ceRNA Crosstalk Research Articles

Comparison and analysis of lncRNA-mediated ceRNA regulation in different molecular subtypes of glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant brain tumor with a poor prognosis. A molecular level classification of GBM can provide insight into accurate patient-specific treatment. Competitive endogenous RNAs (ceRNAs), such as long non-coding RNAs (lncRNAs), play an essential role in the development of tumors and are associated with survival. However, the pattern of lncRNA-mediated ceRNA (LMce) crosstalk in different GBM subtypes is still unclear. In this study, we present a computational cascade to construct LMce networks of different GBM subtypes and investigate the lncRNA-mRNA regulations among them. Our results showed that although most lncRNAs and mRNAs in the different GBM subtype networks were the same, the regulation relationships of these RNAs were different among subtypes. 42.5%, 50.9%, 43.5% and 65.0% lncRNA-mRNA regulatory pairs were classic (CL)-, mesenchymal (MES)-, proneural (PN)- and neural (NE)-specific. In addition, our study identified 61, 132, 24 and 16 modules in which lncRNAs and mRNAs synergically competed with each other for miRNAs as CL-, MES-, PN- and NE-specific. CL- and MES-specific modules were mainly involved in biological functions such as cell proliferation, apoptosis and migration, while PN- and NE-specific modules were mainly related to DNA damage and cell cycle dysregulation. Survival analysis demonstrated that some modules could be potential prognostic markers of patients of CL and MES subtypes. This study uncovered the LMce interaction patterns in different GBM subtypes, identified subtype-specific modules with distinct biological functions, and revealed the potential prognostic markers of patients of different GBM subtypes. These results might contribute to the discovery of the GBM prognostic biomarkers and development of a more accurate therapeutic process.

3' UTR shortening represses tumor-suppressor genes in trans by disrupting ceRNA crosstalk.

Widespread mRNA 3' UTR shortening through alternative polyadenylation 1 promotes tumor growth in vivo 2 . A prevailing hypothesis is that it induces proto-oncogene expression in cis through escaping microRNA-mediated repression. Here we report a surprising enrichment of 3'UTR shortening among transcripts that are predicted to act as competing-endogenous RNAs (ceRNAs) for tumor-suppressor genes. Our model-based analysis of the trans effect of 3' UTR shortening (MAT3UTR) reveals a significant role in altering ceRNA expression. MAT3UTR predicts many trans-targets of 3' UTR shortening, including PTEN, a crucial tumor-suppressor gene 3 involved in ceRNA crosstalk 4 with nine 3'UTR-shortening genes, including EPS15 and NFIA. Knockdown of NUDT21, a master 3' UTR-shortening regulator 2 , represses tumor-suppressor genes such as PHF6 and LARP1 in trans in a miRNA-dependent manner. Together, the results of our analysis suggest a major role of 3' UTR shortening in repressing tumor-suppressor genes in trans by disrupting ceRNA crosstalk, rather than inducing proto-oncogenes in cis.

A FTH1 gene:pseudogene:microRNA network regulates tumorigenesis in prostate cancer.

Non-coding RNAs play a vital role in diverse cellular processes. Pseudogenes, which are non-coding homologs of protein-coding genes, were once considered non-functional evolutional relics. However, recent studies have shown that pseudogene transcripts can regulate their parental transcripts by sequestering shared microRNAs (miRNAs), thus acting as competing endogenous RNAs (ceRNAs). In this study, we utilize an unbiased screen to identify the ferritin heavy chain 1 (FTH1) transcript and multiple FTH1 pseudogenes as targets of several oncogenic miRNAs in prostate cancer (PCa). We characterize the critical role of this FTH1 gene:pseudogene:miRNA network in regulating tumorigenesis in PCa, whereby oncogenic miRNAs downregulate the expression of FTH1 and its pseudogenes to drive oncogenesis. We further show that impairing miRNA binding and subsequent ceRNA crosstalk completely rescues the slow growth phenotype in vitro and in vivo. Our results also demonstrate the reciprocal regulation between the pseudogenes and intracellular iron levels, which are crucial for multiple physiological and pathophysiological processes. In summary, we describe an extensive gene:pseudogene network comprising multiple miRNAs and multiple pseudogenes derived from a single parental gene. The network could be regulated through multiple mechanisms to modulate iron storage in various signaling pathways, the deregulation of which results in PCa development and progression.

Integrative analysis of lncRNAs and miRNAs with coding RNAs associated with ceRNA crosstalk network in triple negative breast cancer.

