Long noncoding RNA HOTAIR promotes breast cancer development through the lncRNA HOTAIR/miR-1/GOLPH3 axis

Background The lncRNA HOTAIR is frequently overexpressed in breast cancer tissues and plays an important role in the development of breast cancer. Here, we investigated the effect of the lncRNA HOTAIR on the biological behaviour of breast cancer cells and its molecular mechanism. Methods We investigated the level of HOTAIR in breast cancer and its clinical pathological characteristics by bioinformatic methods. Then, we evaluated the effects of HOTAIR and miRNA-1 expression on the biological behaviour of breast cancer cells by qPCR, Cell Counting Kit-8 (CCK-8) assay, clonogenic assays, Transwell assay and flow cytometry for cell proliferation, invasion migration and apoptosis, and cell cycle analysis. Finally, the target genes of the lncRNA HOTAIR/miR-1/GOLPH3 regulatory axis were validated by luciferase reports. Results The expression of HOTAIR in breast cancer tissues was significantly higher than that in normal breast tissues (P < 0.05). Silencing of HOTAIR suppressed cell proliferation, invasion and migration, promoted apoptosis and induced G1 phase block in breast cancer (P < 0.0001). We also verified that miR-1 is a target of HOTAIR and that GOLPH3 is a target of miR-1 by luciferase reporter assays (P < 0.001). Conclusions The expression of HOTAIR was significantly elevated in breast cancer tissues. Reducing the expression of HOTAIR inhibited the proliferation, invasion and migration of breast cancer cells and promoted apoptosis, and the mechanism was mainly the effect of the lncRNA HOTAIR/miR-1/GOLPH3 regulatory axis on the biological behaviour of breast cancer cells (AU)

Hypoxia promotes epithelial mesenchymal transition in lung cancer cells via regulating the MIR271 BUB1 pathway

Purpose Lung cancer (LC) is the most common malignancy in the world. It is well that hypoxia is common in lung cancer, which contributes to lung cancer progression and metastasis [1]. miRNA-27a as a repressor factor is a lowly expression within non-small cell lung cancer (NSCLC). However, the molecular mechanism between miR-27a and hypoxia in lung cancer progression remains poorly understood. This study aims to explore hypoxia promotes epithelial-mesenchymal transition in lung cancer cells via regulating the NRF2/miR‑27a/BUB1 pathway. Methods We detect the expression of miR-27a after exposure to hypoxia conditions in lung cancer cells via qPCR. Using MTT assay and colony assay to assess the ability of proliferation in lung cancer cells under hypoxia or transfect miR-27a mimics. The capability of migration and invasion was evaluated by wound healing assay and Boyden-chamber assay. The mRNA and protein expression of EMT markers was respectively detected by qPCR and western blot. We detected NRF2 occupancy at the miR-27a promoter by ChIP-Seq analysis. Meanwhile, the luciferase assay verified BUB1 as a direct target of miR-27a. Results We found hypoxia promotes lung cancer cell proliferation, migration, invasion, and the epithelial-mesenchymal transition (EMT) process by inhibiting the miR-27a expression. miR-27a mimics significantly reduced the promotion effect of hypoxia on the invasion and proliferation of lung cancer cells. NRF2 as regulating the oxidation/anti-oxidation factor was activated under hypoxia conditions. The activation of NRF2 repressed miR-27a expression. On the contrary, the inhibitory effect of hypoxia on miR-27a was reversed when the NFE2L2 gene was silenced. Ectopic expression of NRF2 inhibited miR-27a expression under normoxia. We further validated BUB1 as a direct target of the miR-27a by luciferase assay. Conclusion Hypoxia promotes invasion and epithelial-mesenchymal transition of Lung cancer cells by regulating the NRF2/miR-27a/BUB1 axis (AU)

Long non-coding RNA PVT1 facilitates cell migration and invasion by regulating miR-148a-3p and ROCK1 in breast cancer

