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1.  Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis 
The Lancet. Oncology  2009;11(2):136-146.
Summary
Background
Analyses of microRNA expression profiles have shown that many microRNAs are expressed aberrantly and correlate with tumorigenesis, progression, and prognosis of various haematological and solid tumours. We aimed to assess the relation between microRNA expression and progression and prognosis of gastric cancer.
Methods
353 gastric samples from two independent subsets of patients from Japan were analysed by microRNA microarray. MicroRNA expression patterns were compared between non-tumour mucosa and cancer samples, graded by diffuse and intestinal histological types and by progression-related factors (eg, depth of invasion, metastasis, and stage). Disease outcome was calculated by multivariable regression analysis to establish whether microRNAs are independent prognostic factors.
Findings
In 160 paired samples of non-tumour mucosa and cancer, 22 microRNAs were upregulated and 13 were downregulated in gastric cancer; 292 (83%) samples were distinguished correctly by this signature. The two histological subtypes of gastric cancer showed different microRNA signatures: eight microRNAs were upregulated in diffuse-type and four in intestinal-type cancer. In the progression-related signature, miR-125b, miR-199a, and miR-100 were the most important microRNAs involved. Low expression of let-7g (hazard ratio 2·6 [95% CI 1·3–4·9]) and miR-433 (2·1 [1·1–3·9]) and high expression of miR-214 (2·4 [1·2–4·5]) were associated with unfavourable outcome in overall survival independent of clinical covariates, including depth of invasion, lymph-node metastasis, and stage.
Interpretation
MicroRNAs are expressed differentially in gastric cancers, and histological subtypes are characterised by specific microRNA signatures. Unique microRNAs are associated with progression and prognosis of gastric cancer.
Funding
National Cancer Institute.
doi:10.1016/S1470-2045(09)70343-2
PMCID: PMC4299826  PMID: 20022810
2.  GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer 
Cancer & Metabolism  2014;2:11.
Background
Alterations in glucose metabolism and epithelial-mesenchymal transition (EMT) constitute two important characteristics of carcinoma progression toward invasive cancer. Despite an extensive characterization of each of them separately, the links between EMT and glucose metabolism of tumor cells remain elusive. Here we show that the neuronal glucose transporter GLUT3 contributes to glucose uptake and proliferation of lung tumor cells that have undergone an EMT.
Results
Using a panel of human non-small cell lung cancer (NSCLC) cell lines, we demonstrate that GLUT3 is strongly expressed in mesenchymal, but not epithelial cells, a finding corroborated in hepatoma cells. Furthermore, we identify that ZEB1 binds to the GLUT3 gene to activate transcription. Importantly, inhibiting GLUT3 expression reduces glucose import and the proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. Using a large microarray data collection of human NSCLCs, we determine that GLUT3 expression correlates with EMT markers and is prognostic of poor overall survival.
Conclusions
Altogether, our results reveal that GLUT3 is a transcriptional target of ZEB1 and that this glucose transporter plays an important role in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Moreover, these findings emphasize the development of GLUT3 inhibitory drugs as a targeted therapy for the treatment of patients with poorly differentiated tumors.
doi:10.1186/2049-3002-2-11
PMCID: PMC4122054  PMID: 25097756
Epithelial-mesenchymal transition; Glucose transporter; GLUT3; Non-small cell lung cancer; SLC2A3; ZEB1
3.  The multiMiR R package and database: integration of microRNA–target interactions along with their disease and drug associations 
Nucleic Acids Research  2014;42(17):e133.
microRNAs (miRNAs) regulate expression by promoting degradation or repressing translation of target transcripts. miRNA target sites have been catalogued in databases based on experimental validation and computational prediction using various algorithms. Several online resources provide collections of multiple databases but need to be imported into other software, such as R, for processing, tabulation, graphing and computation. Currently available miRNA target site packages in R are limited in the number of databases, types of databases and flexibility. We present multiMiR, a new miRNA–target interaction R package and database, which includes several novel features not available in existing R packages: (i) compilation of nearly 50 million records in human and mouse from 14 different databases, more than any other collection; (ii) expansion of databases to those based on disease annotation and drug microRNAresponse, in addition to many experimental and computational databases; and (iii) user-defined cutoffs for predicted binding strength to provide the most confident selection. Case studies are reported on various biomedical applications including mouse models of alcohol consumption, studies of chronic obstructive pulmonary disease in human subjects, and human cell line models of bladder cancer metastasis. We also demonstrate how multiMiR was used to generate testable hypotheses that were pursued experimentally.
doi:10.1093/nar/gku631
PMCID: PMC4176155  PMID: 25063298
4.  miR-345 in Metastatic Colorectal Cancer: A Non-Invasive Biomarker for Clinical Outcome in Non-KRAS Mutant Patients Treated with 3rd Line Cetuximab and Irinotecan 
PLoS ONE  2014;9(6):e99886.
