Serum miRNA Profiling for Early PDAC Diagnosis and Prognosis: A Retrospective Study.

BACKGROUND: Tumor stage predicts pancreatic cancer (PDAC) prognosis, but prolonged and short survivals have been described in patients with early-stage tumors. Circulating microRNA (miRNA) are an emerging class of suitable biomarkers for PDAC prognosis. Our aim was to identify whether serum miRNA signatures predict survival of early-stage PDAC. METHODS: Serum RNA from archival 15 stage I-III PDAC patients and 4 controls was used for miRNAs expression profile (Agilent microarrays). PDAC patients with comparable age, gender, diabetes, jaundice and surgery were classified according to survival: less than 14 months (7/15 pts, group A) and more than 22 months (8/15 pts, group B). Bioinformatic data analysis was performed by two-class Significance Analysis of Microarray (SAM) algorithm. Binary logistic regression analyses considering PDAC diagnosis and outcome as dependent variables, and ROC analyses were also performed. RESULTS: 2549 human miRNAs were screened out. At SAM, 76 differentially expressed miRNAs were found among controls and PDAC (FDR = 0.4%), the large majority (50/76, 66%) of them being downregulated in PDAC with respect to controls. Six miRNAs were independently correlated with early PDAC, and among these, hsa-miR-6821-5p was associated with the best ROC curve area in distinguishing controls from early PDAC. Among the 71 miRNAs differentially expressed between groups A and B, the most significant were hsa-miR-3135b expressed in group A only, hsa-miR-6126 and hsa-miR-486-5p expressed in group B only. Eight miRNAs were correlated with the presence of lymph-node metastases; among these, hsa-miR-4669 is of potential interest. hsa-miR-4516, increased in PDAC and found as an independent predictor of survival, has among its putative targets a series of gens involved in key pathways of cancer progression and dissemination, such as Wnt and p53 signalling pathways. CONCLUSIONS: A series of serum miRNAs was identified as potentially useful for the early diagnosis of PDAC, and for establishing a prognosis.

Exploiting pyocyanin to treat mitochondrial disease due to respiratory complex III dysfunction.

Mitochondrial diseases impair oxidative phosphorylation and ATP production, while effective treatment is still lacking. Defective complex III is associated with a highly variable clinical spectrum. We show that pyocyanin, a bacterial redox cycler, can replace the redox functions of complex III, acting as an electron shunt. Sub-µM pyocyanin was harmless, restored respiration and increased ATP production in fibroblasts from five patients harboring pathogenic mutations in TTC19, BCS1L or LYRM7, involved in assembly/stabilization of complex III. Pyocyanin normalized the mitochondrial membrane potential, and mildly increased ROS production and biogenesis. These in vitro effects were confirmed in both DrosophilaTTC19KO and in Danio rerioTTC19KD, as administration of low concentrations of pyocyanin significantly ameliorated movement proficiency. Importantly, daily administration of pyocyanin for two months was not toxic in control mice. Our results point to utilization of redox cyclers for therapy of complex III disorders.

Investigating the epi-miRNome: identification of epi-miRNAs using transfection experiments.

Aim: Growing evidence shows a strong interplay between post-transcriptional regulation, mediated by miRNAs (miRs) and epigenetic regulation. Nevertheless, the number of experimentally validated miRs (called epi-miRs) involved in these regulatory circuitries is still very small. Material & methods: We propose a pipeline to prioritize candidate epi-miRs and to identify potential epigenetic interactors of any given miR starting from miR transfection experiment datasets. Results & conclusion: We identified 34 candidate epi-miRs: 19 of them are known epi-miRs, while 15 are new. Moreover, using an in-house generated gene expression dataset, we experimentally proved that a component of the polycomb-repressive complex 2, the histone methyltransferase enhancer of zeste homolog 2 (EZH2), interacts with miR-214, a well-known prometastatic miR in melanoma and breast cancer, highlighting a miR-214-EZH2 regulatory axis potentially relevant in tumor progression.

Identifying pathways modulating sleep duration: from genomics to transcriptomics.

Recognizing that insights into the modulation of sleep duration can emerge by exploring the functional relationships among genes, we used this strategy to explore the genome-wide association results for this trait. We detected two major signalling pathways (ion channels and the ERBB signalling family of tyrosine kinases) that could be replicated across independent GWA studies meta-analyses. To investigate the significance of these pathways for sleep modulation, we performed transcriptome analyses of short sleeping flies' heads (knockdown for the ABCC9 gene homolog; dSur). We found significant alterations in gene-expression in the short sleeping knockdowns versus controls flies, which correspond to pathways associated with sleep duration in our human studies. Most notably, the expression of Rho and EGFR (members of the ERBB signalling pathway) genes was down- and up-regulated, respectively, consistently with the established role of these genes for sleep consolidation in Drosophila. Using a disease multifactorial interaction network, we showed that many of the genes of the pathways indicated to be relevant for sleep duration had functional evidence of their involvement with sleep regulation, circadian rhythms, insulin secretion, gluconeogenesis and lipogenesis.

miR-146a Exerts Differential Effects on Melanoma Growth and Metastatization.

