC1#2 but not siNC. In addition, down-regulation of CHAC1 counteracted the LPS-induced reactive oxygen species level and malonaldehyde concentrations while restored the LPS-induced glutathione concentrations. Meanwhile, interference of CHAC1 neutralized LPS-induced apoptosis rate, and the relative level of cleaved poly(ADP-ribose) polymerase (PARP)/PARP, and cleaved caspase-3/caspase-3(AU)

NLRX1 increases human retinal pigment epithelial autophagy and reduces H2O2-induced oxidative stress and inflammation by suppressing FUNDC1 phosphorylation and NLRP3 activation

Background: Age-related macular degeneration (AMD) is a leading cause of impaired vision as well as some earlier effects, such as reading and face recognition. Oxidative damage and inflammation of retinal pigment epithelial (RPE) cells are major causes of AMD. Additionally, autophagy in RPE cells can lead to cellular homeostasis under oxidative stress. Nucleotide-binding oligomerization domain (NOD)-like receptor X1 (NLRX1) is a mysterious modulator of the immune system function which inhibits inflammatory response, attenuates reactive oxygen species (ROS) production, and regulates autophagy. This study attempted to explore the role of NLRX1 in oxidative stress, inflammation, and autophagy in AMD. Methods: An in vitro model of AMD was built in human retinal pigment epithelial cell line 19 (ARPE-19) treated with H2O2. The cell viability, NLRX1 expressions, levels of superoxide dismutase (SOD), glutathione (GHS), and ROS, concentrations of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, and monocyte chemoattractant protein-1 (MCP-1), expressions of NLRX1, p62, LC3-II/LC3-I, FUNDC1, and NOD-like receptor protein 3 (NLRP3) inflammasome were expounded by cell counting kit-8, colorimetric, enzyme-linked immunosorbent serologic assay (ELISA), and Western blot assay. Results: H2O2 treatment notably reduced the relative protein expression of NLRX1. Meanwhile, H2O2 incubation decreased cell viability, diminished SOD and GSH concentrations, accompanied with the increased level of ROS, enhanced IL-1β, TNF-α, IL-6, and MCP-1 concentrations, and aggrandized the relative protein expression of p62 with reduced LC3-II/LC3-I ratio. Moreover, these results were further promoted with knockdown of NLRX1 and reversed with overexpression. Mechanically, silencing of NLRX1 further observably enhanced the relative levels of -phosphorylated FUNDC1/FUNDC1 (AU)

Analysis of altered miRNA profiling in the colon of a mouse model with Beta-lactoglobulin allergy

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Coexpresión de NG2/GFAP tras la diferenciación en células transfectadas con las mutaciones de GFAP y en células procedentes de gliomas indiferenciados / NG2 and GFAP co-expression after differentiation in cells transfected with mutant GFAP and in undifferentiated glioma cells

INTRODUCCIÓN: La enfermedad de Alexander es una enfermedad rara causada por mutaciones en el gen que codifica la proteína glial ácida fibrilar (GFAP). En un estudio previo hemos observado que la diferenciación de neuroesferas transfectadas con estas mutaciones genera un tipo celular que comparte la expresión de GFAP y NG2. OBJETIVOS: Determinar el efecto de las mutaciones en marcadores moleculares en comparación con células de glioma diferenciados que expresan simultáneamente GFAP y NG2. MÉTODOS: Se utilizaron muestras de glioblastoma humana (GLM) y neuroesferas procedentes de rata transfectadas con mutaciones de GFAP para el análisis de la expresión tras diferenciación de GFAP y NG2, así como el análisis inmunocitoquímico de diferenciación de ambos tipos celulares y detección de ambas proteínas, junto a nestina, vimentina, Olig2 y caspasa 3 a los 3 y 7 días de diferenciación. RESULTADOS: Tanto las células transfectadas con mutaciones de GFAP como las células procedentes de GLM mostraron un incremento de NG2 y GFAP. Sin embargo, la expresión de células caspasa 3 positiva era marcadamente mayor entre las células transfectadas que entre las células procedentes de GLM. CONCLUSIÓN: Nuestros resultados parecen indicar que la expresión de GFAP no es el único factor que condiciona la muerte celular en la enfermedad de Alexander y que la expresión de caspasa 3 y el potencial papel de la NG2 en incrementar la resistencia a la apoptosis en las células que coexpresan GFAP y NG2 deben ser considerados en la búsqueda de acciones terapéuticas en esta enfermedad