PurposeBreast cancer (BC) is one of the most common malignant tumors for women. The role and potential mechanisms of long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) were explored in BC cell migration and invasion.MethodsPVT1, miR-148a-3p and Rho‑associated, coiled‑coil containing protein kinase 1 (ROCK1) mRNA expressions were detected using real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The ROCK1 protein expression was detected by Western blotting. The relationship of PVT1, miR-148a-3p and ROCK1 was analyzed by Dual Luciferase activity, RNA immunoprecipitation (RIP) and Spearman correlation analysis. Cell invasion and migration were detected by Transwell assay.ResultsUpregulation of PVT1 and ROCK1, and downregulation of miR-148a-3p were observed in BC tissues and cell lines. According to the analysis of Dual Luciferase activity, RIP and Spearman correlation analysis, miR-148a-3p directly binds to PVT1, and ROCK1 is a target of miR-148a-3p. In addition, PVT1 regulated the cells migration and invasion by regulating miR-148a-3p and ROCK1 expression.ConclusionThese data demonstrated that PVT1 was upregulated and facilitated to the cell migration and invasion of BC by the regulation of miR-148a-3p and ROCK1, indicating that PVT1 may be a potential biomarker of BC diagnosis and treatment.

MicroRNA-802 promotes the progression of osteosarcoma through targeting p27 and activating PI3K/AKT pathway

PurposeIncreasing evidences suggest dysfunctions of microRNAs (miRNAs) are playing important part in tumors. Therefore, the role of miR-802 in osteosarcoma (OS) was exploited. The object was to evaluate the effect of miR-802 and verify its influence on p27 Kip1 (p27) in OS.MethodsRT-qPCR experiment was used to detect miR-802 and p27 expression in OS tissues and cells. We explored the function of miR-802 through Transwell assays. The phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase pathway and epithelial–mesenchymal transition (EMT) was detected by Western blot assays. Luciferase assay was used to testify the target of miR-802.ResultsMiR-802 expression was elevated in OS, which was related to poor clinical outcome in OS patients. MiR-802 overexpression promoted OS migration, invasion and EMT. Further, p27 is a direct target of miR-802. P27 elevation counteracted the promotion effect of OS on EMT, migration and invasion induced by miR-802. In addition, miR-802 overexpression inactivated PI3K/AKT pathway via targeting p27 in OS.ConclusionMiR-802 promoted the progress of EMT, migration and invasion in OS via targeting p27. This newly identified miR-802/p27/PI3K/AKT axis may represent potential targets for OS.

miR-22 suppresses cell viability and EMT of ovarian cancer cells via NLRP3 and inhibits PI3K/AKT signaling pathway

Purpose miR-22 plays a great role in inhibiting cell growth, metastasis and enhanced cell apoptosis in several cancers. The purpose of this study was to investigate the functions of miR-22 in ovarian cancer. Methods The proliferative ability was measured using CCK-8 assay. The protein expression associated with EMT and PI3K/AKT signaling biomarkers were measured by western blot. Luciferase assay applied to measure the luciferase activity. Kaplan–Meier method was performed to evaluate the overall survival rate of ovarian cancers. Results miR-22 was low expressed and NLRP3 was overexpressed in ovarian cancer tissues and cells, and downregulation of miR-22 was associated with poor prognosis. The expression of NLRP3 had a negative correlation with miR-22 expression in ovarian cancer. miR-22 promoted cell viability and EMT through directly binding to the 3′-UTR of NLRP3 mRNA and inhibited PI3K/AKT signaling pathway. NLRP3 partially restored functions of miR-22 on cell proliferation and EMT in ovarian cancer. Conclusion miR-22 impaired cell viability and EMT by NLRP3 and inhibited PI3K/AKT signaling pathway in ovarian cancer. The newly identified miR-22/NLRP3/PI3K/AKT axis provides novel insight into the pathogenesis of ovarian cancer (AU)

miR-9 inhibits the metastatic ability of hepatocellular carcinoma via targeting beta galactoside alpha-2.6-sialyltransferase 1

Glycosylation of cell surface proteins regulates critical cellular functions, including invasion and metastasis in cancer cells. Emerging evidence has shown that microRNAs (miRNAs) are involved in regulating both the glycosylation modifications on cell surface and the progression of cancer. In this study, we investigated the role of miR-9 in alfa -2,6-linked sialylation and the metastasis of mouse hepatocellular carcinoma (HCC). According to array-based miRNA expression profiling data of HCC cell lines Hepa1-6, Hca-P, and Hca-F with different lymphatic metastatic capacities, reverse correlation was found between miR-9 expression levels and the metastatic potential in these HCC cells. Additionally, Beta-galactoside alfa -2,6-sialyltransferase 1 (St6gal1) expression level is associated negatively with miR-9 and positively with metastatic potential. Bioinformatics analysis indicated that miR-9 could target St6gal1, which was verified by luciferase reporter assays. miR-9 overexpression reduced expression of St6gal1, which subsequently suppressed HCC cells metastatic potential. Moreover, upregulation of miR-9 could inhibit integrin-Beta1/FAK-mediated cell motility and migration signaling in mouse HCC cells. Together, our results suggest that miR-9 could act as a tumor suppressor and regulate mouse HCC cells migration and invasion by inhibiting the alfa-2,6-linked sialylation. This finding may provide insight into the relationship between abnormal miRNA expression and aberrant cell surface glycosylation during tumor lymphatic metastasis

