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Purpose: The majority of gastric cancer patients are diagnosed with late-stage disease, for which distinct molecular subtypes have been identified that are potentially amenable to targeted therapies. However, there exists no molecular classification system with prognostic power for early-stage gastric cancer (EGC) because the molecular events promoting gastric cancer initiation remain ill-defined.Experimental Design: miRNA microarrays were performed on gastric tissue from the gp130F/F preclinical EGC mouse model, prior to tumor initiation. Computation prediction algorithms were performed on multiple data sets and independent gastric cancer patient cohorts. Quantitative real-time PCR expression profiling was undertaken in gp130F/F-based mouse strains and human gastric cancer cells genetically engineered for suppressed activation of the oncogenic latent transcription factor STAT3. Human gastric cancer cells with modulated expression of the miR-200 family member miR-429 were also assessed for their proliferative response.Results: Increased expression of miR-200 family members is associated with both tumor initiation in a STAT3-dependent manner in gp130F/F mice and EGC (i.e., stage IA) in patient cohorts. Overexpression of miR-429 also elicited contrasting pro- and antiproliferative responses in human gastric cancer cells depending on their cellular histologic subtype. We also identified a miR-200 family–regulated 15-gene signature that integrates multiple key current indicators of EGC, namely tumor invasion depth, differentiation, histology, and stage, and provides superior predictive power for overall survival compared with each EGC indicator alone.Conclusions: Collectively, our discovery of a STAT3-regulated, miR-200 family–associated gene signature specific for EGC, with predictive power, provides a molecular rationale to classify and stratify EGC patients for endoscopic treatment. Clin Cancer Res; 24(6); 1459–72. ©2018 AACR.

These figures and tables contain supporting (and essential) data to confirm the clinical utility of the miR-200 family and signature in early gastric cancer, as well as its regulation by STAT3. Supplementary Figure S1. The association of WHO intestinal-type GC sub-classes and individual TNM stage with the expression of miR-200 family members and miR-21 in TCGA GC patients. Supplementary Figure S2. The association between patient survival and expression of miR- 200s and a complete network map of GSEA results generated from TCGA GC patients. Supplementary Figure S3. miR-429 promotes AGS cells proliferation and clonigenicity. Supplementary Figure S4. The association between patient survival and expression of individual genes from the miR-200 family-associated 15-gene signature. Supplementary Figure S5. STAT3 activation correlates with miR-429 and other miR-200 members in mouse and human gastric epithelial cells. Supplementary Figure S6. Generation of STAT3 knockout human GC lines, and STAT3- independent transcriptional regulation of miR-200 family members. Supplementary Table S1. Clinicopathological features and demographics of GC patients from TCGA cohort used for expression profiling of hsa-miR-429. Supplementary Table S2. Chi-square analysis between the miR-200 family 15-gene signature and conventional key prognostic factors using two independent cohorts. 2 Supplementary Table S3. Multivariate analysis of overall survival comparing the miR-200 family 15-gene signature and other conventional clinical parameters including histology and tumor grade (differentiation) using updated TCGA data.

Purpose: The majority of gastric cancer patients are diagnosed with late-stage disease, for which distinct molecular subtypes have been identified that are potentially amenable to targeted therapies. However, there exists no molecular classification system with prognostic power for early-stage gastric cancer (EGC) because the molecular events promoting gastric cancer initiation remain ill-defined. Experimental Design: miRNA microarrays were performed on gastric tissue from the gp130F/F preclinical EGC mouse model, prior to tumor initiation. Computation prediction algorithms were performed on multiple data sets and independent gastric cancer patient cohorts. Quantitative real-time PCR expression profiling was undertaken in gp130F/F-based mouse strains and human gastric cancer cells genetically engineered for suppressed activation of the oncogenic latent transcription factor STAT3. Human gastric cancer cells with modulated expression of the miR-200 family member miR-429 were also assessed for their proliferative response. Results: Increased expression of miR-200 family members is associated with both tumor initiation in a STAT3-dependent manner in gp130F/F mice and EGC (i.e., stage IA) in patient cohorts. Overexpression of miR-429 also elicited contrasting pro- and antiproliferative responses in human gastric cancer cells depending on their cellular histologic subtype. We also identified a miR-200 family–regulated 15-gene signature that integrates multiple key current indicators of EGC, namely tumor invasion depth, differentiation, histology, and stage, and provides superior predictive power for overall survival compared with each EGC indicator alone. Conclusions: Collectively, our discovery of a STAT3-regulated, miR-200 family–associated gene signature specific for EGC, with predictive power, provides a molecular rationale to classify and stratify EGC patients for endoscopic treatment. Clin Cancer Res; 24(6); 1459–72. ©2018 AACR.