Introduction
MicroRNAs (miRNAs) have important regulatory functions in cellular processes and have shown promising potential as prognostic markers for disease outcome in patients with cancer. The aim of the present study was to find miRNA expression profiles in whole blood that were prognostic for overall survival (OS) in patients with metastatic colorectal cancer (mCRC) treated with cetuximab and irinotecan.
Methods
From 138 patients with mCRC in 3rd line therapy with cetuximab and irinotecan in a prospective phase II study, 738 pretreatment miRNAs were isolated and profiled from whole blood using the TaqMan MicroRNA Array v2.0. Mutation status of KRAS, BRAF, and PI3KCA was known.
Results
After Bonferroni adjustment, 6 miRNAs: (miR-345, miR-143, miR-34a*, miR-628-5p, miR-886-3p and miR-324-3p), were found associated with short OS. miR-345 was the strongest prognostic miRNA, significant in the full cohort and in the non-KRAS mutant population. miR-345, as a continuous variable in the full cohort, resulted in a hazard ratio (HR) of 2.38 per IQR (CI 95%: 1.8–3.1, P-value = 2.86e−07, Bonferroni adjusted, univariable analysis) and a HR = 1.75 per IQR (CI 95%: 1.24–2.48, P-Wald = 1.45e-03) in the multivariable analysis adjusted for gender, age, KRAS, PI3KCA and performance status. miR-345 was prognostic in progression-free survival (PFS) with a HR = 1.63 per IQR (CI 95%: 1.25–2.114, P-Wald = 2.92e-4) in the multivariable analysis. In addition, high miR-345 expression was associated with lack of response to treatment with cetuximab and irinotecan.
Conclusion
We identified miR-345 in whole blood as a potential biomarker for clinical outcome. MiR-345 was a single prognostic biomarker for both OS and PFS in all patients and also in the non-KRAS mutant population.
doi:10.1371/journal.pone.0099886
PMCID: PMC4062472  PMID: 24940606
5.  Therapeutic synergy between microRNA and siRNA in ovarian cancer treatment 
Cancer discovery  2013;3(11):10.1158/2159-8290.CD-13-0159.
Development of improved RNA interference based strategies is of utmost clinical importance. While siRNA-mediated silencing of EphA2, an ovarian cancer oncogene, results in reduction of tumor growth, we present evidence that additional inhibition of EphA2 by a microRNA further ‘boosts’ its anti-tumor effects. We identified miR-520d-3p as a tumor suppressor upstream of EphA2, whose expression correlated with favorable outcomes in two independent patient cohorts comprising of 647 patients. Restoration of miR-520d-3p prominently decreased EphA2 protein levels, and suppressed tumor growth and migration/invasion both in vitro and in vivo. Dual inhibition of EphA2 in vivo using DOPC nano-liposomes loaded with miR-520d-3p and EphA2-siRNA showed synergistic anti-tumor efficiency and greater therapeutic efficacy than either monotherapy alone. This synergy is atleast in part due to miR-520d-3p targeting EphB2, another Eph receptor. Our data emphasize the feasibility of combined miRNA-siRNA therapy, and will have broad implications for innovative gene silencing therapies for cancer and other diseases.
doi:10.1158/2159-8290.CD-13-0159
PMCID: PMC3855315  PMID: 24002999
miR-520d-3p; EphA2; EphB2; ovarian cancer; RNA interference
6.  Usability on the p-medicine infrastructure: an extended usability concept 
ecancermedicalscience  2014;8:399.
Usability testing methods are nowadays integrated into the design and development of health-care software, and the need for usability in health-care information technology (IT) is widely accepted by clinicians and researchers. Usability assessment starts with the identification of specific objectives that need to be tested and continues with the definition of evaluation criteria and monitoring procedures before usability tests are performed to assess the quality of all services and tasks. Such a process is implemented in the p-medicine environment and gives feedback iteratively to all software developers in the project. GCP (good clinical practice) criteria require additional usability testing of the software. For the p-medicine project (www.p-medicine.eu), an extended usability concept (EUC) was developed. The EUC covers topics like ease of use, likeability, and usefulness, usability in trial centres characterised by a mixed care and research environment and by extreme time constraints, confidentiality, use of source documents, standard operating procedures (SOA), and quality control during data handling to ensure that all data are reliable and have been processed correctly in terms of accuracy, completeness, legibility, consistence, and timeliness. Here, we describe the p-medicine EUC, focusing on two of the many key tools: ObTiMA and the Ontology Annotator (OA).
doi:10.3332/ecancer.2014.399
PMCID: PMC3922651  PMID: 24567756
health care; evaluation; good clinical practice
7.  Patterns of Acetaminophen Use Exceeding 4 Grams Daily in a Hospitalized Population at a Tertiary Care Center 
Gastroenterology & Hepatology  2014;10(1):27-34.