MicroRNA-195 inhibits human gastric cancer by directly targeting basic fibroblast growth factor

Purpose. Gastric cancer (GC) is one of the fatal malignancies worldwide with high occurrences but poor outcomes. bFGF has been shown to play significant roles in GC. Yet, whether bFGF affects the development of GC is less studied. Methods. MicroRNA assays, real-time PCR, and western blot were conducted for expression analysis of miR-195-5p and basic fibroblast growth factor (bFGF). Luciferase activity was measured with mutated bFGF 3′-UTR sequence at the 3′ end of the luciferase gene. Two GC cell lines, SNU-1 and KATO-3 overexpressing miR-195-5p and bFGF were subjected to wound healing assay and transwell invasion assay. Mouse GC xenograft model was established and subjected to tumor size analysis. Results. Expression levels of miR-195-5p and bFGF showed negative correlation in human GC tissues. MiR-195-5p directly targeted bFGF 3′-UTR as demonstrated by luciferase activity assay. MiR-195-5p, through downregulating bFGF, inhibited the migration and invasion of SNU-1 and KATO-3 cells, as well as tumorigenesis in a xenograft mouse model, which could be restored by re-introduction of bFGF. Conclusions. MiR-195-5p inhibits tumorigenesis of GC through suppressing bFGF, which supports both miR-195-5p and bFGF as potential therapeutic targets in the treatment of GC (AU)

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Diagnostic value of the dual-luciferase report assay for predicting response to glucocorticoid in children with acute lymphoblastic leukemia

Objective. Resistance to glucocorticoid (GC) is a significant clinical problem in some cases of acute lymphoblastic leukemia (ALL). Current methods of assessing GC resistance are time consuming and have limited reproducibility; in this study, we sought to define a new method of evaluating GC sensitivity and resistance in vitro. Methods. Based on the mechanisms of GC resistance, we hypothesized that the dual-luciferase report (DLR) assay could reflect the transcription effects of GC downstream of the GC-glucocorticoid receptor signaling pathway, thereby allowing the evaluation of reactions to GC. Sixty-two patients with differential GC response were included in this study. The prednisone induction test was used to divide the children with ALL into two groups: GC sensitive (GCS) and GC resistant (GCR). DLR assay was later conducted on those patients to evaluate its value for diagnosis of the GC reactivity. Receiver operating characteristic curves were used to identify the optimal assay cutoff for identifying response to GC. Results. Using the DLR assay analysis, we found that GCR subjects showed significantly lower reporter/control ratios for luciferase, as compared with GCS subjects. The optimal cutoff value for GC response was 0.67, with sensitivity of 77.1% and specificity of 93.3%. The DLR assay results were consistent with prednisone induction test results. Further, the DLR assay was simpler, more sensitive, and less time-consuming than the prednisone induction test. Conclusions. Our study showed that the DLR assay is relatively fast, simple, and sensitive. Accordingly, it could be useful for detecting GC response in children with ALL (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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MicroRNA-585 acts as a tumor suppressor in non-small-cell lung cancer by targeting hSMG-1

Purpose. To investigate the role of miR-585 in the development and progression of non-small-cell lung cancer (NSCLC). Methods. The expression levels of miR-585 in NSCLC cell lines and clinical samples were measured by quantitative PCR. NSCLC cells, A549 and H1299, were stably transfected with lentiviral vectors of miR-585 mimics or negative control. The effects of miR-585 on cell proliferation were detected both in vitro and in vivo. Cell migration and invasion were evaluated using wound healing assay and Transwell assay. Furthermore, luciferase reporter assay was used to identify the direct regulation of hSMG-1 by miR-585. Results. Our results showed that miR-585 was downregulated in NSCLC cell lines and tumor tissues. Ectopic expression of miR-585 inhibited the ability of cell proliferation, migration, and invasion in vitro. In addition, miR-585 also decreased the growth rate of xenografted tumor in nude mice. Mechanically, miR-585 directly targeted the 3′-untranslated region (UTR) of hSMG-1 gene, which likely resulted in a dysfunction of mRNA surveillance and nonsense-mediated mRNA decay. Conclusion. Taken together, miR-585 probably has an inhibitory effect on tumor growth and is a prognostic biomarker of NSCLC (AU)