INTRODUCTION: Alexander disease is a rare disorder caused by mutations in the gene coding for glial fibrillary acidic protein (GFAP). In a previous study, differentiation of neurospheres transfected with these mutations resulted in a cell type that expresses both GFAP and NG2. OBJECTIVE: To determine the effect of molecular marker mutations in comparison to undifferentiated glioma cells simultaneously expressing GFAP and NG2. METHODS: We used samples of human glioblastoma (GBM) and rat neurospheres transfected with GFAP mutations to analyse GFAP and NG2 expression after differentiation. We also performed an immunocytochemical analysis of neuronal differentiation for both cell types and detection of GFAP, NG2, vimentin, Olig2, and aspase-3 at 3 and 7 days from differentiation. RESULTS. Both the cells transfected with GFAP mutations and GBM cells showed increased NG2 and GFAP expression. However, expression of caspase-3-positive cells was found to be considerably higher in transfected cells than in GBM cells. CONCLUSIONS: Our results suggest that GFAP expression is not the only factor associated with cell death in Alexander disease. Caspase-3 expression and the potential role of NG2 in increasing resistance to apoptosis in cells co-expressing GFAP and NG2 should be considered in the search for new therapeutic strategies for the disease

La expresividad variable del síndrome de QT largo de una familia española se explica por la heterocigosis digénica en SCN5A y CACNA1C / Digenic heterozigosity in SCN5A and CACNA1C explains the variable expressivity of the long QT phenotype in a Spanish family

Introducción y objetivos: En 4 miembros de una familia española se identificó una mutación en los canales cardiacos Nav1.5 (p.R1644H) descrita ya y relacionada con el síndrome de QT largo con anterioridad. Sin embargo, solo 1 de los portadores presentaba el intervalo QT prolongado. En los otros 3 individuos se identificó una nueva mutación con cambio de sentido en los canales cardiacos Cav1.2 (p.S1961N). En este trabajo se analizaron las características funcionales de los canales p.S1961N Cav1.2 para averiguar si dicha mutación regula la expresividad del síndrome de QT largo en esta familia. Métodos: La corriente de calcio tipo L (ICaL) se registró mediante la técnica de patch-clamp en células de ovario de hámster chino transfectadas transitoriamente con los canales cardiacos humanos en su forma nativa o mutada. Resultados: La expresión de canales p.S1961N disminuye significativamente la densidad de la ICaL. Al sustituir el ion calcio por bario para suprimir la inactivación dependiente del calcio de los canales Cav1.2, se demostró que la mutación acelera significativamente la inactivación dependiente del voltaje de los canales Cav1.2 y disminuye la constante de tiempo de inactivación. Como consecuencia, la carga total que atraviesa los canales p.S1961N Cav1.2 disminuye significativamente. Los efectos que las mutaciones p.S1961N Cav1.2 y p.R1644H Nav1.5, por separado o en combinación, producen sobre las características de los potenciales de acción (PA) se simularon mediante un modelo matemático de PA ventriculares humanos. Los resultados demuestran que la mutación p.S1961N Cav1.2 abrevia la duración del PA y suprime la prolongación inducida por la mutación p.R1644H de los canales Nav1.5. Conclusiones: La mutación p.S1961N en los canales Cav1.2 disminuye la ICaL, un efecto que podría abreviar la duración de los PA ventriculares humanos. La presencia de esta mutación que disminuye la función de los canales Cav1.2 compensa funcionalmente los efectos producidos por la mutación de los canales Nav1.5 que aumenta su función y prolonga la duración de los PA

Introduction and objectives: A known long QT syndrome-related mutation in Nav1.5 cardiac channels (p.R1644H) was found in 4 members of a Spanish family but only 1 of them showed prolongation of the QT interval. In the other 3 relatives, a novel missense mutation in Cav1.2 cardiac channels was found (p.S1961N). Here, we functionally analyzed p.S1961N Cav1.2 channels to elucidate whether this mutation regulates the expressivity of the long QT syndrome phenotype in this family. Methods: L-type calcium current (ICaL) recordings were performed by using the whole-cell patch-clamp technique in Chinese hamster ovary cells transiently transfected with native and/or p.S1961N Cav1.2 channels. Results: Expression of p.S1961N channels significantly decreased ICaL density. Using Ba as a charge carrier to suppress the Ca-dependent inactivation of Cav1.2 channels, we demonstrated that the mutation significantly accelerates the voltage-dependent inactivation of Cav1.2 channels decreasing the inactivation time constant. As a consequence, the total charge flowing through p.S1961N Cav1.2 channels significantly decreased. The effects of the p.S1961N Cav1.2 and p.R1644H Nav1.5 mutations alone or their combination on the action potential (AP) morphology were simulated using a validated model of the human ventricular AP. The p.S1961N Cav1.2 mutation shortens the AP duration and abrogates the prolongation induced by p.R1644H Nav1.5 channels. Conclusions: The p.S1961N mutation in Cav1.2 channels decreased the ICaL, an effect which might shorten ventricular AP. The presence of the loss-of-function Cav1.2 mutation could functionally compensate the prolonging effects produced by the Nav1.5 gain-of-function mutation