Unintentional acetaminophen-induced hepatotoxicity has been increasingly recognized as a significant problem, prompting increased scrutiny and restrictions from the US Food and Drug Administration on products combining acetaminophen with narcotics. Patterns of acetaminophen use have not previously been reported in the hospitalized patient population, which may be especially vulnerable to liver injury. We aimed to quantify the frequency at which acetaminophen dosing exceeded the recommended maximum of 4 g/day in hospitalized patients. This was a retrospective, single-center, cohort study at a large tertiary care academic hospital. We queried our inpatient electronic medical record database to identify patients admitted between 2008 and 2010 who were receiving cumulative daily acetaminophen doses exceeding 4 g on at least 1 hospital day. Of 43,761 admissions involving acetaminophen administration, the recommended maximum cumulative daily dose of 4 g was exceeded in 1119 (2.6%) cases. Patients who were administered a larger number of acetaminophen-containing medications were more likely to receive doses in excess of the recommended maximum. Alanine aminotransferase (ALT) levels were checked within 14 days following acetaminophen exposure in excess of 4 g in 35 (3.1%) cases. Excessive acetaminophen dosing of hospitalized patients, who may be at increased risk for acetaminophen-induced hepatotoxicity, occurred in a minority of patients. The use of multiple acetaminophen-containing medication formulations contributed to excessive dosing. ALT level monitoring in this group was infrequent, precluding assessment of biochemical evidence of liver injury. This cohort of patients may represent an ideal population for further prospective study with more intensive and longer-term biochemical monitoring to assess for evidence of liver injury.
PMCID: PMC4008956  PMID: 24799836
Acetaminophen; drug-induced liver injury; hepatotoxicity; hospitalized patients; drug safety
8.  Rac1 at the crossroad of actin dynamics and neuroinflammation in Amyotrophic Lateral Sclerosis 
Rac1 is a major player of the Rho family of small GTPases that controls multiple cell signaling pathways, such as the organization of cytoskeleton (including adhesion and motility), cell proliferation, apoptosis and activation of immune cells. In the nervous system, in particular, Rac1 GTPase plays a key regulatory function of both actin and microtubule cytoskeletal dynamics and thus it is central to axonal growth and stability, as well as dendrite and spine structural plasticity. Rac1 is also a crucial regulator of NADPH-dependent membrane oxidase (NOX), a prominent source of reactive oxygen species (ROS), thus having a central role in the inflammatory response and neurotoxicity mediated by microglia cells in the nervous system. As such, alterations in Rac1 activity might well be involved in the processes that give rise to Amyotrophic Lateral Sclerosis (ALS), a complex syndrome where cytoskeletal disturbances in motor neurons and redox alterations in the inflammatory compartment play pivotal and synergic roles in the final disease outcomes. Here we will discuss the genetic and mechanistic evidence indicating the relevance of Rac1 dysregulation in the pathogenesis of ALS.
doi:10.3389/fncel.2014.00279
PMCID: PMC4157560  PMID: 25249940
Amyotrophic Lateral Sclerosis (ALS); Rac1; neuroinflammation; motor neurons; NOX; microglia; reactive oxygen species; spinal muscular atrophy (SMA)
9.  Epigenetic silencing of microRNA-203 is required for EMT and cancer stem cell properties 
Scientific Reports  2013;3:2687.