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Biological function and mechanism of miR-33a in prostate cancer survival and metastasis: via downregulating Engrailed-2

Objective. Recent studies have identified Engrailed-2 (EN-2), a homeobox-containing transcription factor, as a candidate oncogene in prostate cancer (PC). Therapeutic targeting on EN-2, however, is limited because the mechanism underlying EN-2 overexpression in prostatic cancer cells is unknown. This study was to investigate the potential regulatory role of miR-33a on EN-2 expression and explore this signaling axis in ability of prostate cancer survival and metastasis. Methods. The relative expression of miR-33a and EN-2 in paired prostate cancer tissue and adjacent normal tissue as well as in prostate cancer cell lines, PC3 and DU145, was determined using quantitative real-time PCR or western blot, respectively. Cells survival, migration and invasion were evaluated by assays of MTT, TUNEL and Boyden chamber assays, respectively. Direct regulation of EN-2 by miR-33a was examined by luciferase reporter assay. Results. The data showed that miR-33a was upregulated and EN-2 was downregulated in both prostate cancer tissue and prostate cancer cells. miR-33a overexpression suppresses prostate cancer cell survival and metastasis. miR-33a can directly act on EN-2 expression by binding to 3′UTR of its mRNA. Also, miR-33a negatively regulated EN-2 mRNA and protein expression. In pcDNA-EN-2 and miR-33a mimic co-transfected PC3 and DU145 cells, EN-2 overexpression reverses the anti-cell survival and metastasis actions of miR-33a overexpression. The pivotal role of miR-33a in inhibiting prostate tumor growth was confirmed in xenograft models of prostate cancer. Conclusion. Our data suggest that the functional interaction of miR-33a and EN-2 is involved in tumorigenesis of prostate cancer. Also in this process EN-2 serves as a negative responder for miR-33a (AU)

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Let-7b inhibits the malignant behavior of glioma cells and glioma stem-like cells via downregulation of E2F2

Glioblastoma multiforme (GBM), the most common and lethal primary brain tumor in adults characterized by high proliferative ability and mortality rate, contains a small subpopulation of cancer stem-like cells (CSCs), which is responsible for GBM progression and therapeutic resistance. Numerous microRNAs are strongly implicated in the malignancy of glioma. However, their specific functions and roles have yet to be fully demonstrated. In the present study, we revealed that the upregulation of Let-7b, a member of the Let-7 microRNA family, inhibited proliferation, migration, and invasion in glioma cell lines. Using bioinformatics, expression analysis, and luciferase assay, E2F2 was confirmed as a candidate target of Let-7b. Moreover, we also observed that elevated levels of Let-7b resulted in a reduction of tumor sphere growth and stemness of glioma stem-like cells. Furthermore, we found that knockdown of E2F2 expression could reduce the proliferation of glioma and GSCs, while overexpression of E2F2 partially abrogated the inhibitory effect of Let-7b on the proliferation of glioma and GSCs. In conclusion, we suggest that Let-7b could be developed into a promising anticancer target in glioma (AU)

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Retinoic acid modulates PTGDR promoter activity

Background and Objective: Vitamin A has been linked to the development of allergic diseases although its role is not fully understood, Retinoic acid (RA), a metabolite of Vitamin A, has been previously associated with the prostaglandin pathway, and PTGDR, a receptor of PGD2, has been proposed as a candidate gene in allergy and asthma. Considering the role of PTGDR in allergy, the goal of this study was o analyze the effect of RA on the activation of the promoter region of the PTGDR gene. Methods: A549 lung epithelial cells were transfected with 4 combinations of genetic variants of the PTGDR promoter and stimulated with all-trans RA (ATRA); luciferase assays were performed using the Dual Luciferase Reporter System, and real-time quantitative polymerase chain reaction was used to measure the expression of PTGDR, CYP26A1, RARA, RARB, RARG , and RXRA in basal A549 cell cultures and after ATRA treatment. We also performed an in silico analysis. Results: After ATRA treatment increased expression of CYP26A1 (12-fold) and RARB (4-fold) was detected. ATRA activated PTGDR promoter activity in transfected cells (P<.001) and RA response element sequences were identified in silico in this promoter region. Conclusions: RA modulated PTGDR promoter activity. Differential response to RA and to new treatments based on PTGDR modulation could depend on genetic background in allergic asthmatic patients (AU)