Esophageal cancer has a high mortality rate, and its multimodality treatment is often associated with significant rates of severe toxicity. Effort is needed to uncover ways to maximize effectiveness of therapy through identification of predictive markers of response and toxicity. As such, the aim of this study was to identify genes predictive of chemoradiotherapy-induced gastrointestinal toxicity using an immune pathway-targeted approach. Adults with esophageal cancer treated with chemotherapy consisting of 5-fluorouracil and cisplatin and 45–50 Gy radiation were recruited to the study. Pre-therapy-collected whole blood was analyzed for relative expression of immune genes using real-time polymerase chain reaction (RT-PCR). Gene expression was compared between patients who experienced severe regimen-related gastrointestinal toxicity vs. those experiencing mild to moderate toxicity. Blood from 31 patients were analyzed by RT-PCR. Out of 84 immune genes investigated, TNF was significantly elevated (2.05-fold, p = 0.025) in the toxic group (n = 12) compared to the non-toxic group (n = 19). Nausea and vomiting was the most commonly documented severe toxicity. No associations between toxicity and response, age, sex, histology, or treatment were evident. This study supports evidence of TNF as a predictive biomarker in regimen-related gastrointestinal toxicity. Confirming these findings in a larger cohort is warranted.

Deep-sequencing reveals extensive variation in the sequence of endogenously expressed microRNAs (termed 'isomiRs') in human cell lines and tissues, especially in relation to the 3' end. From the immunoprecipitation of the microRNA-binding protein Argonaute and the sequencing of associated small RNAs, we observe extensive 3'-isomiR variation, including for miR-222 where the majority of endogenously expressed miR-222 is extended by 1-5 nt compared to the canonical sequence. We demonstrate this 3' heterogeneity has dramatic implications for the phenotype of miR-222 transfected cells, with longer isoforms promoting apoptosis in a size (but not 3' sequence)-dependent manner. The transfection of longer miR-222 isomiRs did not induce an interferon response, but did downregulate the expression of many components of the pro-survival PI3K-AKT pathway including PIK3R3, a regulatory subunit whose knockdown phenocopied the expression of longer 222 isoforms in terms of apoptosis and the inhibition of other PI3K-AKT genes. As this work demonstrates the capacity for 3' isomiRs to mediate differential functions, we contend more attention needs to be given to 3' variance given the prevalence of this class of isomiR. Refereed/Peer-reviewed

Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine(C-C motif) lig and 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke Refereed/Peer-reviewed

A quantitative, peripherally accessible biomarker for neuropathic pain has great potential to improve clinical outcomes. Based on the premise that peripheral and central immunity contribute to neuropathic pain mechanisms, we hypothesized that biomarkers could be identified from the whole blood of adult male rats, by integrating graded chronic constriction injury (CCI), ipsilateral lumbar dorsal quadrant (iLDQ) and whole blood transcriptomes, and pathway analysis with pain behavior. Correlational bioinformatics identified a range of putative biomarker genes for allodynia intensity, many encoding for proteins with a recognized role in immune/nociceptive mechanisms. A selection of these genes was validated in a separate replication study. Pathway analysis of the iLDQ transcriptome identified Fcγ and Fce signaling pathways, among others. This study is the first to employ the whole blood transcriptome to identify pain biomarker panels. The novel correlational bioinformatics, developed here, selected such putative biomarkers based on a correlation with pain behavior and formation of signaling pathways with iLDQ genes. Future studies may demonstrate the predictive ability of these biomarker genes across other models and additional variables.

Retinoids are promising agents for the treatment/prevention of breast carcinoma. We examined the role of microRNAs in mediating the effects of all-trans-retinoic acid (ATRA), which suppresses the proliferation of estrogen receptor-positive (ERα+) breast carcinoma cells, such as MCF-7, but not estrogen receptor-negative cells, such as MDA-MB-231. We found that pro-oncogenic miR-21 is selectively induced by ATRA in ERα+ cells. Induction of miR-21 counteracts the anti-proliferative action of ATRA but has the potentially beneficial effect of reducing cell motility. In ERα+ cells, retinoid-dependent induction of miR-21 is due to increased transcription of the MIR21 gene via ligand-dependent activation of the nuclear retinoid receptor, RARα. RARα is part of the transcription complex present in the 5′-flanking region of the MIR21 gene. The receptor binds to two functional retinoic acid-responsive elements mapping upstream of the transcription initiation site. Silencing of miR-21 enhances ATRA-dependent growth inhibition and senescence while reverting suppression of cell motility afforded by the retinoid. Up-regulation of miR-21 results in retinoid-dependent inhibition of the established target, maspin. Knockdown and overexpression of maspin in MCF-7 cells indicates that the protein is involved in ATRA-induced growth inhibition and contributes to the ATRA-dependent anti-motility responses. Integration between whole genome analysis of genes differentially regulated by ATRA in MCF-7 and MDA-MB-231 cells, prediction of miR-21 regulated genes, and functional studies led to the identification of three novel direct miR-21 targets: the pro-inflammatory cytokine IL1B, the adhesion molecule ICAM-1 and PLAT, the tissue-type plasminogen activator. Evidence for ICAM-1 involvement in retinoid-dependent inhibition of MCF-7 cell motility is provided.

