IL-33 Is a Cell-Intrinsic Regulator of Fitness during Early B Cell Development.

IL-33 is an IL-1 family member protein that is a potent driver of inflammatory responses in both allergic and nonallergic disease. This proinflammatory effect is mediated primarily by extracellular release of IL-33 from stromal cells and binding of the C-terminal domain of IL-33 to its receptor ST2 on targets such as CD4+ Th2 cells, ILC2, and mast cells. Notably, IL-33 has a distinct N-terminal domain that mediates nuclear localization and chromatin binding. However, a defined in vivo cell-intrinsic role for IL-33 has not been established. We identified IL-33 expression in the nucleus of progenitor B (pro-B) and large precursor B cells in the bone marrow, an expression pattern unique to B cells among developing lymphocytes. The IL-33 receptor ST2 was not expressed within the developing B cell lineage at either the transcript or protein level. RNA sequencing analysis of wild-type and IL-33-deficient pro-B and large precursor B cells revealed a unique, IL-33-dependent transcriptional profile wherein IL-33 deficiency led to an increase in E2F targets, cell cycle genes, and DNA replication and a decrease in the p53 pathway. Using mixed bone marrow chimeric mice, we demonstrated that IL-33 deficiency resulted in an increased frequency of developing B cells via a cell-intrinsic mechanism starting at the pro-B cell stage paralleling IL-33 expression. Finally, IL-33 was detectable during early B cell development in humans and IL33 mRNA expression was decreased in B cell chronic lymphocytic leukemia samples compared with healthy controls. Collectively, these data establish a cell-intrinsic, ST2-independent role for IL-33 in early B cell development.

Systematic dissection of dysregulated transcription factor-miRNA feed-forward loops across tumor types.

Transcription factor and microRNA (miRNA) can mutually regulate each other and jointly regulate their shared target genes to form feed-forward loops (FFLs). While there are many studies of dysregulated FFLs in a specific cancer, a systematic investigation of dysregulated FFLs across multiple tumor types (pan-cancer FFLs) has not been performed yet. In this study, using The Cancer Genome Atlas data, we identified 26 pan-cancer FFLs, which were dysregulated in at least five tumor types. These pan-cancer FFLs could communicate with each other and form functionally consistent subnetworks, such as epithelial to mesenchymal transition-related subnetwork. Many proteins and miRNAs in each subnetwork belong to the same protein and miRNA family, respectively. Importantly, cancer-associated genes and drug targets were enriched in these pan-cancer FFLs, in which the genes and miRNAs also tended to be hubs and bottlenecks. Finally, we identified potential anticancer indications for existing drugs with novel mechanism of action. Collectively, this study highlights the potential of pan-cancer FFLs as a novel paradigm in elucidating pathogenesis of cancer and developing anticancer drugs.

TSGene 2.0: an updated literature-based knowledgebase for tumor suppressor genes.

Tumor suppressor genes (TSGs) are a major type of gatekeeper genes in the cell growth. A knowledgebase with the systematic collection and curation of TSGs in multiple cancer types is critically important for further studying their biological functions as well as for developing therapeutic strategies. Since its development in 2012, the Tumor Suppressor Gene database (TSGene), has become a popular resource in the cancer research community. Here, we reported the TSGene version 2.0, which has substantial updates of contents (e.g. up-to-date literature and pan-cancer genomic data collection and curation), data types (noncoding RNAs and protein-coding genes) and content accessibility. Specifically, the current TSGene 2.0 contains 1217 human TSGs (1018 protein-coding and 199 non-coding genes) curated from over 9000 articles. Additionally, TSGene 2.0 provides thousands of expression and mutation patterns derived from pan-cancer data of The Cancer Genome Atlas. A new web interface is available at http://bioinfo.mc.vanderbilt.edu/TSGene/. Systematic analyses of 199 non-coding TSGs provide numerous cancer-specific non-coding mutational events for further screening and clinical use. Intriguingly, we identified 49 protein-coding TSGs that were consistently down-regulated in 11 cancer types. In summary, TSGene 2.0, which is the only available database for TSGs, provides the most updated TSGs and their features in pan-cancer.