Introducción y Objetivo: La vitamina A se ha relacionado con el desarrollo de las enfermedades alérgicas, si bien su papel no se comprende en su totalidad. El ácido retinoico, un metabolito de la vitamina A, se ha asociado previamente con la ruta de las prostaglandinas. Además, PTGDR, uno de los receptores de PGD2, se ha propuesto como un gen candidato en la alergia y el asma. Considerando el papel de PTGDR en la alergia, el objetivo de este estudio fue analizar el efecto del ácido retinoico sobre la activación de la región promotora del gen PTGDR. Métodos: Se utilizó la línea celular A549 de epitelio de pulmón. Las células fueron transfectadas con cuatro combinaciones de las variantes génicas de PTGDR y fueron estimuladas con ácido retinoico todo-trans (ATRA). Los ensayos de Luciferasa se llevaron a cabo mediante el sistema Dual Luciferase Reporter System. Se realizaron análisis de RT-qPCR para medir la expresión basal de PTGDR, CYP26A1, RARA, RARB, RARG y RXRA de los cultivos de A549 tras el tratamiento con ATRA. Se realizaron también análisis bioinformáticos. Resultados: Se encontraron diferencias significativas en la actividad promotora entre las variantes haplotípicas tras la transfección en la línea celular A549. Tras el tratamiento con ATRA se detectó un incremento de la expresión de CYP26A1 (12 veces) y RARB (4 veces). El ácido retinoico activó la actividad promotora de PTGDR en las células transfectadas (p<0,001). Se identificaron secuencias de Elementos de Respuesta a Ácido Retinoico (RARE) in silico en la región promotora de PTGDR. Conclusiones: El ácido retinoico modula la actividad promotora de PTGDR . Esto podría explicar las diferencias en los efectos del ácido retinoico y en las respuestas a los nuevos tratamientos de la enfermedad alérgica basados en la modulación del receptor PTGDR (AU)

Association between CC motif chemokine ligand 5 (CCL5) polymorphisms and asthma risk: an updated meta-analysis

Background: Findings regarding the associations between the CC motif chemokine ligand 5 (CCL5) -403G/A and -28C/G polymorphisms and asthma risk are controversial. We performed a meta-analysis to determine whether CCL5 polymorphisms are associated with asthma risk. Methods: We searched the Pubmed, Embase, Chinese National Knowledge Infrastructure (CNKI), and Wanfang databases for studies published before June 2013. The strength of associations was calculated using ORs with 95% CIs. Results: Twenty case-control studies were included in this meta-analysis. We did not observe a significant association between the CCL5 -403G/A polymorphism and asthma risk (OR, 1.10; 95% CI, 0.93-1.30; P=.25). The CCL5 -28C/G polymorphism, however, was associated with a significantly elevated asthma risk (OR, 1.17; 95% CI, 1.02-1.33; P=.02). Subgroup analyses found that the CCL5 -28C/G polymorphism was significantly associated with asthma risk in Asians (OR, 1.16; 95% CI, 1.01-1.33; P=.04) and children (OR, 1.29; 95% CI, 1.03-1.63; P=.03). Conclusions: This meta-analysis suggests that the CCL5 -28C/G polymorphism is a risk factor for asthma (AU)