Emergence of CTNNB1 mutation at acquired resistance to KIT inhibitor in metastatic melanoma

Purpose The KIT inhibitor, imatinib, has shown promising efficacy in patients with KIT-mutated melanoma; however, acquisition of resistance to imatinib occurs rapidly in the majority of patients. The mechanisms of acquired resistance to imatinib in melanoma remain unclear. Methods We analyzed biopsy samples from paired baseline and post-treatment tumor lesions in one patient with KIT-mutated melanoma who had had an initial objective tumor regression in response to imatinib treatment followed by disease progression 8 months later. Results Targeted deep sequencing from post-treatment biopsy samples detected an additional mutation in CTNNB1 (S33C) with original KIT L576P mutation. We examined the functional role of the additional CTNNB1 S33C mutation in resistance to imatinib indirectly using the Ba/F3 cell model. Ba/F3 cell lines transfected with both the L576P KIT mutation and the CTNNB1 S33C mutation demonstrated no growth inhibition despite imatinib treatment, whereas growth inhibition was observed in the Ba/F3 cell line transfected with the L576 KIT mutation alone. Conclusions We report the first identification of the emergence of a CTNNB1 mutation that can confer acquired resistance to imatinib. Further investigation into the causes of acquired resistance to imatinib will be essential to improve the prognosis for patients with KIT mutated melanoma (AU)

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MicroRNA-9 promotes the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1

Purpose. The objective of the study was to investigate the role of microRNA-9 (miR-9) targeting forkhead box O1 (FOXO1) in the proliferation, migration, and invasion of breast cancer cells. Methods. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expressions of miR-9 and FOXO1 mRNA in breast cancer tissues, normal breast tissues, breast cancer cell lines, and normal breast epithelial cells. After the up-regulation of miR-9 expression, qRT-PCR and Western blotting were used to determine the expression of FOXO1. The luciferase reporter gene assay was used to validate the target gene. The CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay were used to investigate the changes in the proliferation, migration, and invasion of breast cancer cells, respectively. Results. MicroRNA-9 expression was significantly up-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). FOXO1 mRNA and protein expressions were substantially down-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). There can be a negative correlation between miR-9 and FOXO1 mRNA in breast cancer. Luciferase reporter gene assay indicated that miR-9 can down-regulate FOXO1 expression at a post-transcriptional level through binding specifically to FOXO1 3′UTR. The results of CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay revealed that the inhibition of miR-9 can suppress MCF7 cell proliferation, migration, and invasion. Additionally, the expression of miR-9 increased significantly whilst that of FOXO1 decreased substantially as the disease progressed (P < 0.05). Conclusions. Our study provides evidence that miR-9 can promote the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1 (AU)

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A long noncoding RNA UCA1 promotes proliferation and predicts poor prognosis in glioma

Background. Acting as a proto-oncogene, long noncoding RNAs (lncRNAs) urothelial carcinoembryonic antigen 1 (UCA1) plays a key role in the occurrence and development of several human tumors. However, the expression and biological functions of UCA1 in glioma are less known. This study discussed the expression of UCA1 in glioma and its effect on the proliferation and cell cycle of glioma cells. Method. LncRNA UCA1 expressions in 64 glioma samples (Grade I-II in 22 cases and Grade III-IV in 42 cases, according to WHO criteria) and 10 normal brain samples were detected using real-time fluorescence quantitative PCR. On this basis, the correlations of UCA1 to clinicopathological characteristics and prognosis of glioma were assessed. Then, using qPCR, the lncRNA UCA1 expressions in glioma cell lines and astrocytes were detected. UCA1-overexpressing glioma cell lines U87 and U251 were further detected after siRNA transfection of these two cell lines, and the impact on cell proliferation and cell cycle was assessed with CCK-8 (cell counting kit-8) assay and flow cytometry method (FCM), respectively. The expression of cyclin D1, a cell cycle-related protein, was detected using Western Blot. Result. LncRNA UCA1 expression in the glioma samples was obviously higher as compared with the normal brain samples (P < 0.001), and the expression was correlated significantly with grading of the tumors (P < 0.05). However, lncRNA UCA1 expression was not correlated with age, gender, tumor size and KPS score (P > 0.05). After interference of UCA1 expression by siRNA transfection, the proliferation of both U251 and SHG-44 cells was inhibited (P < 0.05), with more cells arrested in G0/G1 (P < 0.05). Moreover, cyclin D1 expression was also downregulated considerably. Conclusion. LncRNA UCA1 can promote the proliferation and cell cycle progression of glioma cells by upregulating cyclin D1 transcription. So UCA1 may serve as an independent prognostic indicator and a novel therapeutic target for glioma (AU)