Concordant dysregulation of miR-5p and miR-3p arms of the same precursor microRNA may be a mechanism in inducing cell proliferation and tumorigenesis: a lung cancer study.

A precursor microRNA (miRNA) has two arms: miR-5p and miR-3p (miR-5p/-3p). Depending on the tissue or cell types, both arms can become functional. However, little is known about their coregulatory mechanisms during the tumorigenic process. Here, by using the large-scale miRNA expression profiles of five cancer types, we revealed that several of miR-5p/-3p arms were concordantly dysregulated in each cancer. To explore possible coregulatory mechanisms of concordantly dysregulated miR-5p/-3p pairs, we developed a robust computational framework and applied it to lung cancer data. The framework deciphers miR-5p/-3p coregulated protein interaction networks critical to lung cancer development. As a novel part in the method, we uniquely applied the second-order partial correlation to minimize false-positive regulations. Using 279 matched miRNA and mRNA expression profiles extracted from tumor and normal lung tissue samples, we identified 17 aberrantly expressed miR-5p/-3p pairs that potentially modulate the gene expression of 35 protein complexes. Functional analyses revealed that these complexes are associated with cancer-related biological processes, suggesting the oncogenic potential of the reported miR-5p/-3p pairs. Specifically, we revealed that the reduced expression of miR-145-5p/-3p pair potentially contributes to elevated expression of genes in the "FOXM1 transcription factor network" pathway, which may consequently lead to uncontrolled cell proliferation. Subsequently, the regulation of miR-145-5p/-3p in the FOXM1signaling pathway was validated by a cohort of 104 matched miRNA and protein (reverse-phase protein array) expression profiles in lung cancer. In summary, our computational framework provides a novel tool to study miR-5p/-3p coregulatory mechanisms in cancer and other diseases.

Reproducible combinatorial regulatory networks elucidate novel oncogenic microRNAs in non-small cell lung cancer.

While previous studies reported aberrant expression of microRNAs (miRNAs) in non-small cell lung cancer (NSCLC), little is known about which miRNAs play central roles in NSCLC's pathogenesis and its regulatory mechanisms. To address this issue, we presented a robust computational framework that integrated matched miRNA and mRNA expression profiles in NSCLC using feed-forward loops. The network consists of miRNAs, transcription factors (TFs), and their common predicted target genes. To discern the biological meaning of their associations, we introduced the direction of regulation. A network edge validation strategy using three independent NSCLC expression profiling data sets pinpointed reproducible biological regulations. Reproducible regulation, which may reflect the true molecular interaction, has not been applied to miRNA-TF co-regulatory network analyses in cancer or other diseases yet. We revealed eight hub miRNAs that connected to a higher proportion of targets validated by independent data sets. Network analyses showed that these miRNAs might have strong oncogenic characteristics. Furthermore, we identified a novel miRNA-TF co-regulatory module that potentially suppresses the tumor suppressor activity of the TGF-ß pathway by targeting a core pathway molecule (TGFBR2). Follow-up experiments showed two miRNAs (miR-9-5p and miR-130b-3p) in this module had increased expression while their target gene TGFBR2 had decreased expression in a cohort of human NSCLC. Moreover, we demonstrated these two miRNAs directly bind to the 3' untranslated region of TGFBR2. This study enhanced our understanding of miRNA-TF co-regulatory mechanisms in NSCLC. The combined bioinformatics and validation approach we described can be applied to study other types of diseases.

Identifying Targetable Vulnerabilities to Circumvent or Overcome Venetoclax Resistance in Diffuse Large B-Cell Lymphoma.

