13,848 research outputs found
Functional microRNA high throughput screening reveals miR-9 as a central regulator of liver oncogenesis by affecting the PPARA-CDH1 pathway
Full text linkBackground: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths, reflecting the aggressiveness of this type of cancer and the absence of effective therapeutic regimens. MicroRNAs have been involved in the pathogenesis of different types of cancers, including liver cancer. Our aim was to identify microRNAs that have both functional and clinical relevance in HCC and examine their downstream signaling effectors. Methods: MicroRNA and gene expression levels were measured by quantitative real-time PCR in HCC tumors and controls. A TargetScan algorithm was used to identify miR-9 downstream direct targets. Results: A high-throughput screen of the human microRNAome revealed 28 microRNAs as regulators of liver cancer cell invasiveness. MiR-9, miR-21 and miR-224 were the top inducers of HCC invasiveness and also their expression was increased in HCC relative to control liver tissues. Integration of the microRNA screen and expression data revealed miR-9 as the top microRNA, having both functional and clinical significance. MiR-9 levels correlated with HCC tumor stage and miR-9 overexpression induced SNU-449 and HepG2 cell growth, invasiveness and their ability to form colonies in soft agar. Bioinformatics and 3’UTR luciferase analyses identified E-cadherin (CDH1) and peroxisome proliferator-activated receptor alpha (PPARA) as direct downstream effectors of miR-9 activity. Inhibition of PPARA suppressed CDH1 mRNA levels, suggesting that miR-9 regulates CDH1 expression directly through binding in its 3’UTR and indirectly through PPARA. On the other hand, miR-9 inhibition of overexpression suppressed HCC tumorigenicity and invasiveness. PPARA and CDH1 mRNA levels were decreased in HCC relative to controls and were inversely correlated with miR-9 levels. Conclusions: Taken together, this study revealed the involvement of the miR-9/PPARA/CDH1 signaling pathway in HCC oncogenesis
miR-375 mediates the CRF signaling pathway to regulate catecholamine biosynthesis by targeting <i>Sp1</i> in porcine adrenal gland
No full textCorticotropin-releasing-factor (CRF) is a key regulator of catecholamines (CATs) biosynthesis in the adrenal gland. Furthermore, miR-375 has been confirmed to be localized in the mouse adrenal gland. However, the relationships between miR-375 and CRF in regulating CATs biosynthesis remain to be established. This study was designed to investigate the relationship between CRF and miR-375 in the regulation of CATs biosynthesis in the porcine adrenal gland. Eight adult female pigs (four controls; four injected intracerebroventricularly with 50 μg of CRF) were used for the in vivo experiments in this study. The results showed that miR-375 was exclusively localized in porcine adrenal medullary cells. Functional studies showed that miR-375 negatively regulated CATs synthesis in primary cells by affecting the expression of the CATs synthetases tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). CRF up-regulated the expression of CATs synthetase in primary adrenal medullary cells under basal conditions and upon endogenous miR-375 inhibition; the enhanced effects vanished when cellular miR-375 was overexpressed by transfecting miR-375-mic. CRF decreased the expression of miR-375 both in vivo and in vitro. Our in vitro results showed that CRF significantly decreased the expression of miR-375, perhaps by binding to CRFR1. miR-375 functions by directly binding to the 3′-UTR region of specificity protein 1 (Sp1), which is involved in regulating Th and Dbh expression. These data collectively indicate that miR-375 plays an important role in regulating CATs synthesis and mediates the CRF signaling pathway in porcine adrenal medullary cells.</p
Demonstration of the Presence of the "Deleted" MIR122 Gene in HepG2 Cells
No full textMicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells
Importancia clínica de los micrornas de la vía de p53 en cáncer de pulmón no microcítico: miR-34a y miR-16
Full text link[spa] El cáncer de pulmón no célula pequeña es la neoplasia más frecuente en la actualidad en los países industrializados, siendo la primera causa de mortalidad por cáncer en el varón.
Su incidencia continúa aumentando progresivamente, y su tratamiento, pese a los esfuerzos de investigación de los últimos años, sigue siendo poco efectivo en la mayor parte de los casos, situándose la supervivencia global a los cinco años alrededor del 13%.