Introducción: Existen discrepancias entre la asociación del riesgo de padecer asma y diferentes polimorfismos del ligando de la quimiocina CC5 (CCL5). En este trabajo se ha realizado un meta-análisis para determinar si los polimorfismos CCL5-403G / A y CCL5-28C / G se asocian con el riesgo de asma bronquial. Métodos: Se utilizaron diversas bases de datos para realizar las búsquedas de estudios publicados antes de junio de 2013, incluyendo: PubMed, EMBASE, CNKI (Infraestructura del Conocimiento Nacional Chino) y Wanfang Se calcularon los odd ratios combinados (OR) con intervalos de confianza del 95% (IC). Resultados: Se incluyeron un total de 20 estudios de casos y controles. No se encontró una asociación significativa entre el polimorfismo CCL5-403G / A y el riesgo de asma (OR = 1,10, IC del 95%: 0,93 a 1,30, p = 0,25). Por el contrario, el polimorfismo CCL5-28C / G, se asoció con un riesgo significativamente elevado de asma (OR = 1,17, IC del 95%: 1,02 a 1,33, p = 0,02). En los análisis de subgrupos, el riesgo de asma fue significativamente mayor en los asiáticos con el polimorfismo CCL5-28C / G (OR = 1.16, 95% IC 1,01-1,33, P = 0,04) y los niños (OR = 1.29, 95% IC 1,03-1,63, P = 0,03). Conclusiones: Este meta-análisis sugiere que el polimorfismo CCL5-28C / G es un factor de riesgo significativo para padecer asma bronquial (AU)

Estudio funcional del promotor del gen NPC1L1 / Functional analysis of the NPC1L1 gene promoter

Introducción. En la absorción intestinal de colesterol intervienen distintos transportadores, uno de gran importancia y diana del fármaco ezetimiba, la proteína NPC1L1. Se ha demostrado una asociación entre distintas variantes genéticas de NPC1L1, la eficiencia en la absorción de esteroles y la concentración plasmática de colesterol unido a lipoproteínas de baja densidad. El objetivo de este estudio fue identificar y analizar el efecto funcional de los polimorfismos genéticos potencialmente más relevantes del gen NPC1L1sobre su actividad transcripcional. Material y métodos. Como zona de estudio se seleccionó 2 kb de la región promotora del genNPC1L1. Se realizó una búsqueda en bases de datos para localizar variantes descritas en las secuencias seleccionadas. Se diseñaron 3fragmentos, que se amplificaron mediante PCR y posteriormente se secuenciaron. La funcionalidad del polimorfismo –133A>G se determinó mediante ensayos de retardo en gel y medida de la actividadluciferasa. Resultados. El análisis de la zona promotora de102 sujetos normolipidémicos mostró 5 nuevas variantes polimórficas (–1485C>T, –1425C>G,–982G>C, –292T>C y –18C>A) no identificadas previamente. Los resultados de los ensayos de retardo en gel con el polimorfismo –133A>Grevelaron mayor afinidad de unión de las proteínas nucleares con la sonda portadora de la variante–133A. Por otra parte, la actividad del promotor deNPC1L1 con la variante –133G mostró un aumento de 2,5 veces respecto a la variante –133A.Conclusión. Nuestros resultados demuestran que hay diferencias en la afinidad de unión y actividad transcripcional de NPC1L1 en función del polimorfismo –133A>G. Esta variante génica podría contribuir a la variabilidad interindividual de la absorción intestinal de esteroles (AU)

Introduction. Cholesterol absorption is a process which involves the participation of numerous transporters. One of these transporters is NPC1L1,identified as a critical protein for intestinal cholesterol absorption and the molecular target of ezetimibe. It has been shown that NPC1L1 variants are associated with variations in sterol absorption and plasma levels of LDL-C. The aim of this work was to identify and analyse the potential functional effect on the transcriptional activity of NPC1L1gene polymorphisms. Material and methods. The previously describedNPC1L1 promoter variants were located by means of SNP database search. The NPC1L1 promoter was analysed by three PCR reactions followed by automatic capillary sequencing. Functional study of the –133A>G polymorphism was performed by EMSA and Luciferase assays. Results. NPC1L1 promoter analysis in 102normolipemic subjects showed five (..) (AU)

Las aguas y sedimentos del río tunecino Hamdoun presentan riesgo de disrupción endocrina / Water and sediment waste rejected in Hamdoun’sriver are major endocrine disrupting system