The epithelial-mesenchymal transition (EMT) imparts metastatic competence on otherwise non-metastatic cancer cells through decreased inter-cellular adhesions, increased migratory capacity, stem cell properties and anoikis and chemotherapy resistance. In this study, we profiled changes in microRNA expression during EMT in conjunction with changes in DNA methylation at microRNA promoters to discover essential mediators of EMT-imparted stemness properties. MicroRNA-203 (miR-203) expression is repressed following EMT induced by multiple different stimuli and in established claudin-low cell lines as well as the CD44hi/CD24lo stem cell-enriched fraction. Expression of miR-203 in mesenchymal cells compromises migratory and invasive capacity in vitro, and tumor initiation and metastasis in vivo. Unexpectedly, miR-203 expression affects the sphere-forming capacity of neighboring cells by indirectly enhancing expression of DKK1, a secreted inhibitor of Wnt signaling and stemness resulting in suppression of β-catenin protein levels. Our data suggest that restoring miR-203 expression levels may inhibit metastasis and combat deregulated Wnt signaling.
doi:10.1038/srep02687
PMCID: PMC3776231  PMID: 24045437
10.  Association of a MicroRNA/TP53 Feedback Circuitry With Pathogenesis and Outcome of B-Cell Chronic Lymphocytic Leukemia 
Context
Chromosomal abnormalities (namely 13q, 17p, and 11q deletions) have prognostic implications and are recurrent in chronic lymphocytic leukemia (CLL), suggesting that they are involved in a common pathogenetic pathway; however, the molecular mechanism through which chromosomal abnormalities affect the pathogenesis and outcome of CLL is unknown.
Objective
To determine whether the microRNA miR-15a/miR-16-1 cluster (located at 13q), tumor protein p53 (TP53, located at 17p), and miR-34b/miR-34c cluster (located at 11q) are linked in a molecular pathway that explains the pathogenetic and prognostic implications (indolent vs aggressive form) of recurrent 13q, 17p, and 11q deletions in CLL.
Design, Setting, and Patients
CLL Research Consortium institutions provided blood samples from untreated patients (n=206) diagnosed with B-cell CLL between January 2000 and April 2008. All samples were evaluated for the occurrence of cytogenetic abnormalities as well as the expression levels of the miR-15a/miR-16-1 cluster, miR-34b/miR-34c cluster, TP53, and zeta-chain (TCR)–associated protein kinase 70kDa (ZAP70), a surrogate prognostic marker of CLL. The functional relationship between these genes was studied using in vitro gain- and loss-of-function experiments in celllines and primary samples and was validated in a separate cohort of primary CLL samples.
Main Outcome Measures
Cytogenetic abnormalities; expression levels of the miR-15a/miR-16-1 cluster, miR-34 family, TP53 gene, downstream effectors cyclindependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and B-cell CLL/lymphoma 2 binding component 3 (BBC3), and ZAP70 gene; genetic interactions detected by chromatin immunoprecipitation.
Results
In CLLs with13qdeletions the miR-15a/miR-16-1 cluster directly targetedTP53 (mean luciferase activity for miR-15a vs scrambled control, 0.68 relative light units (RLU) [95%confidence interval {CI}, 0.63–0.73]; P=.02;meanfor miR-16 vs scrambled control, 0.62RLU[95%CI, 0.59–0.65]; P=.02) and its downstream effectors. In leukemic cell lines and primary CLL cells, TP53 stimulated the transcription of miR-15/miR-16-1 as well as miR-34b/miR-34c clusters, and the miR-34b/miR-34c cluster directly targeted theZAP70 kinase(meanluciferase activity for miR-34a vs scrambled control, 0.33RLU [95%CI, 0.30–0.36]; P=.02;meanformiR-34bvsscrambledcontrol,0.31RLU [95%CI, 0.30–0.32];P=.01; and mean for miR-34c vs scrambled control, 0.35 RLU [95% CI, 0.33–0.37]; P=.02).
Conclusions
A microRNA/TP53 feedback circuitry is associated with CLL pathogenesis and outcome. This mechanism provides a novel pathogenetic model for the association of 13q deletions with the indolent form of CLL that involves microRNAs, TP53, and ZAP70
doi:10.1001/jama.2010.1919
PMCID: PMC3690301  PMID: 21205967
11.  Profiling HBV integrations in hepatocellular carcinoma 
doi:10.3978/j.issn.2304-3881.2012.10.13
PMCID: PMC3924654  PMID: 24570928
12.  Modulation of MicroRNA-194 and Cell Migration by HER2-Targeting Trastuzumab in Breast Cancer 
PLoS ONE  2012;7(7):e41170.
Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 oncoprotein, can effectively target HER2-positive breast cancer through several mechanisms. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis and apoptosis have been investigated in depth, the effect of trastuzumab on microRNA (miRNA) has not been extensively studied. We have performed miRNA microarray profiling before and after trastuzumab treatment in SKBr3 and BT474 human breast cancer cells that overexpress HER2. We found that trastuzumab treatment of SKBr3 cells significantly decreased five miRNAs and increased three others, whereas treatment of BT474 cells significantly decreased two miRNAs and increased nine. The only change in miRNA expression observed in both cell lines following trastuzumab treatment was upregulation of miRNA-194 (miR-194) that was further validated in vitro and in vivo. Forced expression of miR-194 in breast cancer cells that overexpress HER2 produced no effect on apoptosis, modest inhibition of proliferation, significant inhibition of cell migration/invasion in vitro and significant inhibition of xenograft growth in vivo. Conversely, knockdown of miR-194 promoted cell migration. Increased miR-194 expression markedly reduced levels of the cytoskeletal protein talin2 and specifically inhibited luciferase reporter activity of a talin2 wild-type 3′-untranslated region, but not that of a mutant reporter, indicating that talin2 is a direct downstream target of miR-194. Trastuzumab treatment inhibited breast cancer cell migration and reduced talin2 expression in vitro and in vivo. Knockdown of talin2 inhibited cell migration/invasion. Knockdown of trastuzumab-induced miR-194 expression with a miR-194 inhibitor compromised trastuzumab-inhibited cell migration in HER2-overexpressing breast cancer cells. Consequently, trastuzumab treatment upregulates miR-194 expression and may exert its cell migration-inhibitory effect through miR-194-mediated downregulation of cytoskeleton protein talin2 in HER2-overexpressing human breast cancer cells.
doi:10.1371/journal.pone.0041170
PMCID: PMC3400637  PMID: 22829924
13.  Non-codingRNA sequence variations in human chronic lymphocytic leukemia and colorectal cancer 
Carcinogenesis  2009;31(2):208-215.
Cancer is a genetic disease in which the interplay between alterations in protein-coding genes and non-coding RNAs (ncRNAs) plays a fundamental role. In recent years, the full coding component of the human genome was sequenced in various cancers, whereas such attempts related to ncRNAs are still fragmentary. We screened genomic DNAs for sequence variations in 148 microRNAs (miRNAs) and ultraconserved regions (UCRs) loci in patients with chronic lymphocytic leukemia (CLL) or colorectal cancer (CRC) by Sanger technique and further tried to elucidate the functional consequences of some of these variations. We found sequence variations in miRNAs in both sporadic and familial CLL cases, mutations of UCRs in CLLs and CRCs and, in certain instances, detected functional effects of these variations. Furthermore, by integrating our data with previously published data on miRNA sequence variations, we have created a catalog of DNA sequence variations in miRNAs/ultraconserved genes in human cancers. These findings argue that ncRNAs are targeted by both germ line and somatic mutations as well as by single-nucleotide polymorphisms with functional significance for human tumorigenesis. Sequence variations in ncRNA loci are frequent and some have functional and biological significance. Such information can be exploited to further investigate on a genome-wide scale the frequency of genetic variations in ncRNAs and their functional meaning, as well as for the development of new diagnostic and prognostic markers for leukemias and carcinomas.
doi:10.1093/carcin/bgp209
PMCID: PMC2812567  PMID: 19926640
14.  PDGF induced microRNA alterations in cancer cells 
Nucleic Acids Research  2011;39(10):4035-4047.
Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust changes consisting of let-7d repression by PDGF-AA and miR-146b induction by PDGF-BB. Induction of miR-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos. We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target. We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas. We propose that PDGF-regulated gene transcription involves alterations in non-coding RNAs and provide evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells.
doi:10.1093/nar/gkq1305
PMCID: PMC3105413  PMID: 21266476
15.  Ontogenetic Profile of the Expression of Thyroid Hormone Receptors in Rat and Human Corpora Cavernosa of the Penis 
The Journal of Sexual Medicine  2010;7(4pt1):1381-1390.
Introduction
In the last few years, various studies have underlined a correlation between thyroid function and male sexual function, hypothesizing a direct action of thyroid hormones on the penis.
Aim
To study the spatiotemporal distribution of mRNA for the thyroid hormone nuclear receptors (TR) α1, α2 and β in the penis and smooth muscle cells (SMCs) of the corpora cavernosa of rats and humans during development.
Methods
We used several molecular biology techniques to study the TR expression in whole tissues or primary cultures from human and rodent penile tissues of different ages.
Main Outcome Measure
We measured our data by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) amplification, Northern blot and immunohistochemistry.
Results
We found that TRα1 and TRα2 are both expressed in the penis and in SMCs during ontogenesis without development-dependent changes. However, in the rodent model, TRβ shows an increase from 3 to 6 days post natum (dpn) to 20 dpn, remaining high in adulthood. The same expression profile was observed in humans. While the expression of TRβ is strictly regulated by development, TRα1 is the principal isoform present in corpora cavernosa, suggesting its importance in SMC function. These results have been confirmed by immunohistochemistry localization in SMCs and endothelial cells of the corpora cavernosa.