Triple negative breast cancer (TNBC) is a particular subtype of breast malignant tumor with poorer prognosis than other molecular subtypes. Currently, there is increasing focus on long non-coding RNAs (lncRNAs), which can act as competing endogenous RNAs (ceR-NAs) and suppress miRNA functions involved in post-transcriptional regulatory networks in the tumor. Therefore, to investigate specific mechanisms of TNBC carcinogenesis and improve treatment efficiency, we comprehensively integrated expression profiles, including data on mRNAs, lncRNAs and miRNAs obtained from 116 TNBC tissues and 11 normal tissues from The Cancer Genome Atlas. As a result, we selected the threshold with |log2FC|>2.0 and an adjusted p-value >0.05 to obtain the differentially expressed mRNAs, miRNAs and lncRNAs. Hereafter, weighted gene co-expression network analysis was performed to identify the expression characteristics of dysregulated genes. We obtained five co-expression modules and related clinical feature. By means of correlating gene modules with protein–protein interaction network analysis that had identified 22 hub mRNAs which could as hub target genes. Eleven key dysregulated differentially expressed micro RNAs (DEmiRNAs) were identified that were significantly associated with the 22 hub potential target genes. Moreover, we found that 14 key differentially expressed lncRNAs could interact with the key DEmiRNAs. Then, the ceRNA crosstalk network of TNBC was constructed by utilizing key lncRNAs, key miRNAs, and hub mRNAs in Cytoscape software. We analyzed and described the potential characteristics of biological function and pathological roles of the TNBC ceRNA co-regulatory network; also, the survival analysis was performed for each molecule. These findings revealed that ceRNA crosstalk network could play an important role in the development and progression for TNBC. In addition, we also identified that some molecules in the ceRNA network possess clinical correlation and prognosis.

Systematic review of computational methods for identifying miRNA-mediated RNA-RNA crosstalk.

Posttranscriptional crosstalk and communication between RNAs yield large regulatory competing endogenous RNA (ceRNA) networks via shared microRNAs (miRNAs), as well as miRNA synergistic networks. The ceRNA crosstalk represents a novel layer of gene regulation that controls both physiological and pathological processes such as development and complex diseases. The rapidly expanding catalogue of ceRNA regulation has provided evidence for exploitation as a general model to predict the ceRNAs in silico. In this article, we first reviewed the current progress of RNA-RNA crosstalk in human complex diseases. Then, the widely used computational methods for modeling ceRNA-ceRNA interaction networks are further summarized into five types: two types of global ceRNA regulation prediction methods and three types of context-specific prediction methods, which are based on miRNA-messenger RNA regulation alone, or by integrating heterogeneous data, respectively. To provide guidance in the computational prediction of ceRNA-ceRNA interactions, we finally performed a comparative study of different combinations of miRNA-target methods as well as five types of ceRNA identification methods by using literature-curated ceRNA regulation and gene perturbation. The results revealed that integration of different miRNA-target prediction methods and context-specific miRNA/gene expression profiles increased the performance for identifying ceRNA regulation. Moreover, different computational methods were complementary in identifying ceRNA regulation and captured different functional parts of similar pathways. We believe that the application of these computational techniques provides valuable functional insights into ceRNA regulation and is a crucial step for informing subsequent functional validation studies.

The global view of mRNA-related ceRNA cross-talks across cardiovascular diseases

Competing endogenous RNA (ceRNA) have received wide attention because they are a novel way to regulate genes through sharing microRNAs (miRNAs) that are crucial for complex processes in many diseases. However, no systematic analysis of ceRNA mechanism in cardiovascular disease (CVD) is known. To gain insights into the global properties of ceRNAs in multi-CVDs, we constructed the global view of mRNA-related ceRNA cross-talk in eight major CVDs from ~2,800 samples. We found common features that could be used to uncover similarities among different CVDs and highlighted a common core ceRNA network across CVDs. Comparative analysis of hub ceRNAs in each network revealed three types of hubs, which might play key roles in diverse biological processes. Importantly, by combining CVD-related pathway genes with ceRNA-ceRNA interactions, common modules that might exert functions in specific mechanisms were identified. In addition, our study investigated a potential mechanistic linkage between pathway cross-talk and ceRNA cross-talk. In summary, this study uncovered and systematically characterized global properties of mRNA-related ceRNA cross-talks across CVDs, which may provide a new layer for exploring biological mechanisms and shed new light on cardiology.