Background The genetic changes that drive metaplastic progression from squamous oesophageal mucosa toward intestinal metaplasia and adenocarcinoma are unclear. The aberrant expression of microRNAs (miRNAs) is involved in the development of cancer. This study examined whether miRNAs play a role in the development of oesophageal adenocarcinoma. Methods RNA was extracted from mucosa of normal oesophageal squamous epithelium, normal gastric epithelium, Barrett's oesophagus with intestinal metaplasia and oesophageal adenocarcinoma obtained from 16 individuals. Expression profiles of 377 human miRNAs were determined by microarray analysis and selected miRNAs were analysed further using real-time reverse transcription–polymerase chain reaction (RT–PCR) in tissues from 32 individuals. Results Microarray analyses identified 44 miRNAs likely to have altered expression between various mucosal samples. Of these, miR-21, miR-143, miR-145, miR-194, miR-203, miR-205 and miR-215 were chosen for validation by real-time RT-PCR. Tissue-specific expression profiles were observed, with miR-21, miR-143, miR-145, miR-194 and miR-215 significantly upregulated in columnar tissues compared with normal squamous epithelium. Expression of miR-143, miR-145 and miR-215 was lower in oesophageal adenocarcinoma than in Barrett's oesophagus. Levels of miR-203 and miR-205 were high in normal squamous epithelium and low in columnar epithelia. MiR-205 levels were lower in gastric epithelium than in both Barrett's oesophagus and adenocarcinoma. Conclusion Expression of miRNA might define disease states in oesophageal epithelium. Dysregulation of specific miRNAs could contribute to metaplastic and neoplastic processes in the oesophageal mucosa.

Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague-Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague-Dawley list returned the term "response to hypoxia," absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1alpha were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9041-7) contains supplementary material, which is available to authorized users.

The identification of miRNAs and their target mRNAs and the construction of their regulatory networks may give new insights into biological procedures. This study proposes a computational method to discover the functional miRNA-mRNA regulatory modules (FMRMs), that is, groups of miRNAs and their target mRNAs that are believed to participate cooperatively in post-transcriptional gene regulation under specific conditions. The proposed method identifies negatively regulated patterns of miRNAs and mRNAs which associate with cancer and normal conditions, respectively, in a prostate cancer data set. GO and the literature also suggest that they may relate with prostate cancer. It can potentially identify the biologically relevant chains of 'miRNA target gene condition'. Refereed/Peer-reviewed

Gastrointestinal mucositis involves many changes at the gene level, affecting epithelial/subepithelial interactions and leading to overt damage. The regional specificity and time course of these changes, and how they relate to subsequent mucositis development however remain unknown. The aim of this study was to determine the early time course of gene expression changes along the gastrointestinal tract of the DA rat following chemotherapy. Female DA rats were treated with a single dose of 200 mg/kg irinotecan to induce mucositis, and were killed at short intervals following treatment. Small sections of stomach, jejunum and colon were harvested for analysis of genetic profiles. RNA was hybridised to high density Affymetrix oligonucleotide microarrays. Data analysis was carried out with software package, TimeCourse, freely available through Bioconductor. As early as 1 hr following chemotherapy, expression of hundreds of genes was altered, including those for transcription factors, stress response proteins and protein turnover. These genes are involved in cell proliferation, differentiation and apoptosis along with other cellular processes. At early time points, there was a significant response involving the mitogen-activated protein kinase pathway, cell cycle regulation and cytokine receptor signalling. At later time points, changes to the complement cascade became prominent. We have shown that changes in gene expression following chemotherapy occur by 1 hr, and persist for at least 72 hr after treatment. Many of these changes are highly likely to be specifically related to the subsequent development of gastrointestinal mucositis.