Conclusions
The presence of TRs in the penis provides the biological basis for the direct action of thyroid hormones on this organ. Given this evidence, physicians would be advised to investigate sexual function in men with thyroid disorders. Carosa E, Di Sante S, Rossi S, Castri A, D'Adamo F, Gravina GL, Ronchi P, Kostrouch Z, Dolci S, Lenzi A, and Jannini EA. Ontogenetic profile of the expression of thyroid hormone receptors in rat and human corpora cavernosa of the penis. J Sex Med 2010;7:1381–1390.
doi:10.1111/j.1743-6109.2009.01701.x
PMCID: PMC3017743  PMID: 20141582
Corpora Cavernosa; Thyroid Hormone Receptor; Erectile Dysfunction; Thyroid Disorders and Sexual Dysfunction
16.  MiR-15a and MiR-16 control Bmi-1 expression in ovarian cancer 
Cancer research  2009;69(23):9090-9095.
Oncogenic activation of Bmi-1 is found in a wide variety of epithelial malignancies including ovarian cancer, yet a specific mechanism for over expression of Bmi-1 has not been determined. Thus realizing the immense pathological significance of Bmi-1 in cancer, we wanted to investigate if microRNA aberrations played a role in the regulation of Bmi-1 in ovarian cancer. In this report we identify two microRNAs, miR-15a and miR-16 that are under expressed in ovarian cell lines and in primary ovarian tissues. We demonstrate that these miRNAs directly target the Bmi-1 3’ UTR and significantly correlate with Bmi-1 protein levels in ovarian cancer patients and cell lines. Furthermore, Bmi-1 protein levels are down regulated in response to miR-15a or miR-16 expression and lead to significant reduction in ovarian cancer cell proliferation and clonal growth. These findings suggest the development of therapeutic strategies by restoring miR-15a and miR-16 expression in ovarian cancer and in other cancers that involve up regulation of Bmi-1.
doi:10.1158/0008-5472.CAN-09-2552
PMCID: PMC2859686  PMID: 19903841
MicroRNA; ovarian cancer; Bmi-1; clonal growth; proliferation
17.  Disrupted microRNA expression caused by Mecp2 loss in a mouse model of Rett syndrome 
Epigenetics  2010;5(7):656-663.
MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate post-transcriptional gene expression. They influence a wide range of physiological functions, including neuronal processes, and are regulated by various mechanisms, such as DNA methylation. This epigenetic mark is recognized by transcriptional regulators such as the methyl CpG binding protein Mecp2. Rett syndrome is a complex neurological disorder that has been associated with mutations in the gene coding for Mecp2. Thus, we examined the possible miRNA misregulation caused by Mecp2 absence in a mouse model of Rett syndrome. Using miRNA expression microarrays, we observed that the brain of Rett syndrome mice undergoes a disruption of the expression profiles of miRNAs. Among the significantly altered miRNAs (26%, 65 of 245), overall downregulation of these transcripts was the most common feature (71%), while the remaining 30% were upregulated. Further validation by quantitative RT-PCR demonstrated that the most commonly disrupted miRNAs were miR-146a, miR-146b, miR-130, miR-122a, miR-342 and miR-409 (downregulated) and miR-29b, miR329, miR-199b, miR-382, miR-296, miR-221 and miR-92 (upregulated). Most importantly, transfection of miR-146a in a neuroblastoma cell line caused the downregulation of IL-1 receptor-associated kinase 1 (Irak1) levels, suggesting that the identified defect of miR-146a in Rett syndrome mice brains might be responsible for the observed upregulation of Irak1 in this model of the human disease. Overall, we provide another level of molecular deregulation occurring in Rett syndrome that might be useful for understanding the disease and for designing targeted therapies.
doi:10.4161/epi.5.7.13055
PMCID: PMC3052849  PMID: 20716963
Rett syndrome; Mecp2; microRNAs; DNA methylation; chromatin
18.  Hematopoietic differentiation: a coordinated dynamical process towards attractor stable states 
BMC Systems Biology  2010;4:85.
Background
The differentiation process, proceeding from stem cells towards the different committed cell types, can be considered as a trajectory towards an attractor of a dynamical process. This view, taking into consideration the transcriptome and miRNome dynamics considered as a whole, instead of looking at few 'master genes' driving the system, offers a novel perspective on this phenomenon. We investigated the 'differentiation trajectories' of the hematopoietic system considering a genome-wide scenario.