Research progress in the competing endogenous RNA in gastric cancer pathogenesis

Recent studies demonstrate that the long non-coding RNAs (lncRNAs), pseudogenes, circular RNAs (circRNAs) and so on can be used as competitive endogenous RNAs (ceRNAs), binding to microRNAs (miRNAs), to regulate the expression level of their targets.This novel interplay optimizes traditional liner miRNA→RNA pattern and has become a research hotspot in the scientific community. Dysregulation of ceRNA interplay will influence the body′s normal life activities, leading to the occurrence of diseases, and even tumor formation. This article briefly introduces the key components of ceRNA crosstalk and its research progress in gastric cancer pathogenesis, so as to provide a new thought for cancer research, clinical diagnosis and therapeutics.(Chin J Lab Med, 2017, 40: 637-640) Key words: MicroRNAs; RNA, long noncoding; Pseudogenes

Translating ceRNA Susceptibilities into Correlation Functions

Competition to bind microRNAs induces an effective positive cross talk between their targets, which are therefore known as “competing endogenous RNAs” (ceRNAs). Although such an effect is known to play a significant role in specific situations, estimating its strength from data and experimentally in physiological conditions appears to be far from simple. Here, we show that the susceptibility of ceRNAs to different types of perturbations affecting their competitors (and hence their tendency to cross talk) can be encoded in quantities as intuitive and as simple to measure as correlation functions. This scenario is confirmed by extensive numerical simulations and validated by re-analyzing phosphatase and tensin homolog’s cross-talk pattern from The Cancer Genome Atlas breast cancer database. These results clarify the links between different quantities used to estimate the intensity of ceRNA cross talk and provide, to our knowledge, new keys to analyze transcriptional data sets and effectively probe ceRNA networks in silico.

MicroRNA inhibitors: Natural and artificial sequestration of microRNA

MicroRNA (miRNAs) is post-transcriptional regulator of mRNA. However, the prevalence and activity of miRNA are regulated by other regulators. miRNA inhibitors are natural or artificial RNA transcripts that sequestrate miRNAs and decrease or even eliminate miRNA activity. Competing endogenous RNAs (ceRNAs) are natural and intracellular miRNA inhibitors that compete to bind to shared miRNA recognition elements (MREs) to decrease microRNA availability and relieve the repression of target RNAs. In recent years, studies have revealed that ceRNA crosstalk is involved in many pathophysiological processes and adds a new dimension to miRNA regulation. Artificial miRNA inhibitors are RNA transcripts that are synthesized via chemical and genetic methods. Artificial miRNA inhibitors can be used in miRNA loss-of-function research and gene therapies for certain diseases. In this review, we summarize the recent advances in the two different types of miRNA inhibitors.

LAMC1 mRNA promotes malignancy of hepatocellular carcinoma cells by competing for MicroRNA-124 binding with CD151.

Specific RNAs can function as sinks for endogenous miRNAs, known as competing endogenous RNAs (ceRNAs). Here, we confirm a miR-124 mediated ceRNA crosstalk between LAMC1 and CD151 in hepatocellular carcinoma (HCC). miR-124 negatively regulates LAMC1 expression through two miRNA binding sites within its 3' untranslated region (3'UTR) and suppresses migration and invasion of HCC cells through regulating LAMC1. The wild type LAMC1 miRNA response elements (MREs) facilitate expression of CD151, and this regulation is miR-124 dependent. In clinical hepatic tissues, LAMC1 and CD151 mRNAs exhibit positive correlation. Importantly, LAMC1 MREs promote HCC malignancy by absorbing miR-124 and by assisting CD151 expression. We conclude that LAMC1 mRNA acts as a trans regulator to stimulate CD151 expression by competing for miR-124 binding in HCC cells. © 2017 IUBMB Life, 69(8):595-605, 2017.

Exploring genetic associations with ceRNA regulation in the human genome

Competing endogenous RNAs (ceRNAs) are RNA molecules that sequester shared microRNAs (miRNAs) thereby affecting the expression of other targets of the miRNAs. Whether genetic variants in ceRNA can affect its biological function and disease development is still an open question. Here we identified a large number of genetic variants that are associated with ceRNA's function using Geuvaids RNA-seq data for 462 individuals from the 1000 Genomes Project. We call these loci competing endogenous RNA expression quantitative trait loci or ‘cerQTL’, and found that a large number of them were unexplored in conventional eQTL mapping. We identified many cerQTLs that have undergone recent positive selection in different human populations, and showed that single nucleotide polymorphisms in gene 3΄UTRs at the miRNA seed binding regions can simultaneously regulate gene expression changes in both cis and trans by the ceRNA mechanism. We also discovered that cerQTLs are significantly enriched in traits/diseases associated variants reported from genome-wide association studies in the miRNA binding sites, suggesting that disease susceptibilities could be attributed to ceRNA regulation. Further in vitro functional experiments demonstrated that a cerQTL rs11540855 can regulate ceRNA function. These results provide a comprehensive catalog of functional non-coding regulatory variants that may be responsible for ceRNA crosstalk at the post-transcriptional level.

CeRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

Let-7e modulates the inflammatory response in vascular endothelial cells through ceRNA crosstalk

The inflammatory responses of vascular endothelial cells (VECs) are critical in the development of many cardio-cerebrovascular diseases. Let-7e is an important regulator of endothelial function and inflammation. However, the effects and mechanisms of let-7e on VECs inflammation have not been studied until recently. Thus, we investigated these issues and found that in addition to proliferation, apoptosis and cell adhesion, let-7e was also implicated in the regulation of inflammatory responses through a complex network, including IκBβ and lncRNA lnc-MKI67IP-3. Let-7e promoted NF-κB activation and translocation to the nucleus by inhibiting its target gene (IκBβ) expression and subsequently increased the expression of inflammatory and adhesion molecules. Meanwhile, lnc-MKI67IP-3 acted as a sponge or competing endogenous RNA (ceRNA) for let-7e, suppressing its pro-inflammatory effects, and let-7e decreased lnc-MKI67IP-3 expression, thereby forming a positive feedback loop to aggravate inflammation. Moreover, let-7e, lnc-MKI67IP-3 and IκBβ were also abnormal in oxLDL-treated VECs and atherosclerotic plaques. The present study revealed let-7e as a pro-inflammatory mediator and a novel regulatory mechanism for the NF-κB pathway through ceRNA crosstalk, comprising let-7e and its target IκBβ and the ceRNA lnc-MKI67IP-3. Thus, this molecule might play important roles in the inflammatory responses of VECs and development of atherosclerosis.

On cross-conditional and fluctuation correlations in competitive RNA networks.

In competitive endogenous RNA (ceRNA) networks, different mRNAs targeted by the same miRNA can 'cross-talk' by absorbing miRNAs and relieving repression on the other mRNAs. This creates correlations in mRNA expression even without direct interaction. Most previous theoretical study of cross-talk has focused on correlations in stochastic fluctuations of mRNAs around their steady state values. However, the experimentally known examples of cross-talk do not involve single-cell fluctuations, but rather bulk tissue-level changes between conditions, such as due to differentiation or disease. In our study, we quantify for the first time both fluctuational and cross-conditional cross-talk in chemical kinetic models of miRNA-mRNA interaction networks. We study the parameter regions under which these different types of cross-talk arise, and how they are affected by network structure. We find that while a network may support both fluctuational and cross-conditional cross-talk, the parameter regimes under which each type of cross-talk tends to emerge are rather different. Consistent with previous studies, fluctuational cross-talk occurs when miRNA and mRNA expression levels are 'balanced', whereas cross-conditional cross-talk tends to emerge when average miRNA levels are high and average mRNA levels are low. Conversely, cross-conditional miRNA cross-talk-a little-discussed phenomenon-is greatest when miRNA levels are low and mRNA levels are high. We show that the parameter ranges where cross-talk is maximized can, to some degree, be predicted based on network structure. Indeed, we find that the dominant effect of network structure on correlations happens through the effect of network structure on the overall balance between miRNA and mRNA expression. However, it is not the only effect, as we find that the density of connections between miRNAs and mRNAs in larger networks increases cross-talk without altering the expression balance. Our results deepen the theoretical understanding of cross-talk in ceRNA networks, and have implications for the experimental identification of ceRNA cross-talk phenomena. Simulation software available upon request. tperkins@ohri.ca.

Downregulated pseudogene CTNNAP1 promote tumor growth in human cancer by downregulating its cognate gene CTNNA1 expression.

Accumulating evidence indicates that deregulation of cancer-associated pseudogene is involved in the pathogenesis of cancer. In the study, we demonstrated that pseudogene CTNNAP1, for the CTNNA1 gene, was dysregulated in colorectal cancer and the degree of dysregulation was remarkably associated with tumor node metastasis (TNM) stage (P<0.05). The mechanistic experiments revealed that pseudogene CTNNAP1 played a pivotal role in the regulation of its cognate gene CTNNA1 by competition for microRNA-141. Moreover, gain-of-function approaches showed that overexpression of CTNNAP1 or CTNNA1 significantly inhibited cell proliferation and tumor growth in vitro and in vivo by inducing G0/G1 cell cycle arrest. Our findings add a new regulatory circuit via competing endogenous RNA (ceRNA) cross-talk between pseudogene CTNNAP1 and its cognate gene CTNNA1, and provide new insights into potential diagnostic biomarker for monitoring human colorectal cancer.