Clinical trials with single-agent venetoclax/ABT-199 (anti-apoptotic BCL2 inhibitor) revealed that diffuse large B-cell lymphoma (DLBCL) is not solely dependent on BCL2 for survival. Gaining insight into pathways/proteins that increase venetoclax sensitivity or unique vulnerabilities in venetoclax-resistant DLBCL would provide new potential treatment avenues. Therefore, we generated acquired venetoclax-resistant DLBCL cells and evaluated these together with intrinsically venetoclax-resistant and -sensitive DLBCL lines. We identified resistance mechanisms, including alterations in BCL2 family members that differed between intrinsic and acquired venetoclax resistance and increased dependencies on specific pathways. Although combination treatments with BCL2 family member inhibitors may overcome venetoclax resistance, RNA-sequencing and drug/compound screens revealed that venetoclax-resistant DLBCL cells, including those with TP53 mutation, had a preferential dependency on oxidative phosphorylation. Mitochondrial electron transport chain complex I inhibition induced venetoclax-resistant, but not venetoclax-sensitive, DLBCL cell death. Inhibition of IDH2 (mitochondrial redox regulator) synergistically overcame venetoclax resistance. Additionally, both acquired and intrinsic venetoclax-resistant DLBCL cells were similarly sensitive to inhibitors of transcription, B-cell receptor signaling, and class I histone deacetylases. These approaches were also effective in DLBCL, follicular, and marginal zone lymphoma patient samples. Our results reveal there are multiple ways to circumvent or overcome the diverse venetoclax resistance mechanisms in DLBCL and other B-cell lymphomas and identify critical targetable pathways for future clinical investigations.

SIRT6 loss causes intervertebral disc degeneration in mice by promoting senescence and SASP status.

Intervertebral disc degeneration is a major risk factor contributing to chronic low back and neck pain. While the etiological factors for disc degeneration vary, age is still one of the most important risk factors. Recent studies have shown the promising role of SIRT6 in mammalian aging and skeletal tissue health, however its role in the intervertebral disc health remains unexplored. We investigated the contribution of SIRT6 to disc health by studying the age-dependent spinal phenotype of mice with conditional deletion of Sirt6 in the disc ( Acan CreERT2 ; Sirt6 fl/fl ). Histological studies showed a degenerative phenotype in knockout mice compared to Sirt6 fl/fl control mice at 12 months which became pronounced at 24 months. RNA-Seq analysis of NP and AF tissues, quantitative histone analysis, and in vitro multiomics employing RNA-seq with ATAC-seq revealed that SIRT6-loss resulted in changes in acetylation and methylation status of specific Histone 3 lysine residues, thereby affecting DNA accessibility and transcriptomic landscape. A decrease in autophagy and an increase in DNA damage were also noted in Sirt6 -deficient cells. Further mechanistic insights revealed that loss of SIRT6 increased senescence and SASP burden in the disc characterized by increased p21, γH2AX, IL-6, and TGF-ß abundance. Taken together our study highlights the contribution of SIRT6 in modulating DNA damage, autophagy and cell senescence, and its importance in maintaining disc health during aging thereby underscoring it as a potential therapeutic target to treat intervertebral disc degeneration.

Neuroendocrine gene subsets are uniquely dysregulated in prostate adenocarcinoma.

Prostate cancer has heterogeneous growth patterns, and its prognosis is the poorest when it progresses to a neuroendocrine phenotype. Using bioinformatic analysis, we evaluated RNA expression of neuroendocrine genes in a panel of five different cancer types: prostate adenocarcinoma, breast cancer, kidney chromophobe, kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. Our results show that specific neuroendocrine genes are significantly dysregulated in these tumors, suggesting that they play an active role in cancer progression. Among others, synaptophysin (SYP), a conventional neuroendocrine marker, is upregulated in prostate adenocarcinoma (PRAD) and breast cancer (BRCA). Our analysis shows that SYP is enriched in small extracellular vesicles (sEVs) derived from plasma of PRAD patients, but it is absent in sEVs derived from plasma of healthy donors. Similarly, classical sEV markers are enriched in sEVs derived from plasma of prostate cancer patients, but weakly detectable in sEVs derived from plasma of healthy donors. Overall, our results pave the way to explore new strategies to diagnose these diseases based on the neuroendocrine gene expression in patient tumors or plasma sEVs.

Decoding the lncRNAome Across Diverse Cellular Stresses Reveals Core p53-effector Pan-cancer Suppressive lncRNAs.