UNLABELLED: Malignant melanoma is the most aggressive form of skin cancer; therefore, it is crucial to disclose its underlying molecular mechanisms. MicroRNAs (miRNAs) are small endogenous noncoding RNAs able to posttranscriptionally downregulate the expression of direct target genes. Using a melanoma progression model, miR-146a was identified as a key double-acting player in melanoma malignancy. In fact, miR-146a is able to enhance tumor growth, while it suppresses dissemination. It was determined that miR-146a coordinated melanoma cell growth by its direct targets lunatic fringe (LFNG) and NUMB, which operate on the NOTCH/PTEN/Akt pathway; while inhibition of metastasis formation was linked to decreased expression of ITGAV and ROCK1. Relevantly, miR-146a expression correlated with melanoma recurrence and was enriched in both patient-derived melanoma and cutaneous metastasis specimens, while its direct targets were depleted. However, miR-146a levels drop in circulating tumor cells (CTCs), suggesting the necessity for miR-146a expression to fluctuate during tumor progression in order to favor tumor growth and allow dissemination. This study reconciles the contradictory biologic functions of miR-146a in melanoma progression and unravels distinct molecular mechanisms that need to be considered for therapeutic interventions. IMPLICATIONS: miR-146a controls melanoma progression in a dual way, promoting growth and inhibiting dissemination; however, it is poorly expressed in CTCs, resulting in overall tumor spreading and distant-site colonization. Mol Cancer Res; 14(6); 548-62. ©2016 AACR.

MicroRNA-27a Contributes to Rhabdomyosarcoma Cell Proliferation by Suppressing RARA and RXRA.

BACKGROUND: Rhabdomyosarcomas (RMS) are rare but very aggressive childhood tumors that arise as a consequence of a regulatory disruption in the growth and differentiation pathways of myogenic precursor cells. According to morphological criteria, there are two major RMS subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS) with the latter showing greater aggressiveness and metastatic potential with respect to the former. Efforts to unravel the complex molecular mechanisms underlying RMS pathogenesis and progression have revealed that microRNAs (miRNAs) play a key role in tumorigenesis. METHODOLOGY/PRINCIPAL FINDINGS: The expression profiles of 8 different RMS cell lines were analyzed to investigate the involvement of miRNAs in RMS. The miRNA population from each cell line was compared to a reference sample consisting of a balanced pool of total RNA extracted from those 8 cell lines. Sixteen miRNAs whose expression discriminates between translocation-positive ARMS and negative RMS were identified. Attention was focused on the role of miR-27a that is up-regulated in the more aggressive RMS cell lines (translocation-positive ARMS) in which it probably acts as an oncogene. MiR-27a overexpressing cells showed a significant increase in their proliferation rate that was paralleled by a decrease in the number of cells in the G1 phase of the cell cycle. It was possible to demonstrate that miR-27a is implicated in cell cycle control by targeting the retinoic acid alpha receptor (RARA) and retinoic X receptor alpha (RXRA). CONCLUSIONS: Study results have demonstrated that miRNA expression signature profiling can be used to classify different RMS subtypes and suggest that miR-27a may have a therapeutic potential in RMS by modulating the expression of retinoic acid receptors.

Involvement of microRNAs in the regulation of muscle wasting during catabolic conditions.

Loss of muscle proteins and the consequent weakness has important clinical consequences in diseases such as cancer, diabetes, chronic heart failure, and in aging. In fact, excessive proteolysis causes cachexia, accelerates disease progression, and worsens life expectancy. Muscle atrophy involves a common pattern of transcriptional changes in a small subset of genes named atrophy-related genes or atrogenes. Whether microRNAs play a role in the atrophy program and muscle loss is debated. To understand the involvement of miRNAs in atrophy we performed miRNA expression profiling of mouse muscles under wasting conditions such as fasting, denervation, diabetes, and cancer cachexia. We found that the miRNA signature is peculiar of each catabolic condition. We then focused on denervation and we revealed that changes in transcripts and microRNAs expression did not occur simultaneously but were shifted. Indeed, whereas transcriptional control of the atrophy-related genes peaks at 3 days, changes of miRNA expression maximized at 7 days after denervation. Among the different miRNAs, microRNA-206 and -21 were the most induced in denervated muscles. We characterized their pattern of expression and defined their role in muscle homeostasis. Indeed, in vivo gain and loss of function experiments revealed that miRNA-206 and miRNA-21 were sufficient and required for atrophy program. In silico and in vivo approaches identified transcription factor YY1 and the translational initiator factor eIF4E3 as downstream targets of these miRNAs. Thus miRNAs are important for fine-tuning the atrophy program and their modulation can be a novel potential therapeutic approach to counteract muscle loss and weakness in catabolic conditions.