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High expression of long non-coding RNA ATB indicates a poor prognosis and regulates cell proliferation and metastasis in non-small cell lung cancer

Background and aim. Long non-coding RNAs (lncRNAs) have been demonstrated to act as a critical regulator in the processes of tumor biology. In this study, whether lncRNA-ATB is a potential indicator for non-small cell lung cancer (NSCLC) was investigated and its biological function in NSCLC was also determined. Methods. The expression levels of lncRNA-ATB in NSCLC tissues and cell lines were measured. A549 cell line was explored to investigate the functions of lncRNA-ATB in NSCLC. Results. Real-time PCR results showed that lncRNA-ATB expression was up-regulated in both in NSCLC tissues and cell lines. High lncRNA-ATB expression in tumor tissue was associated with larger tumor size, lymph node metastasis, and distant metastasis in patients with NSCLC, respectively. In addition, the patients with high expression of lncRNA-ATB presented a lower survival probability. In vitro experiments showed that down-regulation of lncRNA-ATB promoted the cell apoptosis, whereas inhibited the cell viability, cell migration, and cell invasion. Conclusion. High expression of lncRNA-ATB indicated a poor prognosis and led to the cell proliferation and metastasis in NSCLC (AU)

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Nanopartículas sólidas lipídicas para terapia génica / Solid lipid nanoparticles for gene therapy

La terapia génica consiste en la administración de ácidos nucleicos con el fin de modular la expresión de proteínas específicas y revertir así una enfermedad. Es una nueva área de la medicina con gran potencial para el tratamiento de enfermedades tanto hereditarias como adquiridas. Hasta ahora, los sistemas virales de administración de ácidos nucleicos han resultado eficaces, pero presentan importantes problemas de seguridad. Los vectores no virales, en cambio, son más seguros pero menos eficaces, aunque su eficacia ha aumentado significativamente en los últimos años. Esta revisión recoge la contribución de nuestro grupo de investigación al diseño de vectores no virales basados en nanopartículas sólidas lipídicas (SLNs) para terapia génica. Hemos estudiado la relación entre factores de la formulación con los procesos de internalización y disposición intracelular del material genético, que condicionan la eficacia de transfección, y por primera vez demostramos la capacidad de las SLNs para inducir la síntesis de una proteína tras su administración endovenosa a ratones. Esta revisión también recoge nuestros trabajos sobre la aplicación de las SLNs en el tratamiento de enfermedades infecciosas y enfermedades raras, como la retinosquisis juvenil ligada al cromosoma X, enfermedad en la que la retina está desestructurada debido a la deficiencia de la proteína retinosquisina. La administración de SLNs con el gen que codifica esta proteína en un modelo animal de esta enfermedad indujo la recuperación estructural de la retina. Los trabajos aquí recogidos muestran el gran potencial de las SLNs como sistemas de administración de ácidos nucleicos (AU)

Gene therapy is a rapidly advancing field with great potential for the treatment of genetic and acquired systemic diseases. This therapy requires the introduction of foreign genetic material in the target cells to modify a genetic sequence. Viral vectors are the most effective, but their application is limited by their immunogenicity, oncogenicity and the small size of the DNA they can transport. Non-viral vectors, however, are safer, lowered cost, and more reproducible and do not present DNA size limit. The main problem of nonviral systems is their low transfection efficiency, although it has improved during the last years. This review presents the contribution of our research group to the design and evaluation of SLNs based non-viral vectors for gene transfer. We report our studies about the relationship between formulation factors and cell uptake and intracellular trafficking of the genetic material, very important for transfection. We have shown, for the first time, the ability to induce transgene protein expression of the SLNs after endovenous administration to mice. This revision also reports our work about the potential application of SLNs to the treatment of infectious and rare diseases, as the X-linked juvenile retinoschisis, a retinal disorder due to a deficiency in the protein retinoschisin, and characterized by poor eyesight and degeneration of the retina. The intraocular injection of SLNs bearing the gene that encodes retinoschisin to diseased mice led to the partial recovery of the retina. All together, our results show the potential of SLNs as a system for gene delivery (AU)