// James Neil Fisher 1, * , Mineko Terao 1, * , Maddalena Fratelli 1, * , Mami Kurosaki 1 , Gabriela Paroni 1 , Adriana Zanetti 1 , Maurizio Gianni 1 , Marco Bolis 1 , Monica Lupi 2 , Anna Tsykin 3 , Gregory J. Goodall 3 , Enrico Garattini 1 1 Laboratory of Molecular Biology, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy 2 Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy 3 Centre for Cancer Biology, SA Pathology, Adelaide, Australia and Department of Medicine, University of Adelaide, Adelaide, Australia * These authors have contributed equally to this work Correspondence to: Enrico Garattini, e-mail: egarattini@marionegri.it Keywords: retinoic acid, microRNA, breast cancer, lapatinib, network analysis Received: December 19, 2014 Accepted: April 04, 2015 Published: April 18, 2015 ABSTRACT SKBR3 -cells, characterized by ERBB2 / RARA co-amplification, represent a subgroup of HER2 + breast-cancers sensitive to all-trans retinoic acid (ATRA) and Lapatinib. In this model, the two agents alone or in combination modulate the expression of 174 microRNAs (miRs). These miRs and predicted target-transcripts are organized in four interconnected modules ( Module-1 to -4 ). Module-1 and Module-3 consist of ATRA/Lapatinib up-regulated and potentially anti-oncogenic miRs, while Module-2 contains ATRA/Lapatinib down-regulated and potentially pro-oncogenic miRs. Consistent with this, the expression levels of Module-1/-3 and Module-2 miRs are higher and lower, respectively, in normal mammary tissues relative to ductal-carcinoma- in-situ , invasive-ductal-carcinoma and metastases. This indicates associations between tumor-progression and the expression profiles of Module-1 to -3 miRs. Similar associations are observed with tumor proliferation-scores, staging, size and overall-survival using TCGA (The Cancer Genome Atlas) data. Forced expression of Module-1 miRs, (miR-29a-3p; miR-874-3p) inhibit SKBR3 -cell growth and Module-3 miRs (miR-575; miR-1225-5p) reduce growth and motility. Module-2 miRs (miR-125a; miR-193; miR-210) increase SKBR3 cell growth, survival and motility. Some of these effects are of general significance, being replicated in other breast cancer cell lines representing the heterogeneity of this disease. Finally, our study demonstrates that HIPK2-kinase and the PLCXD1-phospholipase-C are novel targets of miR-193a-5p/miR-210-3p and miR-575/miR-1225-5p, respectively.

Angiogenesis is a complex multicellular process requiring the orchestration of many events including migration, alignment, proliferation, lumen formation, remodeling, and maturation. Such complexity indicates that not only individual genes but also entire signaling pathways will be crucial in angiogenesis. To define an angiogenic blueprint of regulated genes, we utilized our well-characterized three-dimensional collagen gel model of in vitro angiogenesis, in which the majority of cells synchronously progress through defined morphological stages culminating in the formation of capillary tubes. We developed a comprehensive three-tiered approach using microarray analysis, which allowed us to identify genes known to be involved in angiogenesis and genes hitherto unlinked to angiogenesis as well as novel genes and has proven especially useful for genes where the magnitude of change is small. Of interest is the ability to recognize complete signaling pathways that are regulated and genes clustering into ontological groups implicating the functional importance of particular processes. We have shown that consecutive members of the mitogen-activated protein kinase and leukemia inhibitory factor signaling pathways are altered at the mRNA level during in vitro angiogenesis. Thus, at least for the mitogen-activated protein kinase pathway, mRNA changes as well as the phosphorylation changes of these gene products may be important in the control of blood vessel morphogenesis. Furthermore, in this study, we demonstrated the power of virtual Northern blot analysis, as an alternative to quantitative RT-PCR, for measuring the magnitudes of differential gene expression.

The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR-200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR-200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration. Synopsis miR-200 microRNAs are involved in the maintenance of epithelial integrity. Direct, transcriptome-wide target detection and validation identifies genes functionally grouped as regulators of Rho GTPase signaling, invadopodia formation, metalloprotease activity and cell adhesion, which together regulate cell motility, migration and cancer metastasis. The global profile of miR-200 targets in breast cancer cells reveals a network of cytoskeletal regulators miR-200 is found to control invadopodia, focal adhesions and Rho GTPase signaling. Canonical seed-3'UTR target site interactions are dominant, but target sites in coding regions and non-canonical interactions are also detected. Target genes identified in cell lines negatively correlate with miR-200 across human breast cancer samples as well. miR-200 microRNAs are involved in the maintenance of epithelial integrity. Direct, transcriptome-wide target detection and validation identifies genes functionally grouped as regulators of Rho GTPase signaling, invadopodia formation, metalloprotease activity and cell adhesion, which together regulate cell motility, migration and cancer metastasis. Refereed/Peer-reviewed

Sphingosine kinase 1 (SK1) is a lipid kinase that catalyses the formation of sphingosine-1-phosphate (S1P). Considerable evidence has implicated elevated cellular SK1 in tumour development, progression and disease severity. In particular, SK1 has been shown to enhance cell survival and proliferation and induce neoplastic transformation. Although S1P has been found to have both cellsurface G-protein-coupled receptors and intracellular targets, the specific downstream pathways mediating oncogenic signalling by SK1 remain poorly defined. Here, using a gene expression array approach, we have demonstrated a novel mechanism whereby SK1 regulates cell survival, proliferation and neoplastic transformation through enhancing expression of transferrin receptor 1 (TFR1). We showed that elevated levels of SK1 enhanced total as well as cell-surface TFR1 expression, resulting in increased transferrin uptake into cells. Notably, we also found that SK1 activation and localization to the plasma membrane, which are critical for its oncogenic effects, are necessary for regulation of TFR1 expression specifically through engagement of the S1P G-protein coupled receptor, S1P2. Furthermore, we showed that blocking TFR1 function with a neutralizing antibody inhibits SK1-induced cell proliferation, survival and neoplastic transformation of NIH3T3 fibroblasts. Similar effects were observed following antagonism of S1P2. Together these findings suggest that TFR1 has an important role in SK1-mediated oncogenesis. Refereed/Peer-reviewed