Despite long non-coding RNAs (lncRNAs) emerging as key contributors to malignancies, their transcriptional regulation, tissue-type expression under different conditions, and functions remain largely unknown. Developing a combined computational and experimental framework, which integrates pan-cancer RNAi/CRISPR screens, and genomic, epigenetic, and expression profiles (including single-cell RNA sequencing), we report across multiple cancers, core p53-transcriptionally regulated lncRNAs, which were thought to be primarily cell/tissue-specific. These lncRNAs were consistently directly transactivated by p53 with different cellular stresses in multiple cell types and associated with pan-cancer cell survival/growth suppression and patient survival. Our prediction results were verified through independent validation datasets, our own patient cohort, and cancer cell experiments. Moreover, a top predicted p53-effector tumor-suppressive lncRNA (we termed PTSL) inhibited cell proliferation and colony formation by modulating the G2 regulatory network, causing G2 cell-cycle arrest. Therefore, our results elucidated previously unreported, high-confidence core p53-targeted lncRNAs that suppress tumorigenesis across cell types and stresses. Significance: Identification of pan-cancer suppressive lncRNAs transcriptionally regulated by p53 across different cellular stresses by integrating multilayered high-throughput molecular profiles. This study provides critical new insights into the p53 tumor suppressor by revealing the lncRNAs in the p53 cell-cycle regulatory network and their impact on cancer cell growth and patient survival.

Genomic Landscape and Potential Regulation of RNA Editing in Drug Resistance.

Adenosine-to-inosine RNA editing critically affects the response of cancer therapies. However, comprehensive identification of drug resistance-related RNA editing events and systematic understanding of how RNA editing mediates anticancer drug resistance remain unclear. Here, 7157 differential editing sites (DESs) are identified from 98 127 informative RNA editing sites in tumor tissues, many of which are validated in cancer cell lines. Diverse editing patterns of DESs are discovered in resistant samples, which could not be fully explained by adenosine deaminase acting on RNA enzymes. Some RNA-binding proteins are identified that potentially regulate these editing events. Notably, the DESs are significantly enriched in 3'-untranslated regions (3'-UTRs). The impact of DESs in 3'-UTR on the microRNA (miRNA) regulations is explored, and some triplets (DES, miRNA, and gene) that may contribute to drug resistance are identified. In addition, it is determined that the functions of genes enriched with DESs are associated with drug resistance, such as apoptosis, drug metabolism, and DNA synthesis involved in DNA repair. An online resource (http://www.jianglab.cn/REDR/) to support convenient retrieval of DESs is also built. The findings reveal the landscape and potential regulatory mechanism of RNA editing in drug resistance, providing new therapeutic targets for reversing drug resistance.

Targeted MDM2 Degradation Reveals a New Vulnerability for p53-Inactivated Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) frequently inactivate p53, increasing their aggressiveness and therapy resistance. We identified an unexpected protein vulnerability in p53-inactivated TNBC and designed a new PROteolysis TArgeting Chimera (PROTAC) to target it. Our PROTAC selectively targets MDM2 for proteasome-mediated degradation with high-affinity binding and VHL recruitment. MDM2 loss in p53 mutant/deleted TNBC cells in two-dimensional/three-dimensional culture and TNBC patient explants, including relapsed tumors, causes apoptosis while sparing normal cells. Our MDM2-PROTAC is stable in vivo, and treatment of TNBC xenograft-bearing mice demonstrates tumor on-target efficacy with no toxicity to normal cells, significantly extending survival. Transcriptomic analyses revealed upregulation of p53 family target genes. Investigations showed activation and a required role for TAp73 to mediate MDM2-PROTAC-induced apoptosis. Our data, challenging the current MDM2/p53 paradigm, show MDM2 is required for p53-inactivated TNBC cell survival, and PROTAC-targeted MDM2 degradation is an innovative potential therapeutic strategy for TNBC and superior to existing MDM2 inhibitors. SIGNIFICANCE: p53-inactivated TNBC is an aggressive, therapy-resistant, and lethal breast cancer subtype. We designed a new compound targeting an unexpected vulnerability we identified in TNBC. Our MDM2-targeted degrader kills p53-inactivated TNBC cells, highlighting the requirement for MDM2 in TNBC cell survival and as a new therapeutic target for this disease. See related commentary by Peuget and Selivanova, p. 1043. This article is highlighted in the In This Issue feature, p. 1027.