miRNA involvement in angiogenesis in age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Late-stage AMD is characterized by choroidal neovascularization (CNV). miR-93 appears to play a role in regulating vascular endothelial growth factor-A (VEGF-A), a known factor involved in neovascularization. Understanding its biological significance might enable development of therapeutic interventions for diseases like AMD. We aimed to determine the role of miR-93 in AMD using a laser-induced CNV mouse model. CNV was induced by laser photocoagulation in C57BL/6 mice. The CNV mice were transfected with scrambled miR or miR-93 mimic. The treatment effect was assessed by fundus photography and fluorescein angiography and confirmed by choroidal flatmount. The expression of miR-93 and VEGF-A in ocular tissues was analysed by quantitative polymerase chain reaction (qPCR) and Western blot. The overexpression effects of miR-93 were also proved on human microvascular endothelial cells (HMECs). Significantly decreased expression of miR-93 was observed by qPCR analysis in CNV mice compared to untreated mice (p < 0.05). VEGF-A messenger RNA (mRNA) and protein expression were upregulated with CNV; these changes were ameliorated by restoration of miR-93 (p < 0.05). CNV was reduced after miR-93 transfection. Transfection of miR-93 reduced the proliferation of HMECs (p < 0.01), but no significant changes were observed in 2D capillary-like tube formation (p > 0.05) and migration (p > 0.05) compared with that in the untreated cells. miR-93 has been shown to be a negative modulator of angiogenesis in the eye. All together, these results highlight the therapeutic potential of miR-93 and suggest that it may contribute as a putative therapeutic target for AMD in humans (AU)

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Estudio funcional de los polimorfismos del promotor del gen de la esclerostina / Functional study of promoter gene polymorphisms of sclerostin

La esclerostina, codificada por el gen SOST, es un inhibidor de la vía Wnt, y por ello tiene una influencia negativa sobre la masa ósea. Algunos polimorfismos del promotor de SOST se han asociado a diferencias en la densidad mineral ósea (DMO) en varios estudios, pero se desconocen cuáles son los mecanismos moleculares implicados. El objetivo de este estudio fue comprobar las consecuencias funcionales de uno de esos polimorfismos in vitro . Para ello se clonó la región promotora proximal del gen SOST, con diferentes alelos del polimorfismo rs851054, y se transfectaron en células HEK-293T, SAOS-2 y HOSTE85. En ningún caso se observaron diferencias significativas en la actividad transcripcional entre los vectores con el alelo A y los vectores con el alelo G. La co-transfección de vectores de expresión de los factores de transcripción RUNX2 y OSX estimuló claramente la actividad transcripcional (2,5±0,9 veces sobre el valor basal para el alelo A y 1,9±0,8 veces para el alelo G; en ambos casos, p<0,05), sin que hubiera, sin embargo, diferencias entre los alelos. Tampoco se hallaron diferencias en la fijación a proteínas nucleares analizadas en experimentos de retardo de la movilidad electroforética. En conclusión, la región situada antes del inicio de la traducción del gen SOST tiene una potente actividad promotora, que es aumentada por los factores RUNX2 y OSX. Las variantes frecuentes de esta región se han asociado con la DMO, pero los mecanismos implicados son aún desconocidos, puesto que los alelos analizados no muestran diferencias en la actividad transcripcional in vitro (AU)

Sclerostin, encoded by the SOST gene, inhibits the Wnt pathway and, consequently, tends to decrease bone mass. Some polymorphisms of the SOST promoter have been associated with bone mineral density (BMD), but the molecular mechanisms involved are unknown. The aim of this study was to study the functional role of one polymorphism in vitro. We cloned the proximal promoter region of SOST gene, containing different alleles at the rs851054 SNP, in luciferase reporter vectors and transfected them into the cell lines HEK-293T, SAOS-2 and HOS-TE85. We did not find significant differences in the transcriptional activity of vectors with either the A or the G allele of the SNP. The co-transfection of vectors expressing RUNX2 and OSX markedly increased the transcriptional activity of the SOST promoter constructs (A allele, 2.5±0.9 fold, p<0.05; G allele, 1.9±0.8 fold, p<0.05), without significant differences between the rs851054 alleles. Moreover, no allele differences were detected in EMSAs. In conclusion, the DNA region upstream of the TSS of the SOST gene has a strong promoter activity that is enhanced by RUNX2 and OSX. Frequent allelic variants in this region have been associated with BMD, but the mechanisms involved remain to be elucidated because no functional differences between alleles were detected in vitro (AU)

MicroRNA-197 influences 5-fluorouracil resistance via thymidylate synthase in colorectal cancer