Hyperactivation of the latent transcription factor signal transducer and activator of transcription (STAT)3 is frequently observed in inflammation-associated gastric cancer. However the full spectrum of downstream gene networks modulated by STAT3 during the pathogenesis of gastric tumourigenesis remains ill-defined. The gp130 F / F gastric cancer mouse model which recapitulates the hyperactive STAT3 signaling and tumor progression observed in human disease was therefore employed to identify novel STAT3-regulated microRNAs (miRNAs) associated with the onset of gastric tumours. Using a miRNA array approach coupled with miRNA real-time quantitative PCR validation, we identified that 2 miRNAs (miR-146b and miR-429) are consistently up in stomach tissue from gp130 F / F mice at 4 weeks (prior to tumour initiation) and 3 months (established tumour formation) of age compared to age-matched wild type mice. Consistent with our previous demonstration that interleukin (IL)-11/STAT3 signaling plays a causal role during gastric tumourigenesis in gp130 F / F mice, normalization of gastric STAT3 activation and IL-11 expression in gp130 F / F mice lacking the IL-11 ligand-binding receptor subunit (IL-11Ra) also normalized the expression of these miRNAs. We also found that in response to IL-11 treatment of wild-type mice, miR-146b and miR-429 are significantly increased along with STAT3 activation. Furthermore, these 2 miRs are upregulated in human intestinal-type gastric cancer biopsies characterized by increased STAT3 signaling. Collectively, our data reveal 2 novel STAT3-regulated miRNAs in gastric tumourigenesis, and therefore suggest that the regulation of specific miRNAs may represent an additional mechanism by which STAT3 promotes oncogenesis in the stomach.

Cellular junctions are essential to the normal functioning of the endothelium and control angiogenesis, tissue leak, and inflammation. From a screen of micro RNAs (miRNAs) altered in in vitro angiogenesis, we selected a subset predicted to target junctional molecules. MiR-27a was rapidly downregulated upon stimulation of in vitro angiogenesis, and its level of expression is reduced in neovessels in vivo. The downregulation of miR-27a was essential for angiogenesis because ectopic expression of miR-27a blocked capillary tube formation and angiogenesis. MiR-27a targets the junctional, endothelial-specific cadherin, VE-cadherin. Consistent with this, vascular permeability to vascular endothelial growth factor in mice is reduced by administration of a general miR-27 inhibitor. To determine that VE-cadherin was the dominant target of miR-27a function, we used a novel technology with "Blockmirs," inhibitors that bind to the miR-27 binding site in VE-cadherin. The Blockmir CD5-2 demonstrated specificity for VE-cadherin and inhibited vascular leak in vitro and in vivo. Furthermore, CD5-2 reduced edema, increased capillary density, and potently enhanced recovery from ischemic limb injury in mice. The Blockmir technology offers a refinement in the use of miRNAs, especially for therapy. Further, targeting of endothelial junctional molecules by miRNAs has clinical potential, especially in diseases associated with vascular leak.

Background: Transcription factors (TFs) and microRNAs (miRNAs) are primary metazoan gene regulators. Regulatory mechanisms of the two main regulators are of great interest to biologists and may provide insights into the causes of diseases. However, the interplay between miRNAs and TFs in a regulatory network still remains unearthed. Currently, it is very difficult to study the regulatory mechanisms that involve both miRNAs and TFs in a biological lab. Even at data level, a network involving miRNAs, TFs and genes will be too complicated to achieve. Previous research has been mostly directed at inferring either miRNA or TF regulatory networks from data. However, networks involving a single type of regulator may not fully reveal the complex gene regulatory mechanisms, for instance, the way in which a TF indirectly regulates a gene via a miRNA. Conclusions: We have designed a framework to infer gene regulatory networks involving both TFs and miRNAs from multiple sources of data, including gene expression data, target information, and sample categories. Results on the EMT data sets have shown that the proposed approach is able to produce compact and meaningful gene regulatory networks that are highly relevant to the biological conditions of the data sets. This framework has the potential for application to other heterogeneous datasets to reveal the complex gene regulatory relationships. Results: We propose a framework to learn from heterogeneous data the three-component regulatory networks, with the presence of miRNAs, TFs, and mRNAs. This method firstly utilises Bayesian network structure learning to construct a regulatory network from multiple sources of data: gene expression profiles of miRNAs, TFs and mRNAs, target information based on sequence data, and sample categories. Then, in order to produce more meaningful results for further biological experimentation and research, the method searches the learnt network to identify the interplay between miRNAs and TFs and applies a network motif finding algorithm to further infer the network. We apply the proposed framework to the data sets of epithelial-to-mesenchymal transition (EMT). The results elucidate the complex gene regulatory mechanism for EMT which involves both TFs and miRNAs. Several discovered interactions and molecular functions have been confirmed by literature. In addition, many other discovered interactions and bio-markers are of high statistical significance and thus can be good candidates for validation by experiments. Moreover, the results generated by our method are compact, involving a small number of interactions which have been proved highly relevant to EMT. Refereed/Peer-reviewed