La alta tasa de recaída, incluso en estadios iniciales susceptibles de cirugías radicales, justifica el interés de estudiar marcadores pronósticos de supervivencia y recaída. Esto nos ayudaría a distinguir grupos de riesgo, cuyo interés radica en ayudarnos a conocer la necesidad y efecto de los tratamientos adyuvantes, con quimioterapia y radioterapia así como abrir la puerta a posibles nuevas armas terapéuticas.
Dada la alta incidencia de alteraciones en la vía de p53 en el cáncer de pulmón no célula pequeña, y su probable valor pronóstico, nuestra hipótesis es que existen alteraciones en los niveles de expresión de los miRNAs directamente regulados por p53, así como en los reguladores intermedios de la función de p53, con posible valor pronóstico en recaída y supervivencia de pacientes operados de cáncer de pulmón no célula pequeña o no microcítico(CPNCP).
Tratamos pues de identificar el papel que juegan los miRNAs como marcadores útiles en el pronóstico del CPNCP en estadios iniciales, tras cirugía radical. De forma más específica determinar el posible papel pronóstico en recaída y en supervivencia de los miRNAs de la familia de miR-34: miR-34a, miR-34b y miR-34c, cuya transcripción es activada por p53. También definir el posible papel pronóstico en recaída y en supervivencia de los miRNAs miR-16 y miR-143, cuyos niveles finales en la célula, están modulados por p53. Así como, conocer si existen posibles interacciones entre miR-34a, miR-34b, miR-34c, miR-16 y miR-143 a nivel pronóstico.
Para ello, hemos planteado un estudio retrospectivo, con las muestras de tejido tumoral y normal pareado de 70 pacientes, diagnosticados y tratado, de cáncer de pulmón no microcítico estadios I-III, entre Febrero de 1996 y Septiembre de 2002, en el Hospital Clínic de Barcelona. Se realizaron la extracción y cuantificación de RNA. Determinación de los niveles de los microRNAs mencionados. Determinación del estado de metilación de la región promotora del gen MIR34A. Determinación de la presencia de mutaciones de P53 entre los exones 5-8.
Tras estos estudios los resultado obtenidos se han publicado en dos artículos. El primero de ellos en la revista “Carcinogenesis” y el segundo en la revista “Journal of Surgical Oncology”.
En el primer artículo, titulado “miR-34a as a prognostic marker of relapse in surgically resected non-small-cell lung cancer”, relacionamos los niveles de miR-34a con la recaída tumoral, estableciendo tres grupos en función de esta tasa de recaída: el grupo con niveles bajos de miR34a, con un 67% de recaída, los que tenían niveles altos, con un 43% de recaída y los que tenían los niveles más altos con un 0% de recaída. Además, en el análisis univariado para riesgo de recaída, el estado mutacional de P53, los niveles de miR-34a
y el estadio IA vs el resto, se correlacionaron con probabilidad de recaída. Se realizaron dos análisis mutilvariados, incluyendo o no el estado mutacional de P53, permaneciendo en ambos análisis la expresión de miR-34a, como factor independiente para recaída tumoral. Se observó que los pacientes con mutaciones de P53 presentaban una media de expresión de miR-34a más baja. Se observó que el subgrupo de pacientes en el que los niveles de expresión de miR-34a baja coincidían con presencia de mutaciones de P53, presentaban una alta tasa de recaída. En los pacientes sin mutaciones de de P53, existe una diferencia significativa en los niveles de expresión de miR-34a entre los pacientes que presentaban la región promotora de MIR34A metilada vs los que no. Por todo esto, encontramos que miR-34a se demuestra como un nuevo marcador biológico con significación en el pronóstico de la recaída de pacientes sometidos a cirugía del CPNCP, abriendo la posibilidad de una futura herramienta en el algoritmo de decisiones terapéuticas.
En el segundo artículo, titulado “Prognostic Implications of miR-16 Expression Levels in Resected Non-Small-Cell Lung Cancer”, desde la hipótesis de que P53 activa la transcripción de la familia de miRNAs de miR-34 y regula la maduración de miR-16 y miR143, se estudiaron los niveles de expresión de miR-143 y miR-16 en el tejido normal y tumoral pareado de cada paciente de la serie, antes mencionada, de 70 pacientes.