Purpose. The response rate of first-line fluoropyrimidine-based regimens for metastatic colorectal cancer (mCRC) is generally less than 50 %. The down-regulation of miR-197 in colorectal cancer cells after exposure to 5-fluorouracil might be related to the mechanism of resistance to fluoropyrimidine-based chemotherapy. So we investigated the regulatory mechanism of miR-197 on 5-FU sensitivity. Methods. Dual luciferase reporter gene construct and dual luciferase reporter assay were used to identify the target of miR-197. TYMS expression was evaluated by immunohistochemistry staining. 5-Fu resistance of colorectal cancer cell lines was detected by MTS assay. The expression of miR-197 was detected by real time PCR. Results. A luciferase assay and western blot analysis confirmed that miR-197 directly binds to and negatively regulates TYMS expression. Overexpressing miR-197 could increase the sensitivity of colorectal cancer cells to 5-fluorouracil (5-FU). The expression of miR-197 negatively correlated with TYMS expression in cancerous tissues from patients with stage IV colorectal cancer. Conclusion. miR-197 mediates the response of colorectal cancer cells to 5-FU by regulating TYMS expression (AU)

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Sodium/iodide symporter gene transfection increases radionuclide uptake in human cisplatin-resistant lung cancer cells

The sodium/iodide symporter (NIS) is involved in iodide uptake and has been used for the diagnosis and treatment of thyroid cancer. Transfection of the NIS gene in A549 human lung cancer cells can induce radioactive iodine (131I) and radioactive technetium (99mTc) uptake. The aim of the present study was to assess the role of NIS in 99mTc and 131I uptake by the A549/DDP human cisplatin-resistant lung cancer cell line. To do so, recombinant adenovirus, adenovirus-enhanced green fluorescent protein-human NIS (Ad-eGFP-hNIS) and Ad-eGFP-rat NIS (Ad-eGFP-rNIS) vectors were established. These vectors were transfected into A549/DDP cells and xenograft tumors in nude mice. Assessment of 99mTc and 131I uptake was performed. Results showed that the transfection efficiency of Ad-eGFP-hNIS and Ad-eGFP-rNIS in A549/DDP cells was at least 90 % in all experiments, and that the uptake ability of 99mTc and 131I was highly enhanced (14–18 folds for 99mTc, and 12–16 folds for 131I). However, the radionuclide concentration in transfected NIS genes’ A549/DDP cells reached a plateau within 30–60 min, indicating that NIS transport led rapidly to 99mTc and 131I saturation in cells. In xenograft tumor models, uptake of 99mTcO4 − was obviously higher in the hNIS and rNIS groups compared with controls. In conclusion, these results support the hypothesis that A549/DDP cells can effectively uptake 99mTc and 131I when transfected with the hNIS and rNIS gene. The rNIS or hNIS gene could be used as an effective method for the effective delivery of radioactive products to specific tissues for imagery and/or treatment (AU)

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Demografía, características clínicas y genéticas de pacientes con carcinoma medular de tiroides en los últimos 16 años en Castilla-La Mancha / Demographic, clinical, and genetic characteristics of patients with medullary thyroid cancer in the past 16 years in Castilla-La Mancha

OBJETIVO: El carcinoma medular de tiroides es un tumor de baja prevalencia cuyo pronóstico es peor que el del cáncer diferenciado de tiroides debido su mayor agresividad. El objetivo de este trabajo es describir las características demográficas, clínicas y genéticas de los pacientes atendidos en el área sanitaria de la Comunidad de Castilla-La Mancha durante 16 años. PACIENTES Y MÉTODOS: Los datos se recogieron mediante revisión de historias clínicas. RESULTADOS: Se revisaron las historias clínicas de 58 pacientes con una edad media al diagnóstico de 51 años (intervalo de 6 a 82 años) y un 63,8% de mujeres. La prevalencia fue de 2,84 casos por 100.000 habitantes, con una gran variabilidad entre áreas (de 0 a 5,4 casos por 100.000 habitantes). Los casos familiares representaron el 34,5% del total, siendo la mutación más frecuente la C634Y. El motivo más frecuente de diagnóstico fue la palpación de un bultoma cervical (70,6%); se solicitó ecografía al diagnóstico en 56 de 58 casos, y la calcitonina en 8 de 58 casos. La multicentricidad del tumor fue descrita en el 59 y 50% de los casos de síndrome de neoplasia endocrina múltiple tipo 2A y 2B, respectivamente, y en ningún caso esporádico. El 52% de los pacientes presentaba un estadio avanzado al diagnóstico (III o IV). La mediana de seguimiento fue de 36 meses (rango intercuartílico 14-210), con la pérdida de 11 pacientes durante el seguimiento. CONCLUSIONES: El diagnóstico de carcinoma medular de tiroides en Castilla-La Mancha se basa en la ecografía cervical, pero no en la calcitonina. Existe una alta prevalencia de este carcinoma, tanto familiar como esporádico, y una importante variabilidad en el tipo de mutación del protooncogén rearranged during transfection comparadas con las del resto de la población española