The DNA-damage response to γ-radiation is affected by miR-27a in A549 cells.

Perturbations during the cell DNA-Damage Response (DDR) can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs), small non-coding RNAs that act as post-transcriptional regulators of gene expression. The oncogenic miR-27a is over-expressed in several tumors and, in the present study, we investigated its interaction with ATM, the gene coding for the main kinase of DDR pathway. Experimental validation to confirm miR-27a as a direct regulator of ATM was performed by site-direct mutagenesis of the luciferase reporter vector containing the 3'UTR of ATM gene, and by miRNA oligonucleotide mimics. We then explored the functional miR-27a/ATM interaction under biological conditions, i.e., during the response of A549 cells to ionizing radiation (IR) exposure. To evaluate if miR-27a over-expression affects IR-induced DDR activation in A549 cells we determined cell survival, cell cycle progression and DNA double-strand break (DSB) repair. Our results show that up-regulation of miR-27a promotes cell proliferation of non-irradiated and irradiated cells. Moreover, increased expression of endogenous mature miR-27a in A549 cells affects DBS rejoining kinetics early after irradiation.

Identification of p130Cas/ErbB2-dependent invasive signatures in transformed mammary epithelial cells.

Understanding transcriptional changes during cancer progression is of crucial importance to develop new and more efficacious diagnostic and therapeutic approaches. It is well known that ErbB2 is overexpressed in about 25% of human invasive breast cancers. We have previously demonstrated that p130Cas overexpression synergizes with ErbB2 in mammary cell transformation and promotes ErbB2-dependent invasion in three-dimensional (3D) cultures of human mammary epithelial cells. Here, by comparing coding and non-coding gene expression profiles, we define the invasive signatures associated with concomitant p130Cas overexpression and ErbB2 activation in 3D cultures of mammary epithelial cells. Specifically, we have found that genes involved in amino acids synthesis (CBS, PHGDH), cell motility, migration (ITPKA, PRDM1), and angiogenesis (HEY1) are upregulated, while genes involved in inflammatory response (SAA1, S100A7) are downregulated. In parallel, we have shown that the expression of specific miRNAs is altered. Among these, miR-200b, miR-222, miR-221, miR-R210, and miR-424 are upregulated, while miR-27a, miR-27b, and miR-23b are downregulated. Overall, this study presents, for the first time, the gene expression changes underlying the invasive behavior following p130Cas overexpression in an ErbB2 transformed mammary cell model.

miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1.

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse-associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR-148b, down-regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR-148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR-148b in the control of breast cancer progression.

Analysis of miRNA and mRNA expression profiles highlights alterations in ionizing radiation response of human lymphocytes under modeled microgravity.

BACKGROUND: Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of "Response to DNA damage" is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. CONCLUSIONS/SIGNIFICANCE: On the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL.

High IGFBP2 expression correlates with tumor severity in pediatric rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common childhood sarcoma and is identified as either the embryonal or alveolar (ARMS) subtype. In approximately 75% of cases, ARMSs are characterized by specific chromosomal translocations that involve PAX and FKHR genes. ARMS gene expression signatures vary, depending on the presence or absence of the translocations. Insulin-like growth factor-binding protein 2 (IGFBP2) is strongly overexpressed in translocation-negative RMS. Because IGFBP2 is associated with tumorigenesis, we investigated its functional role in RMS. An analysis of IGFBP2 distribution in RMS cell lines revealed a strong accumulation in the Golgi complex, in which morphological characteristics appeared peculiarly modified. After silencing IGFBP2 expression, our microarray analysis revealed mostly cell cycle and actin cytoskeleton gene modulations. In parallel, IGFBP2-silenced cells showed reduced cell cycle and rates of invasion and decreased seeding in the lungs after tail vein injections in immunodeficient mice. An analysis of IGFBP2 mRNA and protein localization in human tumors showed abnormal protein accumulation in the Golgi complex, mostly in PAX/FKHR-negative RMS. Moreover, an analysis of patients with RMS revealed the presence of conspicuous circulating levels of IGFBP2 proteins in children with highly aggressive RMS tumors. Taken together, our data provide evidence that IGFBP2 contributes to tumor progression and that it could be used as a marker to better classify clinical and biological risks in RMS.

microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C.