Así los pacientes se clasificaron de acuerdo a los niveles de expresión de miR-16(alto, normal y bajo). Aquellos pacientes con niveles normales tuvieron la mejor evolución, mientras que aquellos con niveles más altos tuvieron la peor. La supervivencia libre de enfermedad (SLE) era de 22,4 meses para los pacientes con niveles altos de miR-16, 71,8 meses para los de niveles normales y de 55,8 meses para los de niveles bajos. La supervivencia global (SG) fue de 23,9 meses para los que tenían niveles altos de miR-16, de 97,6 meses para los que tenían niveles normales y 63,5 meses para aquellos con niveles bajos. No se observó correlación entre los niveles de miR-143 y la evolución clínica de los pacientes. El análisis multivariado mostró a miR-16 como factor pronóstico independiente de SLE Y SG. En un análisis secundario, examinamos la correlación potencial entre la expresión de miR-16 y miR-34a y el estado mutacional de P53.No hubo correlación entre el estado mutacional de P53 y miR-16, pero si se observó una interacción entre miR-16 y miR-34a. En los pacientes con niveles altos de miR-34a, se observaron diferencias entre SLE Y SG, de acuerdo a los niveles de expresión de miR-16, mientras que en los pacientes con niveles bajos de miR-34a presentaban un pobre pronóstico, independientemente de los niveles de expresión de miR-16. Estos resultados nos indican el posible valor pronóstico de miR-16 en pacientes intervenidos de CPNCP, además del posible sinergismo entre miR-34a y miR16.[eng] Non small cell lung cancer is more common today, in industrialized countries and is the leading cause of cancer death in men. Its incidence continues to increase gradually, and their treatment, despite the research efforts of recent years, it remains ineffective in most cases, putting the overall survival at five years about 13%. The high rate of relapse, even in early stages susceptible to radical surgery, justifies the interest in studying prognostic markers of survival and relapse. This would help us distinguish risk groups, whose interest lies in helping to meet the need and effect of adjuvant treatment with chemotherapy and radiotherapy as well as opening the door to possible new therapeutic tools. Given the high incidence of alterations in the p53 pathway in non small cell lung cancer, and likely its prognostic role, our hypothesis is that there are alterations in the expression levels of miRNAs directly regulated by p53, as well as in intermediate regulators of p53 function with potential prognostic value on relapse and survival of patients undergoing non small cell lung cancer (NSCLC). Then, we try to identify the role of miRNAs as useful markers for the prognosis of NSCLC in initial stages, after radical surgery. More specifically determine the possible prognostic role in relapse and survival of the family miRNAs miR-34: miR-34a, miR-34b and miR-34c, whose transcription is activated by p53. Also define the possible prognostic role in relapse and survival of the miRNAs miR-16 and miR-143, whose final levels in the cell are modulated by p53. And, determine whether there are possible interactions between miR-34a, miR-34b, miR-34c, miR-16 and miR-143 as regards prognosis. To do this, we have proposed a retrospective study, with the tumor tissue and paired normal in 70 patients, diagnosed and treated of non small cell lung cancer stages I-III, between February 1996 and September 2002, at the Hospital Clínic, in Barcelona. We performed RNA extraction and quantification. Determining levels of microRNAs mentioned. Determining the status of methylation of the promoter region of the gene MIR34A. Determining the presence of p53 mutations between exons 5-8. Following, the results obtained from these studies have been published in two articles. The first in the journal "Carcinogenesis" and the second in the "Journal of Surgical Oncology." In the first article, entitled "miR-34a as a Prognostic marker of relapse in surgically resected non-small-cell lung cancer", we relate the levels of miR-34a with the tumor relapse, establishing three groups according to the rate of relapse: the group with low levels of miR34a, with 67% relapse, those with high levels, with 43% relapse and those with the highest levels with a 0% relapse. Furthermore, in univariate analysis for risk of relapse, the mutational status of p53, the levels of miR-34a and stage IA vs the rest, were correlated with likelihood of relapse. Two multivariate analyzes were performed, including or not the P53 mutational status, remaining in both analyzes the expression of miR-34a, as an independent factor for tumor relapse. It was observed that patients with p53 mutations had a mean expression of miR-34a lower. It was noted that the subgroup of patients in which the expression levels of miR-34a low coincided with the presence of p53 mutations, had a high rate of relapse. In patients without mutations of p53, there is a significant difference in expression levels of miR-34a between the patients with the methylated promoter region MIR34A vs. those without. For all this, we found that miR-34a is shown as a novel biomarker with prognostic significance in patients relapsed NSCLC undergoing surgery, opening the possibility of a future tool in the therapeutic decision algorithm. In the