4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.

Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.

Systematic lncRNA mapping to genome-wide co-essential modules uncovers cancer dependency on uncharacterized lncRNAs.

Quantification of gene dependency across hundreds of cell lines using genome-scale CRISPR screens has revealed co-essential pathways/modules and critical functions of uncharacterized genes. In contrast to protein-coding genes, robust CRISPR-based loss-of-function screens are lacking for long noncoding RNAs (lncRNAs), which are key regulators of many cellular processes, leaving many essential lncRNAs unidentified and uninvestigated. Integrating copy number, epigenetic, and transcriptomic data of >800 cancer cell lines with CRISPR-derived co-essential pathways, our method recapitulates known essential lncRNAs and predicts proliferation/growth dependency of 289 poorly characterized lncRNAs. Analyzing lncRNA dependencies across 10 cancer types and their expression alteration by diverse growth inhibitors across cell types, we prioritize 30 high-confidence pan-cancer proliferation/growth-regulating lncRNAs. Further evaluating two previously uncharacterized top proliferation-suppressive lncRNAs (PSLR-1, PSLR-2) showed they are transcriptionally regulated by p53, induced by multiple cancer treatments, and significantly correlate to increased cancer patient survival. These lncRNAs modulate G2 cell cycle-regulating genes within the FOXM1 transcriptional network, inducing a G2 arrest and inhibiting proliferation and colony formation. Collectively, our results serve as a powerful resource for exploring lncRNA-mediated regulation of cellular fitness in cancer, circumventing current limitations in lncRNA research.

CTpathway: a CrossTalk-based pathway enrichment analysis method for cancer research.

BACKGROUND: Pathway enrichment analysis (PEA) is a common method for exploring functions of hundreds of genes and identifying disease-risk pathways. Moreover, different pathways exert their functions through crosstalk. However, existing PEA methods do not sufficiently integrate essential pathway features, including pathway crosstalk, molecular interactions, and network topologies, resulting in many risk pathways that remain uninvestigated. METHODS: To overcome these limitations, we develop a new crosstalk-based PEA method, CTpathway, based on a global pathway crosstalk map (GPCM) with >440,000 edges by combing pathways from eight resources, transcription factor-gene regulations, and large-scale protein-protein interactions. Integrating gene differential expression and crosstalk effects in GPCM, we assign a risk score to genes in the GPCM and identify risk pathways enriched with the risk genes. RESULTS: Analysis of >8300 expression profiles covering ten cancer tissues and blood samples indicates that CTpathway outperforms the current state-of-the-art methods in identifying risk pathways with higher accuracy, reproducibility, and speed. CTpathway recapitulates known risk pathways and exclusively identifies several previously unreported critical pathways for individual cancer types. CTpathway also outperforms other methods in identifying risk pathways across all cancer stages, including early-stage cancer with a small number of differentially expressed genes. Moreover, the robust design of CTpathway enables researchers to analyze both bulk and single-cell RNA-seq profiles to predict both cancer tissue and cell type-specific risk pathways with higher accuracy. CONCLUSIONS: Collectively, CTpathway is a fast, accurate, and stable pathway enrichment analysis method for cancer research that can be used to identify cancer risk pathways. The CTpathway interactive web server can be accessed here http://www.jianglab.cn/CTpathway/ . The stand-alone program can be accessed here https://github.com/Bioccjw/CTpathway .

miRcorrNet: machine learning-based integration of miRNA and mRNA expression profiles, combined with feature grouping and ranking.