A quantitative, peripherally accessible biomarker for neuropathic pain has great potential to improve clinical outcomes. Based on the premise that peripheral and central immunity contribute to neuropathic pain mechanisms, we hypothesized that biomarkers could be identified from the whole blood of adult male rats, by integrating graded chronic constriction injury (CCI), ipsilateral lumbar dorsal quadrant (iLDQ) and whole blood transcriptomes, and pathway analysis with pain behavior. Correlational bioinformatics identified a range of putative biomarker genes for allodynia intensity, many encoding for proteins with a recognized role in immune/nociceptive mechanisms. A selection of these genes was validated in a separate replication study. Pathway analysis of the iLDQ transcriptome identified Fcγ and Fcε signaling pathways, among others. This study is the first to employ the whole blood transcriptome to identify pain biomarker panels. The novel correlational bioinformatics, developed here, selected such putative biomarkers based on a correlation with pain behavior and formation of signaling pathways with iLDQ genes. Future studies may demonstrate the predictive ability of these biomarker genes across other models and additional variables.

Motivation: MicroRNAs (miRNAs) are small non-coding RNAs that cause mRNA degradation and translational inhibition. They are important regulators of development and cellular homeostasis through their control of diverse processes. Recently, great efforts have been made to elucidate their regulatory mechanism, but the functions of most miRNAs and their precise regulatory mechanisms remain elusive. With more and more matched expression profiles of miRNAs and mRNAs having been made available, it is of great interest to utilize both expression profiles to discover the functional regulatory networks of miRNAs and their target mRNAs for potential biological processes that they may participate in. Results: We present a probabilistic graphical model to discover functional miRNA regulatory modules at potential biological levels by integrating heterogeneous datasets, including expression profiles of miRNAs and mRNAs, with or without the prior target binding information. We applied this model to a mouse mammary dataset. It effectively captured several biological process specific modules involving miRNAs and their target mRNAs. Furthermore, without using prior target binding information, the identified miRNAs and mRNAs in each module show a large proportion of overlap with predicted miRNA target relationships, suggesting that expression profiles are crucial for both target identification and discovery of regulatory modules. Contact: bing.liu@unisa.edu.au; jiuyong.li@unisa.edu.au Supplementary information: Supplementary data are available at Bioinformatics online.

Background microRNAs (miRNAs) regulate target gene expression by controlling their mRNAs post-transcriptionally. Increasing evidence demonstrates that miRNAs play important roles in various biological processes. However, the functions and precise regulatory mechanisms of most miRNAs remain elusive. Current research suggests that miRNA regulatory modules are complicated, including up-, down-, and mix-regulation for different physiological conditions. Previous computational approaches for discovering miRNA-mRNA interactions focus only on down-regulatory modules. In this work, we present a method to capture complex miRNA-mRNA interactions including all regulatory types between miRNAs and mRNAs. Results We present a method to capture complex miRNA-mRNA interactions using Bayesian network structure learning with splitting-averaging strategy. It is designed to explore all possible miRNA-mRNA interactions by integrating miRNA-targeting information, expression profiles of miRNAs and mRNAs, and sample categories. We also present an analysis of data sets for epithelial and mesenchymal transition (EMT). Our results show that the proposed method identified all possible types of miRNA-mRNA interactions from the data. Many interactions are of tremendous biological significance. Some discoveries have been validated by previous research, for example, the miR-200 family negatively regulates ZEB1 and ZEB2 for EMT. Some are consistent with the literature, such as LOX has wide interactions with the miR-200 family members for EMT. Furthermore, many novel interactions are statistically significant and worthy of validation in the near future. Conclusions This paper presents a new method to explore the complex miRNA-mRNA interactions for different physiological conditions using Bayesian network structure learning with splitting-averaging strategy. The method makes use of heterogeneous data including miRNA-targeting information, expression profiles of miRNAs and mRNAs, and sample categories. Results on EMT data sets show that the proposed method uncovers many known miRNA targets as well as new potentially promising miRNA-mRNA interactions. These interactions could not be achieved by the normal Bayesian network structure learning.

Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the βc subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene βc Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34+CD38−CD123+ leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.