second article, entitled "Prognostic Implications of miR-16 Expression Levels in Resected Non-Small-Cell Lung Cancer", from the hypothesis that p53 activates transcription of the family of miRNAs miR-34 and regulates the maturation of miR-16 to miR-143, we studied the expression levels of miR-143 and miR-16 in the paired normal and tumor tissue of each patient in the series, mentioned above, in 70 patients. So patients were classified according to the expression levels of miR-16 (high, normal and low). Patients with normal levels had the best clinical course, while those with higher levels had the worst. The disease-free survival (DFS) was 22.4 months for patients with high levels of miR-16, 71.8 months for normal levels of 55.8 months for low levels. Overall survival (OS) was 23.9 months for those with high levels of miR-16, from 97.6 months for those with normal levels and 63.5 months for those with low levels. No correlation was observed between the levels of miR-143 and the clinical course of patients. Multivariate analysis showed miR-16 as an independent prognostic factor for DFS and OS. In a secondary analysis, we examined the potential correlation between the expression of miR-16 and miR-34a and the mutational status of P53. There was no correlation between p53 mutational status and miR-16, but observed an interaction between miR-16 and miR-34a. In patients with high levels of miR-34a, there were differences between DFS and OS, according to the expression levels of miR-16, whereas in patients with low levels of miR-34a had a poor prognosis, regardless of expression levels of miR-16. These results indicate the possible prognostic value of miR-16 in patients operated for NSCLC, as well as possible synergism between miR-34a and miR1
MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis
Full text linkMicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of
multiple sclerosis
Changsheng Du1,5, Chang Liu1,5, Jiuhong Kang1,2, Guixian Zhao3, Zhiqiang Ye4, Shichao Huang1, Zhenxin Li3, Zhiying Wu3 & Gang Pei1,2
Interleukin 17 (IL-17)-producing T helper cells (TH-17 cells) are increasingly recognized as key participants in various autoimmune diseases, including multiple sclerosis. Although sets of transcription factors and cytokines are known to regulate TH-17 differentiation, the role of noncoding RNA is poorly understood. Here we identify a TH-17 cell–associated microRNA,
miR-326, whose expression was highly correlated with disease severity in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis (EAE). In vivo silencing of miR-326 resulted in fewer TH-17 cells and mild EAE, and its overexpression led to more TH-17 cells and severe EAE. We also found that miR-326 promoted TH-17 differentiation by targeting Ets-1, a negative regulator of TH-17 differentiation. Our data show a critical role for microRNA in TH-17 differentiation and the pathogenesis of multiple sclerosis
miR-101, miR-548b, miR-554, and miR-1202 are reliable prognosis predictors of the miRNAs associated with cancer immunity in primary central nervous system lymphoma
No full textMicroRNAs (miRNAs) inhibit protein function by silencing the translation of target mRNAs. However, in primary central nervous system lymphoma (PCNSL), the expression and functions of miRNAs are inadequately known. Here, we examined the expression of 847 miRNAs in 40 PCNSL patients with a microarray and investigated for the miRNA predictors associated with cancer immunity-related genes such as T helper cell type 1/2 (Th-1/Th-2) and regulatory T cell (T-reg) status, and stimulatory and inhibitory checkpoint genes, for prognosis prediction in PCNSL. The aim of this study is to find promising prognosis markers based on the miRNA expression in PCNSL. We detected 334 miRNAs related to 66 cancer immunity-related genes in the microarray profiling. Variable importance measured by the random survival forest analysis and Cox proportional hazards regression model elucidated that 11 miRNAs successfully constitute the survival formulae dividing the Kaplan-Meier curve of the respective PCNSL subgroups. On the other hand, univariate analysis shortlisted 23 miRNAs for overall survival times, with four miRNAs clearly dividing the survival curves—miR-101/548b/554/1202. These miRNAs regulated Th-1/Th-2 status, T-reg cell status, and immune checkpoints. The miRNAs were also associated with gene ontology terms as Ras/MAP-kinase, ubiquitin ligase, PRC2 and acetylation, CDK, and phosphorylation, and several diseases including acquired immunodeficiency syndrome, glioma, and those related to blood and hippocampus with statistical significance. In conclusion, the results demonstrated that the four miRNAs comprising miR-101/548b/554/1202 associated with cancer immunity can be a useful prognostic marker in PCNSL and would help us understand target pathways for PCNSL treatments.</div
Rede uitgesproken op 17 september 1956 ter gelegenheid van de erepromotie van Dr. Th. von Karman door de promotor Dr. C.B. Biezeno
No full textRede uitgesproken door prof. C.B. Biezeno ter gelegenheid van de erepromotie aan de TH Delft van dr. Theodore von Karman.Precision and Microsystems EngineeringMechanical, Maritime and Materials Engineerin
miR-155 inhibition sensitizes CD4+ Th cells for TREG mediated suppression.