OBJECTIVE: Medullary thyroid cancer is a rare tumor that is more aggressive and has a worse prognosis than differentiated thyroid cancer. The purpose of this study was to report the demographic, clinical, and genetic characteristics of patients seen in the health care system of the community of Castilla-La Mancha over a 16-year period. PATIENTS AND METHODS: Data were collected through a review of patients' medical records. RESULTS: The medical records of 58 patients (mean age at diagnosis, 51 years; range, 6-82 years; 63.8% women) were reviewed. Prevalence rate was 2.84 cases per 100,000 inhabitants, with a high variability between areas (range, 0-5.4 cases per 100,000 inhabitants). Familial cases accounted for 34.5% of all medullary thyroid cancers, and the most common mutation was C634Y. The condition was most commonly diagnosed following palpation of a cervical lump (70.6%). At diagnosis, 56 of 58 patients underwent ultrasound and 8 of 58 patients were tested for serum calcitonin. Tumor multicentricity was reported in 59 and 50% of patients with multiple endocrine neoplasia syndrome type 2A and 2B, respectively, and in no sporadic cases. Fifty-two percent of patients had an advanced stage (III or IV) at diagnosis. Median follow-up was 36 months (interquartile range, 14-210); 11 patients were lost to follow-up. CONCLUSIONS: In Castilla-La Mancha, medullary thyroid cancer is diagnosed by cervical ultrasound, rather than calcitonin assay. There is a high prevalence of both familial and sporadic medullary thyroid cancer, and a significant variability in the type of proto-oncogen rearranged during transfection mutation as compared to the rest of the Spanish population

Expression of grape class IV chitinase in Spodoptera frugiperda (Sf9) insect cells

INTRODUCTION: Most of pathogenesis related (PR) proteins possess complicated structures; hence their active recombinant forms are usually produced in eukaryotic systems. In this study, we employed an insect cell line to express a recombinant form of a previously identified grape PR3 allergen categorised as class IV chitinase. METHODS: Grape chitinase cDNA was amplified by RT-PCR and inserted into pFastBacHTA using restriction enzymes. The recombinant pFastBacHTA was applied for the transformation of Escherichia coli DH10Bac cells. The purified recombinant bacmid was used for transfection of Sf9 cells. Finally, the IgE-immunoreactivity of purified recombinant protein was evaluated using grape allergic patient's sera. Moreover, polyclonal anti-6His-tag and monoclonal anti-chitinase antibodies were used for further assessment of recombinant protein. RESULTS: SDS-PAGE analysis of the transfected Sf9 cells showed expression of a monomeric 25 kDa and a dimeric 50 kDa recombinant protein. Western blotting revealed considerable IgE reactivity of the recombinant protein with grape allergic patients' sera. Furthermore, confirmatory assays showed specific reactivity of the recombinant protein with anti-His tag and anti-chitinase antibodies. CONCLUSION: This study showed that, in contrast to E. coli, insect cells are suitable hosts for the production of a soluble and IgE-reactive recombinant form of grape class IV chitinase. This recombinant allergen could be used for component resolved diagnosis of grape allergy or other immunodiagnostic purposes

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Alternate PAX3 and PAX7 C-terminal isoforms in myogenic differentiation and sarcomagenesis

OBJECTIVE: Pax3 and Pax7 are closely related genes that are involved in commitment of cells to a myogenic lineage during skeletal muscle development and regeneration. Several Pax3 and Pax7 transcripts are expressed from the genes, generating different isoforms with potentially distinct DNA binding and transactivation properties. The aim of this study was to investigate the implication of Pax3 and Pax7 C-terminal isoforms during myogenic differentiation and tumorigenesis, since fusions involving these genes are commonly associated with alveolar rhabdomyosarcoma (ARMS). METHODS: Uncommitted (mouse mesenchymal stem cells, MSCs) and committed (C2C12) myogenic precursor cells were stably transfected with PAX3/FKHR and PAXC7/ FKHR fusion genes. We analysed gene and protein expression comparing the newly generated cells with the parental cells, to determine the functional importance of Pax3 and Pax7 C-terminal isoforms. RESULTS: We found that the transcript Pax3c was expressed at low levels in undifferentiated C2C12 and MSCs cells, but its expression levels increased considerably at later stages of differentiation. However, expression levels of Pax3d transcript increased only slightly after differentiation. Pax7 transcripts, present before differentiation in committed C2C12 cells, but absent in uncommitted MSCs, increased noticeably in MSCs after differentiation. We also found that the presence of PAX/FKHR fusions prevented both C2C12 and MSC cells from terminal myogenic differentiation and increased the expression of discrete endogenous Pax3/7 transcripts, in particular Pax3d and Pax7B. CONCLUSIONS: Our results suggest that both Pax3 and Pax7 transcripts are required for commitment of cells to the myogenic lineage, with each transcript having a distinct role. More specifically, the Pax3c isoform may be required for terminal myogenic differentiation whereas the Pax3d isoform may be involved in undifferentiated cell maintenance and/or proliferation (AU)