A better understanding of disease development and progression mechanisms at the molecular level is critical both for the diagnosis of a disease and for the development of therapeutic approaches. The advancements in high throughput technologies allowed to generate mRNA and microRNA (miRNA) expression profiles; and the integrative analysis of these profiles allowed to uncover the functional effects of RNA expression in complex diseases, such as cancer. Several researches attempt to integrate miRNA and mRNA expression profiles using statistical methods such as Pearson correlation, and then combine it with enrichment analysis. In this study, we developed a novel tool called miRcorrNet, which performs machine learning-based integration to analyze miRNA and mRNA gene expression profiles. miRcorrNet groups mRNAs based on their correlation to miRNA expression levels and hence it generates groups of target genes associated with each miRNA. Then, these groups are subject to a rank function for classification. We have evaluated our tool using miRNA and mRNA expression profiling data downloaded from The Cancer Genome Atlas (TCGA), and performed comparative evaluation with existing tools. In our experiments we show that miRcorrNet performs as good as other tools in terms of accuracy (reaching more than 95% AUC value). Additionally, miRcorrNet includes ranking steps to separate two classes, namely case and control, which is not available in other tools. We have also evaluated the performance of miRcorrNet using a completely independent dataset. Moreover, we conducted a comprehensive literature search to explore the biological functions of the identified miRNAs. We have validated our significantly identified miRNA groups against known databases, which yielded about 90% accuracy. Our results suggest that miRcorrNet is able to accurately prioritize pan-cancer regulating high-confidence miRNAs. miRcorrNet tool and all other supplementary files are available at https://github.com/malikyousef/miRcorrNet.

SFSSClass: an integrated approach for miRNA based tumor classification.

BACKGROUND: MicroRNA (miRNA) expression profiling data has recently been found to be particularly important in cancer research and can be used as a diagnostic and prognostic tool. Current approaches of tumor classification using miRNA expression data do not integrate the experimental knowledge available in the literature. A judicious integration of such knowledge with effective miRNA and sample selection through a biclustering approach could be an important step in improving the accuracy of tumor classification. RESULTS: In this article, a novel classification technique called SFSSClass is developed that judiciously integrates a biclustering technique SAMBA for simultaneous feature (miRNA) and sample (tissue) selection (SFSS), a cancer-miRNA network that we have developed by mining the literature of experimentally verified cancer-miRNA relationships and a classifier uncorrelated shrunken centroid (USC). SFSSClass is used for classifying multiple classes of tumors and cancer cell lines. In a part of the investigation, poorly differentiated tumors (PDT) having non diagnostic histological appearance are classified while training on more differentiated tumor (MDT) samples. The proposed method is found to outperform the best known accuracy in the literature on the experimental data sets. For example, while the best accuracy reported in the literature for classifying PDT samples is approximately 76.5%, the accuracy of SFSSClass is found to be approximately 82.3%. The advantage of incorporating biclustering integrated with the cancer-miRNA network is evident from the consistently better performance of SFSSClass (integration of SAMBA, cancer-miRNA network and USC) over USC (eg., approximately 70.5% for SFSSClass versus approximately 58.8% in classifying a set of 17 MDT samples from 9 tumor types, approximately 91.7% for SFSSClass versus approximately 75% in classifying 12 cell lines from 6 tumor types and approximately 82.3% for SFSSClass versus approximately 41.2% in classifying 17 PDT samples from 11 tumor types). CONCLUSION: In this article, we develop the SFSSClass algorithm which judiciously integrates a biclustering technique for simultaneous feature (miRNA) and sample (tissue) selection, the cancer-miRNA network and a classifier. The novel integration of experimental knowledge with computational tools efficiently selects relevant features that have high intra-class and low inter-class similarity. The performance of the SFSSClass is found to be significantly improved with respect to the other existing approaches.

TargetMiner: microRNA target prediction with systematic identification of tissue-specific negative examples.