Results
We developed serum-free liquid suspension unilineage cultures of cord blood (CB) CD34+ hematopoietic progenitor cells through erythroid (E), megakaryocytic (MK), granulocytic (G) and monocytic (Mo) pathways. These cultures recapitulate physiological hematopoiesis, allowing the analysis of almost pure unilineage precursors starting from initial differentiation of HPCs until terminal maturation. By analyzing the expression profile of protein coding genes and microRNAs in unilineage CB E, MK, G and Mo cultures, at sequential stages of differentiation and maturation, we observed a coordinated, fully interconnected and scalable character of cell population behaviour in both transcriptome and miRNome spaces reminiscent of an attractor-like dynamics. MiRNome and transcriptome space differed for a still not terminally committed behaviour of microRNAs.
Conclusions
Consistent with their roles, the transcriptome system can be considered as the state space of a cell population, while the continuously evolving miRNA space corresponds to the tuning system necessary to reach the attractor. The behaviour of miRNA machinery could be of great relevance not only for the promise of reversing the differentiated state but even for tumor biology.
doi:10.1186/1752-0509-4-85
PMCID: PMC2904736  PMID: 20553595
19.  MicroRNA Fingerprints Identify miR-150 as a Plasma Prognostic Marker in Patients with Sepsis 
PLoS ONE  2009;4(10):e7405.
Background
The physiopathology of sepsis continues to be poorly understood, and despite recent advances in its management, sepsis is still a life-threatening condition with a poor outcome. If new diagnostic markers related to sepsis pathogenesis will be identified, new specific therapies might be developed and mortality reduced. Small regulatory non-coding RNAs, microRNAs (miRNAs), were recently linked to various diseases; the aim of our prospective study was to identify miRNAs that can differentiate patients with early-stage sepsis from healthy controls and to determine if miRNA levels correlate with the severity assessed by the Sequential Organ Failure Assessment (SOFA) score.
Methodology/Principal Findings
By using genome-wide miRNA profiling by microarray in peripheral blood leukocytes, we found that miR-150, miR-182, miR-342-5p, and miR-486 expression profiles differentiated sepsis patients from healthy controls. We also proved by quantitative reverse transcription-polymerase chain reaction that miR-150 levels were significantly reduced in plasma samples of sepsis patients and correlated with the level of disease severity measured by the SOFA score, but were independent of the white blood counts (WBC). We found that plasma levels of tumor necrosis factor alpha, interleukin-10, and interleukin-18, all genes with sequence complementarity to miR-150, were negatively correlated with the plasma levels of this miRNA. Furthermore, we identified that the plasma levels ratio for miR-150/interleukin-18 can be used for assessing the severity of the sepsis.
Conclusions/Significance
We propose that miR-150 levels in both leukocytes and plasma correlate with the aggressiveness of sepsis and can be used as a marker of early sepsis. Furthermore, we envision miR-150 restoration as a future therapeutic option in sepsis patients.
doi:10.1371/journal.pone.0007405
PMCID: PMC2756627  PMID: 19823581
20.  Pegylated interferon 2a and 2b in combination with ribavirin for the treatment of chronic hepatitis C in HIV infected patients 
Coinfection with hepatitis C virus (HCV) and HIV is an increasingly recognized clinical dilemma, particularly since the advent of highly active antiretroviral therapy. Several studies of this population have demonstrated both more rapid progression of liver disease and poorer overall prognosis compared to HCV monoinfected patients. Consensus guidelines, based primarily on the results of 4 major randomized trials, recommend treatment with peginterferon and ribavirin for 48 weeks in coinfected patients. However, this current standard of care is associated with lower response rates to therapy than those seen in monoinfected patients. Important predictors of response include HCV genotype, pretreatment HCV RNA level, and presence of rapid virologic response (RVR) and early virologic response (EVR). Use of weight-based ribavirin dosing appears to be safe and enhances the likelihood of sustained virologic response (SVR). Adverse effects most commonly encountered are anemia and weight loss. Mitochondrial toxicity can occur in the setting of concomitant nucleoside reverse transcriptase inhibitor use, especially didanosine, abacavir, and zidovudine, and these should be discontinued before initiation of ribavirin therapy. Discontinuation of therapy should be considered in patients failing to demonstrate EVR, though ongoing trials are investigating a potential role for maintenance therapy in these patients. Peginterferon combined with weight-based ribavirin is appropriate and safe for treatment of HCV in HIV – HCV coinfected patients. This review summarizes the data supporting these recommendations.