TSC1-TSC2 complex on the crossroad of pancreatic β cell signaling. Role on cell proliferation, death and survival / Papel del complejo TSC1-TSC2 en la señalización de la célula β pancreática: prolifreración, muerte y supervivencia celular

TSC1-TSC2 complex has emerged as a signal interaction core within the cell. This complex integrates both nutrient and growth factor signaling and is a critical negative regulator of mTORC1.mTORC1 signaling leads to increased protein biosynthesis, which is essential for cell proliferation. Other cellular events such as endoplasmic reticulum stress and autophagy are intimately linked with TSC/mTORC1 pathway and play an important role in pancreatic βcell death or survival. We found that either insulin, glucose independent signaling or the energetic status of the cell are able to modulateTSC2 phosphorylation in pancreatic β cell lines. To show the central role of TSC2 for these cells, we conducted siRNA-mediatedTSC2 silencing. Downregulation of TSC2 leads to an increase inmTORC1/p70S6K signaling, this produces resistance to insulin action. However, specific expression of insulin receptor isoform A restored insulin signalling under these conditions. Moreover, we have explored other processes related to the TSC/mTORC1 pathway andtheir effect on cell death or survival (AU)

El complejo TSC1-TSC2 ha emergido como un núcleo de integración de la señalización de factores de crecimiento y del estado energético celular. Este complejo funciona como un regulador negativo de la actividad de mTORC1. La señalización a través de mTORC1 dirige la síntesis proteica, esencial para la proliferación celular. Otros procesos como el estrés de retículo y la autofagia están íntimamente ligados a la ruta TSC/mTORC1 y juegan un importante papel en la muerte o supervivencia de las células β pancreáticas. Encontramos que tanto la insulina, como la acción independiente de la glucosa o el estatus energético celular modulan la fosforilación de TSC2 en líneas celulares β pancreáticas. Para demostrar el papel central de TSC2 en la regulación de estas células, hemos silenciado su expresión mediante transfección con ARNi. Una menor expresión de TSC2 lleva a un aumento de la señalización demTORC1/p70S6K, lo cual produjo un estado de resistencia a la acción de la insulina. Sin embargo, la expresión selectiva de la isoforma A del receptor de insulina consigue salvar esta resistencia. Por otro lado, hemos explorado otros procesos modulados por la ruta como la autofagiao el estrés de retículo, y su efecto en la supervivencia o muerte celular (AU)

Diagnóstico y terapia del cáncer mediante magnetosomas / Cancer therapy and diagnosis by magnetosomes

Los magnetosomas (magnetoliposomas) han permitido en los últimos años un gran avance en la terapia del cáncer, gracias a su capacidad para transportar de forma eficiente diferentes agentes quimioterápicos hasta la masa tumoral. Estos sistemas no sólo se usan en el transporte de fármacos sino que también presentan importantes posibilidades en el diagnóstico de esta severa enfermedad, en terapia génica (transfección magnética o magnetofección) y en hipertermia. Sin embargo, su destino biológico, una vez que son administrados, se encuentra fuertemente condicionado por su geometría y propiedades fisicoquímicas. El presente trabajo se centra en la investigación de los principales aspectos relacionados con el diseño y utilización clínica de los magnetosomas. En concreto, estrategias de formulación, biocompatibilidad, toxicidad y situación actual a nivel preclínico(AU)

During the last years, magnetosomes (magnetoliposomes) have contributed to a significant improvement in cancer therapy, thanks to their capability to transport very efficiently the antitumor drug dose to the malignant cells. Interestingly, these systems also offer very promising possibilities in cancer diagnosis, gene therapy (magnetic transfection or magnetofection), and hyperthermia. However, the biological fate of magnetosomes, upon administration, is markedly conditioned by their geometry and physicochemical properties. This work is focussed on the investigation of the main aspects related to the engineering and clinical use of liposomes. Concretely, formulation strategies, biocompatibility, toxicity, and current state of the art at the preclinical level(AU)