MOTIVATION: Prediction of microRNA (miRNA) target mRNAs using machine learning approaches is an important area of research. However, most of the methods suffer from either high false positive or false negative rates. One reason for this is the marked deficiency of negative examples or miRNA non-target pairs. Systematic identification of non-target mRNAs is still not addressed properly, and therefore, current machine learning approaches are compelled to rely on artificially generated negative examples for training. RESULTS: In this article, we have identified approximately 300 tissue-specific negative examples using a novel approach that involves expression profiling of both miRNAs and mRNAs, miRNA-mRNA structural interactions and seed-site conservation. The newly generated negative examples are validated with pSILAC dataset, which elucidate the fact that the identified non-targets are indeed non-targets.These high-throughput tissue-specific negative examples and a set of experimentally verified positive examples are then used to build a system called TargetMiner, a support vector machine (SVM)-based classifier. In addition to assessing the prediction accuracy on cross-validation experiments, TargetMiner has been validated with a completely independent experimental test dataset. Our method outperforms 10 existing target prediction algorithms and provides a good balance between sensitivity and specificity that is not reflected in the existing methods. We achieve a significantly higher sensitivity and specificity of 69% and 67.8% based on a pool of 90 feature set and 76.5% and 66.1% using a set of 30 selected feature set on the completely independent test dataset. In order to establish the effectiveness of the systematically generated negative examples, the SVM is trained using a different set of negative data generated using the method in Yousef et al. A significantly higher false positive rate (70.6%) is observed when tested on the independent set, while all other factors are kept the same. Again, when an existing method (NBmiRTar) is executed with the our proposed negative data, we observe an improvement in its performance. These clearly establish the effectiveness of the proposed approach of selecting the negative examples systematically. AVAILABILITY: TargetMiner is now available as an online tool at www.isical.ac.in/ approximately bioinfo_miu

Pan-cancer analysis reveals cooperativity of both strands of microRNA that regulate tumorigenesis and patient survival.

Recently, both 5p and 3p miRNA strands are being recognized as functional instead of only one, leaving many miRNA strands uninvestigated. To determine whether both miRNA strands, which have different mRNA-targeting sequences, cooperate to regulate pathways/functions across cancer types, we evaluate genomic, epigenetic, and molecular profiles of >5200 patient samples from 14 different cancers, and RNA interference and CRISPR screens in 290 cancer cell lines. We identify concordantly dysregulated miRNA 5p/3p pairs that coordinately modulate oncogenic pathways and/or cell survival/growth across cancers. Down-regulation of both strands of miR-30a and miR-145 recurrently increased cell cycle pathway genes and significantly reduced patient survival in multiple cancers. Forced expression of all four strands show cooperativity, reducing cell cycle pathways and inhibiting lung cancer cell proliferation and migration. Therefore, we identify miRNA whose 5p/3p strands function together to regulate core tumorigenic processes/pathways and reveal a previously unknown pan-cancer miRNA signature with patient prognostic power.

MicroRNA and transcription factor co-regulatory networks and subtype classification of seminoma and non-seminoma in testicular germ cell tumors.

Recent studies have revealed that feed-forward loops (FFLs) as regulatory motifs have synergistic roles in cellular systems and their disruption may cause diseases including cancer. FFLs may include two regulators such as transcription factors (TFs) and microRNAs (miRNAs). In this study, we extensively investigated TF and miRNA regulation pairs, their FFLs, and TF-miRNA mediated regulatory networks in two major types of testicular germ cell tumors (TGCT): seminoma (SE) and non-seminoma (NSE). Specifically, we identified differentially expressed mRNA genes and miRNAs in 103 tumors using the transcriptomic data from The Cancer Genome Atlas. Next, we determined significantly correlated TF-gene/miRNA and miRNA-gene/TF pairs with regulation direction. Subsequently, we determined 288 and 664 dysregulated TF-miRNA-gene FFLs in SE and NSE, respectively. By constructing dysregulated FFL networks, we found that many hub nodes (12 out of 30 for SE and 8 out of 32 for NSE) in the top ranked FFLs could predict subtype-classification (Random Forest classifier, average accuracy ≥90%). These hub molecules were validated by an independent dataset. Our network analysis pinpointed several SE-specific dysregulated miRNAs (miR-200c-3p, miR-25-3p, and miR-302a-3p) and genes (EPHA2, JUN, KLF4, PLXDC2, RND3, SPI1, and TIMP3) and NSE-specific dysregulated miRNAs (miR-367-3p, miR-519d-3p, and miR-96-5p) and genes (NR2F1 and NR2F2). This study is the first systematic investigation of TF and miRNA regulation and their co-regulation in two major TGCT subtypes.