Mechanisms controlling the balance between proliferation and self-renewal versus growth suppression and differentiation during normal and leukemic myelopoiesis are not understood. We have used the bi-potent FDB1 myeloid cell line model, which is responsive to myelopoietic cytokines and activated mutants of the granulocyte macrophage-colony stimulating factor (GM-CSF) receptor, having differential signaling and leukemogenic activity. This model is suited to large-scale gene-profiling, and we have used a factorial time-course design to generate a substantial and powerful data set. Linear modeling was used to identify gene-expression changes associated with continued proliferation, differentiation, or leukemic receptor signaling. We focused on the changing transcription factor profile, defined a set of novel genes with potential to regulate myeloid growth and differentiation, and demonstrated that the FDB1 cell line model is responsive to forced expression of oncogenes identified in this study. We also identified gene-expression changes associated specifically with the leukemic GM-CSF receptor mutant, V449E. Signaling from this receptor mutant down-regulates CCAAT/enhancer-binding protein α (C/EBPα) target genes and generates changes characteristic of a specific acute myeloid leukemia signature, defined previously by gene-expression profiling and associated with C/EBPα mutations.

The advent of microarray technology has significantly changed the way we can quantitatively measure and observe gene expression at the mRNA level within a given biological sample of interest, allowing for the monitoring of tens to hundreds of thousands of genes within a single experiment. The two main array platforms are spotted two-colour arrays and one-colour in situ-synthesized arrays. Microarrays are used for a wide range of applications including gene annotation, investigation of gene-gene interactions, elucidation of gene regulatory networks and gene-expression profiling of Saccharomyces cerevisiae and other fungal organisms. Academic researchers and both the pharmaceutical and agricultural industries have an enormous interest in developing microarrays both as diagnostic tools and for use in basic research into how pathogens, such as fungi, interact with their host. Microarray experiments generate vast quantities of raw gene expression data, therefore good experimental design and statistical analysis is required for the extraction of accurate and useful information regarding the expression of genes. In this review we firstly provide an overview of the arrival and development of microarray technology. We then focus on the issues surrounding experimental design and the processing of microarray images, followed by a discussion on methods for cleaning and normalizing raw gene expression data and a final discussion of the importance statistical analysis plays in identifying differentially expressed genes.

In a recent report (Paroni et al, Oncogene, 31:3431-43, 2012), we demonstrated that 25% of all ERBB2+ breast cancers present with a co-amplification of the RARA locus, encoding the nuclear retinoic acid receptor, RARα. ERBB2+/RARA+ breast cancer cell lines, like SKBR3 and AU565, are sensitive to the anti-proliferative and cyto-differentiating effects of all-trans retinoic acid (ATRA), whereas the ERBB2+/RARA− counterparts are refractory. Simultaneous targeting of RARα with ATRA and ERBB2 with lapatinib in SKBR3 and AU565 cells leads to synergistic interactions as to growth inhibition and cyto-differentiation. In addition, combinations of ATRA and lapatinib exert an apoptotic effect, which is not observed with the single components. This suggests that ERBB2+/RARA+ breast cancer patients may benefit from lapatinib+ATRA regimens and represent the rationale of an ongoing clinical trial. The use of lapatinib and ATRA for the stratified therapy of ERBB2+/RARA+ patients requires knowledge of the molecular mechanisms underlying the cross-talk between the two compounds. We evaluated the effects of lapatinib and/or ATRA on the expression profiles of more than 1,000 microRNAs in SKBR3 cells using an appropriate microarray platform. Both lapatinib and ATRA caused up- and down-regulation of numerous microRNAs. The combination of the two compounds enhanced the action of the single components of the mixture and resulted in the up- and down-regulation of novel target microRNAs as well. We integrated these results with microRNA-target-gene prediction algorithms and whole-genome gene-expression data looking for significant correlations between microRNA and potential/established target mRNA levels. Network analysis led to the identification of two highly interconnected microRNA/target-mRNA modules controlled in opposite directions by lapatinib+ATRA. The first one consists of six microRNAs (miR-1207-5-p, miR-874, miR-134, miR-762, miR-1181 and miR-320c) which are up-regulated by lapatinib+ATRA and control a common set of down-regulated target mRNAs in a coordinate fashion. Most of the mRNAs of the module control cell proliferation in a positive fashion. The second network consists of seven microRNAs (miR-203, miR-125a-5p, miR-193a-5p, miR-210, miR-362-5p, miR-532-5p and miR-342-3p) which are down-regulated by lapatinib+ATRA and control a series of up-regulated target-mRNAs. In this case, the majority of regulated genes is involved in negative regulation of cell proliferation. Coordinated regulation of microRNA sets controlling a common group of genes and functions represents a new concept that deserves further studies. Currently, we are in the process of establishing the functional significance of the identified microRNA modules for the anti-tumor effects of lapatinib and ATRA. Citation Format: Enrico Garattini, James N. Fisher, Gabriela Paroni, Floriana Centritto, Gregory J. Goodall, Anna Tsykin, Maddalena Fratelli, Mineko Terao. Combinations of lapatinib and all-trans retinoic acid control the coordinate expression of microRNA modules and target-mRNAs in ERBB2+/RARA+ breast carcinoma cells: Significance for the antitumor activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3088. doi:10.1158/1538-7445.AM2013-3088