No full textBackgroundIn humans and mice naturally occurring CD4(+)CD25(+) regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.Principal findingsDNA-Microarray analyses of human as well as murine conventional CD4(+) Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4(+) Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4(+) Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.ConclusionInvestigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4(+) Th cells to nTreg cell-mediated suppression
Identification of the target genes of miR-206 that are regulated by TH.
No full text<p>A. Levels of miR-206 in AML 12 cells stably transfected to ectopically express miR-206. AML12 cells were transfected with pEGP-mmu-mir-206 expression vector or pEGP-mir null control vector and selected with puromycin and green florescence protein by microscopy. The expression of miR-206 was examined with the Taqman miRNA Assay (n = 5). B. The expression of genes that are putative targets of miR-206 in the two cell types shown in figure 4A was examined with RT-PCR (n = 3). C. The effects of TH on the expression of miR-206 in the transfected cells. The miR-206 transfected cells were treated with 10 nm T3 for 24 hours. MiR-206 expression was analyzed with Taqman miRNA Assay (n = 3). D. The effect of TH on the expression of the miR-206 target genes in the transfected cells. The miR-206 transfected cells were treated with 10 nm T3 for 24 hours. The expression of target genes was examined with RT-PCR (n = 3). Data are presented as mean ± SE. A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.</p
MiR-124, miR-125b and miR-181a/a*affect subspecification of lt-NES cell-derived neurons.
No full text<p>(<b>A</b>, <b>B</b>) Immunostainings for β-III tubulin plus TH <b>(A)</b> or GAD65/67 (<b>B</b>) in lt-NES cells (I3 cell line) transduced with LVTHM-ctr or LVTHM-miR-124, -miR-125b and -miR-181a/a*, respectively, and differentiated for 15 days. Scale bars = 100 µm. (<b>C</b>, <b>D</b>) Histograms showing the fold change in the number of TH-positive neurons (<b>C</b>) and GAD65/67-positive neurons (<b>D</b>) relative to the number of β-III tubulin-positive neurons in lt-NES cells transduced with LVTHM-ctr or LVTHM-miR-124, -miR-125b, -miR-153, -miR-181a/a* and -miR-324-5p/3p, compared to untransduced cells (equal to 1, dashed line). Data are presented as mean + SEM (n = 3; **, p≤0.01). (<b>E</b>) qRT-PCR analyses of NURR1, DAT, TH and GAD1 expression in 15 days differentiated lt-NES cell cultures overexpressing LVTHM-ctr, -miR-124, -miR-125b, and -miR-181a/a*, respectively. Data are normalized to 18S rRNA reference levels and presented as average changes + SEM relative to expression levels in LVTHM-ctr transduced lt-NES cells (equal to 1, n≥3; *, p≤0.05; **, p≤0.01; ***, p≤0.0001). Abbreviations: ctr, control; DAT, dopamine transporter; GAD, glutamic acid decarboxylase; lt-NES, long-term self-renewing neuroepithelial-like stem cells; NURR1, Nuclear receptor related 1 protein; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; rRNA, ribosomal RNA; TH, tyrosine-hydroxylase.</p
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