Characterization of long non-coding RNA-associated ceRNA network to reveal potential prognostic lncRNA biomarkers in human ovarian cancer.

Accumulating evidence has underscored the important roles of long non-coding RNAs (lncRNAs) acting as competing endogenous RNAs (ceRNAs) in cancer initiation and progression. In this study, we used an integrative computational method to identify miRNA-mediated ceRNA crosstalk between lncRNAs and mRNAs, and constructed global and progression-related lncRNA-associated ceRNA networks (LCeNETs) in ovarian cancer (OvCa) based on “ceRNA hypothesis”. The constructed LCeNETs exhibited small world, modular architecture and high functional specificity for OvCa. Known OvCa-related genes tended to be hubs and occurred preferentially in the functional modules. Ten lncRNA ceRNAs were identified as potential candidates associated with stage progression in OvCa using ceRNA-network driven method. Finally, we developed a ten-lncRNA signature which classified patients into high- and low-risk subgroups with significantly different survival outcomes. Our study will provide novel insight for better understanding of ceRNA-mediated gene regulation in progression of OvCa and facilitate the identification of novel diagnostic and therapeutic lncRNA ceRNAs for OvCa.

Competing endogenous RNA networks: tying the essential knots for cancer biology and therapeutics.

A recently discovered dimension of post-transcriptional gene regulation involves co-regulatory crosstalk between RNA transcripts, which compete for common pools of microRNA (miRNA) molecules. These competing endogenous RNAs (ceRNAs), or natural miRNA sponges, have an active role in regulating miRNA availability within the cell and form intertwined regulatory networks. Recent reports have implicated diverse RNA species including protein-coding messenger RNAs and non-coding RNAs as ceRNAs in human development and diseases including human cancer. In this review, we discuss the most recent discoveries that implicate natural miRNA decoys in human cancer biology, as well as exciting advances in the study of ceRNA networks and dynamics. The structure and topology of intricate genome-scale ceRNA networks can be predicted computationally, and their dynamic response to fluctuations in ceRNA and miRNA levels can be studied via mathematical modeling. Additionally, the development of new methods to quantitatively determine absolute expression levels of miRNA and ceRNA molecules have expanded the capacity to accurately study the efficiency of ceRNA crosstalk in diverse biological models. These major milestones are of critical importance to identify key components of ceRNA regulatory networks that could aid the development of new approaches to cancer diagnostics and oligonucleotide-based therapeutics.

Endogenous miRNA and Target Concentrations Determine Susceptibility to Potential ceRNA Competition

Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of invivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict invivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ?3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition invivo.

RNA-Based Regulation: Dynamics and Response to Perturbations of Competing RNAs

The observation that, through a titration mechanism, microRNAs (miRNAs) can act as mediators of effective interactions among their common targets (competing endogenous RNAs or ceRNAs) has brought forward the idea (i.e., the ceRNA hypothesis) that RNAs can regulate each other in extended cross-talk networks. Such an ability might play a major role in posttranscriptional regulation to shape a cell’s protein repertoire. Recent work focusing on the emergent properties of the cross-talk networks has emphasized the high flexibility and selectivity that may be achieved at stationarity. On the other hand, dynamical aspects, possibly crucial on the relevant timescales, are far less clear. We have carried out a dynamical study of the ceRNA hypothesis on a model of posttranscriptional regulation. Sensitivity analysis shows that ceRNA cross-talk is dynamically extended, i.e., it may take place on timescales shorter than those required to achieve stationarity even in cases where no cross-talk occurs in the steady state, and is possibly amplified. In addition, in the case of large, transfection-like perturbations, the system may develop a strongly nonlinear, threshold response. Finally, we show that the ceRNA effect provides a very efficient way for a cell to achieve fast positive shifts in the level of a ceRNA when necessary. These results indicate that competition for miRNAs may indeed provide an elementary mechanism to achieve system-level regulatory effects on the transcriptome over physiologically relevant timescales.

The multilayered complexity of ceRNA crosstalk and competition

Recent reports have described an intricate interplay among diverse RNA species, including protein-coding messenger RNAs and non-coding RNAs such as long non-coding RNAs, pseudogenes and circular RNAs. These RNA transcripts act as competing endogenous RNAs (ceRNAs) or natural microRNA sponges - they communicate with and co-regulate each other by competing for binding to shared microRNAs, a family of small non-coding RNAs that are important post-transcriptional regulators of gene expression. Understanding this novel RNA crosstalk will lead to significant insight into gene regulatory networks and have implications in human development and disease.