Diversos estudios recientes sostienen que muchos productos químicos antropogénicos, presentes en el medio ambiente, imitan la acción de hormonas endógenas. Estos disruptores endocrinos pueden originar múltiples efectos adversos en la fauna, como la feminización de peces, la pérdida de capacidad reproductiva, defectos congénitos y, a veces, pueden estar en el origen de algunos tipos de cáncer en el ser humano. La aparición de intersexos en peces de varios ríos europeos se ha atribuido a la exposición a sustancias químicas estrogénicas presentes en los efluentes de estaciones de tratamiento de aguas residuales. Para profundizar en el efecto ambiental de estos contaminantes, hemos investigado la actividad estrogénica, de receptor de hidrocarburo arílico y de receptor X de pregnano, de muestras de agua y sedimentos del río Hamdoun, tomadas aguas arriba y aguas abajo de la zona de vertidos procedentes del área industrial de la región central de Túnez. Mediante un ensayo in vitro de células cancerosas bioluminiscentes que expresan el gen de la luciferasa bajo el control de ciertos elementos con acción hormonal, hemos detectado escasa actividad estrogénica en agua y sedimentos por encima de la zona de vertidos; sin embargo, encontram os fuerte actividad es trogénica, de receptor de hidrocarburo arílico y de receptor X de pregnano en agua y s edimentos río abajo. Con experimentos de com petición demostramos que, predominantemente, las muestras de agua y de sedimentos con actividad estrogénica contienen compuestos con alta y baja afinidad con el ER " recombinante, respectivamente. Estos resultados indican que el agua del río y los sedimentos constituyen un importante sumidero y pueden ser fuente potencial de contaminantes disruptores endocrinos(AU)

In recent years, many studies supported that anthropogenic chemicals occurring in the environment have been shown to mimic the action of endogenous hormones. These endocrine-disrupting chemicals can potentially lead to a host of adverse effects on wildlife, such as the feminization of fish, the lack of reproduction success, birth defects and sometimes they can be the origin of some kind of cancers in human. The occurrence of intersex fish in a number of European rivers has been attributed to the exposure to estrogenic chemicals present in sewage treatment work effluents. To further understand the environmental effect of these contaminants, the estrogenic, aryl hydrocarbon receptor and pregnane X receptor activities of water and sediments were investigated in this study. The water and sediment samples were obtained from upstream and downstream outfalls of the Hamdoun River located in proximity of the industrial area in the centre region of Tunisia. Using an in vitro assay with bioluminescent cancer cells expressing luciferase gene under different hormonal responsive element control, we detected a much lower level of estrogenic activity in water and sediment upstream, however, we found out a strong estrogenic, aryl hydrocarbon receptor and pregnane X receptor activities in water and sediment downstream this river. By using competition experiments, we demonstrated that estrogenic activity found contained mainly compounds with a strong and lower affinitiy in water and sediment respectively with the recombinant ER ". These results suggest that the river water and sediments are a major sink and could be a potential source of endocrine-disrupting chemicals contaminants(AU)

Transferencia génica mediante virus adeno-asociados en un modelo de daño articular en rodilla de rata / Gene transfer mediated by adeno-associated virus vector in a rat knee model of articular damage

El cartílago articular es el tejido encargado de disminuirla fricción entre las superficies articulares durante el movimiento.Su limitada capacidad de regeneración hace quelas lesiones osteocondrales posean un mal pronóstico ypuedan acabar generando una artrosis en la articulación.La terapia génica para este tipo de patologías resulta muyprometedora, puesto que actualmente, no hay ningún procedimientocapaz de restablecer el tejido dañado. En nuestroestudio hemos puesto a punto un modelo de transferenciagénica a los tejidos de la articulación de la rodillade rata, inyectando intraarticularmente vectores derivadosde virus adeno-asociados, capaces de inducir la expresiónde luciferasa. Los resultados muestran cómo la inducciónde la expresión resulta significativa a partir de los dos mesesde evolución en todos los tejidos articulares (cartílagoarticular, menisco, membrana sinovial y hueso subcondral).La presencia de daño, tanto de tipo mecánico comoautoinmune (artritis inducida por colágeno) no modifica laexpresión de proteína por parte del vector

Articular cartilage is a tissue that decreases friction onarticular surfaces during movement. Its limited regenerativecapacity makes osteocondral lessions to extend andgenerate osteoarthritis in the joint. Gene therapy is apromising alternative to these patologies, since there areno proceedings actually, that restablishes damaged tissue.In our study, we have developed a model of gene transferto articular tissues injecting intraarticularly an adeno-associatedderived vector that induces expression of luciferase.Results obtained show a significant induction of proteinexpression two months after injecting vectors, in all articulartissues (articular cartilage, meniscus, synovium andsubcondral bone). The presence of lessions, mechanic orautoimmune (collagen induced arthritis) do not modify theexpression of protein induced by the vector