The MYB oncogene encodes a transcription factor, Myb, which is essential for normal haemopoiesis and also for the proliferation of most acute leukaemias (reviewed in ref. 1). While a number of Myb target genes have been reported previously, these do not completely account for key elements of MYB’s activity, including its pro-leukaemic and differentiation-suppressing functions. We hypothesised that this reflects the fact that previous screens may have not been sufficiently comprehensive and/or employed the most appropriate cell systems. Thus we have embarked upon a program to identify and validate Myb targets critical for these functions. Here we report results from extensive expression profiling studies using a conditionally myb-transformed myeloid cell line, ERMYB (2). We have used ~44,000-element Illumina Beadchips in conjunction with a kinetic profiling strategy that selects candidates based on rapid, statistically-significant and consistent responses to both activation and inactivation of Myb. This has resulted in the identification of a substantial number of candidate Myb-activated and -repressed genes (381 and 502, respectively). In addition, we have used this cell system to identify candidate Myb-regulated microRNAs. Inspection of the list of candidate Myb-activated genes revealed several previously-described Myb targets including myc, bcl2, gstm1 and mpo, providing additional confidence in our approach. Our focus to date has been on novel candidates that may mediate myb’s ability to enhance proliferation, suppress differentiation and possibly suppress apoptosis. Q-RT-PCR was used as an initial validation step for a number of such targets; to date 13/14 genes identified by array screening have been confirmed by this method in ERMYB cells. A second approach to validation is to confirm correlation with Myb over-expression in a second cell system (FDB-1) (3). As in primary cells, enforced Myb expression can suppress differentiation and promote proliferation of these cells in the presence of GM-CSF (4). Amongst the Myb-activated genes are gfi1 and nucleostemin/gnl3, which are involved in stem-cell functions, cellular proliferation and in the case of gfi1, lineage-specific functions. Strikingly, candidate Myb-repressed genes include several important positive regulators of haemopoietic differentiation and/or negative regulators of proliferation, namely gata3, sfpi1/pu.1, cebpb, junb, klf’s-3,-6 -13 and btg1. Most of these genes have evolutionarily conserved internal or proximal candidate Myb binding sites. Our progress in validating these by chromatin immunoprecipitation will be presented. Finally, we have identified a number of microRNAs that are potentially regulated by Myb. These include members of the miR-17–92 cluster and mir-146b, which appear to be activated and repressed by Myb, respectively. These have been validated by Q-PCR for both the mature miR and the precursor pri-miR transcript. Interestingly, the mir-17–92 cluster has been strongly implicated in oncogenesis and cell cycle regulation (5), while miR-146a/b may have tumour suppressor activity.

Osteoarthritis (OA) is characterized by alterations to subchondral bone as well as articular cartilage. Changes to bone in OA have also been identified at sites distal to the affected joint, which include increased bone volume fraction and reduced bone mineralization. Altered bone remodelling has been proposed to underlie these bone changes in OA. To investigate the molecular basis for these changes, we performed microarray gene expression profiling of bone obtained at autopsy from individuals with no evidence of joint disease (control) and from individuals undergoing joint replacement surgery for either degenerative hip OA, or fractured neck of femur (osteoporosis [OP]). The OP sample set was included because an inverse association, with respect to bone density, has been observed between OA and the low bone density disease OP. Compugen human 19K-oligo microarray slides were used to compare the gene expression profiles of OA, control and OP bone samples. Four sets of samples were analyzed, comprising 10 OA-control female, 10 OA-control male, 10 OA-OP female and 9 OP-control female sample pairs. Print tip Lowess normalization and Bayesian statistical analyses were carried out using linear models for microarray analysis, which identified 150 differentially expressed genes in OA bone with t scores above 4. Twenty-five of these genes were then confirmed to be differentially expressed (P0.01) by real-time PCR analysis. A substantial number of the top-ranking differentially expressed genes identified in OA bone are known to play roles in osteoblasts, osteocytes and osteoclasts. Many of these genes are targets of either the WNT (wingless MMTV integration) signalling pathway (TWIST1, IBSP, S100A4, MMP25, RUNX2 and CD14) or the transforming growth factor (TGF)-beta/bone morphogenic protein (BMP) signalling pathway (ADAMTS4, ADM, MEPE, GADD45B, COL4A1 and FST). Other differentially expressed genes included WNT (WNT5B, NHERF1, CTNNB1 and PTEN) and TGF-beta/BMP (TGFB1, SMAD3, BMP5 and INHBA) signalling pathway component or modulating genes. In addition a subset of genes involved in osteoclast function (GSN, PTK9, VCAM1, ITGB2, ANXA2, GRN, PDE4A and FOXP1) was identified as being differentially expressed in OA bone between females and males. Altered expression of these sets of genes suggests altered bone remodelling and may in part explain the sex disparity observed in OA.