Malignant melanoma is fatal in its metastatic stage. It is therefore essential to unravel the molecular mechanisms that govern disease progression to metastasis. MicroRNAs (miRs) are endogenous non-coding RNAs involved in tumourigenesis. Using a melanoma progression model, we identified a novel pathway controlled by miR-214 that coordinates metastatic capability. Pathway components include TFAP2C, homologue of a well-established melanoma tumour suppressor, the adhesion receptor ITGA3 and multiple surface molecules. Modulation of miR-214 influences in vitro tumour cell movement and survival to anoikis as well as extravasation from blood vessels and lung metastasis formation in vivo. Considering that miR-214 is known to be highly expressed in human melanomas, our data suggest a critical role for this miRNA in disease progression and the establishment of distant metastases.

New miRNA labeling method for bead-based quantification.

BACKGROUND: microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies. RESULTS: Here we have applied with an innovative approach, the Luminex xMAP technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies. CONCLUSIONS: We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex xMAP technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.

Defining the gene expression signature of rhabdomyosarcoma by meta-analysis.

BACKGROUND: Rhabdomyosarcoma is a highly malignant soft tissue sarcoma in childhood and arises as a consequence of regulatory disruption of the growth and differentiation pathways of myogenic precursor cells. The pathogenic pathways involved in this tumor are mostly unknown and therefore a better characterization of RMS gene expression profile would represent a considerable advance. The availability of publicly available gene expression datasets have opened up new challenges especially for the integration of data generated by different research groups and different array platforms with the purpose of obtaining new insights on the biological process investigated. RESULTS: In this work we performed a meta-analysis on four microarray and two SAGE datasets of gene expression data on RMS in order to evaluate the degree of agreement of the biological results obtained by these different studies and to identify common regulatory pathways that could be responsible of tumor growth. Regulatory pathways and biological processes significantly enriched has been investigated and a list of differentially meta-profiles have been identified as possible candidate of aggressiveness of RMS. CONCLUSION: Our results point to a general down regulation of the energy production pathways, suggesting a hypoxic physiology for RMS cells. This result agrees with the high malignancy of RMS and with its resistance to most of the therapeutic treatments. In this context, different isoforms of the ANT gene have been consistently identified for the first time as differentially expressed in RMS. This gene is involved in anti-apoptotic processes when cells grow in low oxygen conditions. These new insights in the biological processes responsible of RMS growth and development demonstrate the effective advantage of the use of integrated analysis of gene expression studies.

Gene expression profiling identifies potential relevant genes in alveolar rhabdomyosarcoma pathogenesis and discriminates PAX3-FKHR positive and negative tumors.

We analyzed the expression signatures of 14 tumor biopsies from children affected by alveolar rhabdomyosarcoma (ARMS) to identify genes correlating to biological features of this tumor. Seven of these patients were positive for the PAX3-FKHR fusion gene and 7 were negative. We used a cDNA platform containing a large majority of probes derived from muscle tissues. The comparison of transcription profiles of tumor samples with fetal skeletal muscle identified 171 differentially expressed genes common to all ARMS patients. The functional classification analysis of altered genes led to the identification of a group of transcripts (LGALS1, BIN1) that may be relevant for the tumorigenic processes. The muscle-specific microarray platform was able to distinguish PAX3-FKHR positive and negative ARMS through the expression pattern of a limited number of genes (RAC1, CFL1, CCND1, IGFBP2) that might be biologically relevant for the different clinical behavior and aggressiveness of the 2 ARMS subtypes. Expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR.

A leukemia-enriched cDNA microarray platform identifies new transcripts with relevance to the biology of pediatric acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: Microarray gene expression profiling has been widely applied to characterize hematologic malignancies, has attributed a molecular signature to leukemia subclasses and has allowed new subclasses to be distinguished. We set out to use microarray technology to identify novel genes relevant for leukemogenesis. To this end we used a unique leukemia-enriched cDNA microarray platform. DESIGN AND METHODS: The systematic sequencing of cDNA libraries of normal and leukemic bone marrow allowed us to increase the number of genes to yield a new release of a previously generated cDNA microarray. Using this platform we analyzed the expression profiles of 4,670 genes in bone marrow samples from 18 pediatric patients with acute lymphoblastic leukemia (ALL). RESULTS: Expression profiling consistently distinguished the leukemia patients into three groups, those with T-ALL, B-ALL and B-ALL with MLL/AF4 rearrangement, in agreement with the clinical classification. Our platform identified 30 genes that best discriminate these three subtypes. Using mini-array technology these 30 genes were validated in another cohort of 17 patients. In particular we identified two novel genes not previously reported: endomucin (EMCN) and ubiquitin specific protease 33 (USP33) that appear to be over-expressed in B-ALL relative to their expression in T-ALL. INTERPRETATION AND CONCLUSIONS: Microarray technology not only allows the distinction between disease subclasses but also offers a chance to identify new genes involved in leukemogenesis. Our approach of using a unique platform has proven to be fruitful in identifying new genes and we suggest exploration of other malignancies using this approach.