PMCID: PMC2621394  PMID: 19209261
hepatitis C; human immunodeficiency virus; peginterferon; ribavirin
21.  RANK signals from CD4+3− inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla 
The Journal of Experimental Medicine  2007;204(6):1267-1272.
Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4+3− inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4+3− cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire+ mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80−Aire− mTEC progenitors into CD80+Aire+ mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire+ mTECs and highlight a previously unrecognized role for CD4+3−RANKL+ inducer cells in intrathymic self-tolerance.
doi:10.1084/jem.20062497
PMCID: PMC2118623  PMID: 17502664
22.  Compatible solutes from hyperthermophiles improve the quality of DNA microarrays 
BMC Biotechnology  2007;7:82.
Background
DNA microarrays are among the most widely used technical platforms for DNA and RNA studies, and issues related to microarrays sensitivity and specificity are therefore of general importance in life sciences. Compatible solutes are derived from hyperthermophilic microorganisms and allow such microorganisms to survive in environmental and stressful conditions. Compatible solutes show stabilization effects towards biological macromolecules, including DNA.
Results
We report here that compatible solutes from hyperthermophiles increased the performance of the hybridization buffer for Affymetrix GeneChip® arrays. The experimental setup included independent hybridizations with constant RNA over a wide range of compatible solute concentrations. The dependence of array quality and compatible solute was assessed using specialized statistical tools provided by both the proprietary Affymetrix quality control system and the open source Bioconductor suite.
Conclusion
Low concentration (10 to 25 mM) of hydroxyectoine, potassium mannosylglycerate and potassium diglycerol phosphate in hybridization buffer positively affected hybridization parameters and enhanced microarrays outcome. This finding harbours a strong potential for the improvement of DNA microarray experiments.
doi:10.1186/1472-6750-7-82
PMCID: PMC2248183  PMID: 18036223
23.  Hepatitis C risk assessment, testing and referral for treatment in urban primary care: Role of race and ethnicity 
AIM: To determine rates of hepatitis C (HCV) risk factor ascertainment, testing, and referral in urban primary care practices, with particular attention to the effect of race and ethnicity.
METHODS: Retrospective chart review from four primary care sites in Philadelphia; two academic primary care practices and two community clinics was performed. Demographics, HCV risk factors, and other risk exposure information were collected.
RESULTS: Four thousand four hundred and seven charts were reviewed. Providers documented histories of injection drug use (IDU) and transfusion for less than 20% and 5% of patients, respectively. Only 55% of patients who admitted IDU were tested for HCV. Overall, minorities were more likely to have information regarding a risk factor documented than their white counterparts (79% vs 68%, P < 0.0001). Hispanics were less likely to have a risk factor history documented, compared to blacks and whites (P < 0.0001). Overall, minorities were less likely to be tested for HCV than whites in the presence of a known risk factor (23% vs 35%, P = 0.004). Among patients without documentation of risk factors, blacks and Hispanics were more likely to be tested than whites (20% and 24%, vs 13%, P < 0.005, respectively).
CONCLUSION: (1) Documentation of an HCV risk factor history in urban primary care is uncommon, (2) Racial differences exist with respect to HCV risk factor ascertainment and testing, (3) Minority patients, positive for HCV, are less likely to be referred for subspecialty care and treatment. Overall, minorities are less likely to be tested for HCV than whites in the presence of a known risk factor.
doi:10.3748/wjg.v13.i7.1074
PMCID: PMC4146870  PMID: 17373742
Hepatitis C; Minority groups; Urban health; Primary health care; Risk assessment
24.  TOM: a web-based integrated approach for identification of candidate disease genes 
Nucleic Acids Research  2006;34(Web Server issue):W285-W292.
The massive production of biological data by means of highly parallel devices like microarrays for gene expression has paved the way to new possible approaches in molecular genetics. Among them the possibility of inferring biological answers by querying large amounts of expression data. Based on this principle, we present here TOM, a web-based resource for the efficient extraction of candidate genes for hereditary diseases. The service requires the previous knowledge of at least another gene responsible for the disease and the linkage area, or else of two disease associated genetic intervals. The algorithm uses the information stored in public resources, including mapping, expression and functional databases. Given the queries, TOM will select and list one or more candidate genes. This approach allows the geneticist to bypass the costly and time consuming tracing of genetic markers through entire families and might improve the chance of identifying disease genes, particularly for rare diseases. We present here the tool and the results obtained on known benchmark and on hereditary predisposition to familial thyroid cancer. Our algorithm is available at .
doi:10.1093/nar/gkl340
PMCID: PMC1538851  PMID: 16845011

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