G C A T T A C G G C A T genes Article CircIMPACT: An R Package to Explore Circular RNA Impact on Gene Expression and Pathways Alessia Buratin 1,2 , Enrico Gaffo 2 , Anna Dal Molin 2 and Stefania Bortoluzzi 2,3,* 1 Department of Biology, University of Padova, 35131 Padova, Italy; [email protected] 2 Department of Molecular Medicine, University of Padova, 35131 Padova, Italy; [email protected] (E.G.); [email protected] (A.D.M.) 3 Interdepartmental Research Center for Innovative Biotechnologies (CRIBI), University of Padova, 35131 Padova, Italy * Correspondence: [email protected] Abstract: Circular RNAs (circRNAs) are transcripts generated by back-splicing. CircRNAs might regulate cellular processes by different mechanisms, including interaction with miRNAs and RNA- binding proteins. CircRNAs are pleiotropic molecules whose dysregulation has been linked to human diseases and can drive cancer by impacting gene expression and signaling pathways. The detection of circRNAs aberrantly expressed in disease conditions calls for the investigation of their functions. Here, we propose CircIMPACT, a bioinformatics tool for the integrative analysis of circRNA and gene expression data to facilitate the identification and visualization of the genes whose expression varies according to circRNA expression changes. This tool can highlight regulatory axes potentially governed by circRNAs, which can be prioritized for further experimental study. The usefulness of CircIMPACT is exemplified by a case study analysis of bladder cancer RNA-seq data. The link between circHIPK3 and heparanase (HPSE) expression, due to the circHIPK3-miR558-HPSE Citation: Buratin, A.; Gaffo, E.; Dal regulatory axis previously determined by experimental studies on cell lines, was successfully detected. Molin, A.; Bortoluzzi, S. CircIMPACT: CircIMPACT is freely available at GitHub. An R Package to Explore Circular RNA Impact on Gene Expression and Keywords: circular RNA; gene expression; regulatory axes; pathways Pathways. Genes 2021, 12, 1044. https://doi.org/10.3390/ genes12071044 1. Introduction Academic Editor: Björn Voß Circular RNAs (circRNAs) are a class of abundant and stable RNAs that result from the ligation of a downstream splice donor to an upstream splice acceptor [1]. The progres- Received: 1 June 2021 sive discovery of circRNA functions, involvement in biological processes, and oncogenic Accepted: 2 July 2021 potential made them attractive molecules for both fundamental and cancer research [2]. Published: 6 July 2021 CircRNAs regulate cellular processes by acting with different mechanisms (Figure1 ), mostly involving sequence-specific binding with other nucleic acids or proteins. Of note, Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in one prominent mechanism whereby circRNAs are believed to function is by sponging published maps and institutional affil- miRNA, thus regulating the expression of miRNA-target genes, working as competitive iations. endogenous RNAs (ceRNAs) [3]. Aberrant DNA methylation and histone modifications can be controlled by circRNAs that regulate key epigenetic “writers” like DNMT1 [4] and EZH2 [5] methyltransferases. Regulatory functions on gene transcription were also described for circRNAs [6]. Other circRNAs modulate the activity of RNA-binding proteins (RBPs), a large class of molecules involved in a multitude of processes, including the Copyright: © 2021 by the authors. control of cell cycle progression [7] and splicing [8], among others. In addition, since most Licensee MDPI, Basel, Switzerland. This article is an open access article circRNAs originate from the circularization of coding gene exons, circRNA biogenesis distributed under the terms and can compete with linear RNA splicing [8]. Beyond exerting functions typical of long conditions of the Creative Commons non-coding RNAs, circRNAs can be translated into peptides [9,10]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Genes 2021, 12, 1044. https://doi.org/10.3390/genes12071044 https://www.mdpi.com/journal/genes Genes 2021, 12, x FOR PEER REVIEW 2 of 13 Evidence on circRNA functions added another level of complexity in the network of diverse players in regulating cellular processes, impacting normal and pathogenic or malignant phenotypes. Transcriptomics data has been proven useful to unearth the effects of protein and RNA dysfunctions, and their contribution to the pathogenesis and global deregulation of gene expression in cancer. This type of analysis falls in the field of expression data reverse engineering, which has been extensively used to infer regulatory Genes 2021, 12, 1044 networks involving transcriptional regulators [11], miRNAs [12], and combinations2 of 13 thereof [13,14]. Starting from the hypothesis that circRNAs affect gene expression, then gene expression profiles could be used to infer circRNA biological functions (Figure 1). Figure 1. CircRNAs regulate cell behavior with different mechanisms. The integrated analysis of Figure 1. CircRNAs regulate cell behavior with different mechanisms. The integrated analysis circRNA and linear gene expression profiles can predict circRNA functionsby identifying the of circRNA and linear gene expression profiles can predict circRNA functionsby identifying the biological processes and pathways impacted by circRNA expression variation. biological processes and pathways impacted by circRNA expression variation. EvidenceCircRNAs on can circRNA be detected functions and addedquantifi anothered using level RNA-seq of complexity [15] and in theappropriate network ofsoftware diverse tools players [16,17]. in regulating Typically, cellular studies processes, aiming at impactingidentifying normal circRNA and roles pathogenic in disease or malignantand cancer phenotypes. face two consecutive Transcriptomics challenges. data has The been first proven is to usefulefficiently to unearth prioritize the the ef- fectscircRNAs of protein that discriminate and RNA dysfunctions, between normal and an theird malignant contribution cells or to that the pathogenesishave the potential and globalto define deregulation new and possibly of gene relevant expression disease in cancer. subtypes. This Subsequently, type of analysis the falls involvement in the field of ofcircRNAs expression in dataspecific reverse pathological engineering, mechan whichisms has beenor biological extensively processes used toinfer should regula- be toryidentified. networks These involving challenges transcriptional can be tackled regulators by circRNA [11 ],screening, miRNAs for [12 ],instance, and combinations by massive thereofcircRNA [13 silencing,14]. Starting or overexpression from the hypothesis studies, thatand circRNAswith experimental affect gene investigation expression, of then the genemechanisms expression involved, profiles once could the be usedcircRNAs to infer whose circRNA expression biological impacts functions significant (Figure1). cell featuresCircRNAs have been can identified. be detected and quantified using RNA-seq [15] and appropriate soft- wareComputational tools [16,17]. Typically, predictions studies were aiming extensivel at identifyingy used to circRNAidentify circRNA roles in disease correlation and cancerwith diseases face two [18] consecutive and infer challenges. circRNA functi The firstons. isMost to efficiently of the efforts prioritize were the put circRNAs toward thatpredicting discriminate the miRNA-sponging between normal andactivity malignant of circRNAs cells or and that inferring have the potentialcircRNA-miRNA- to define newgene andregulatory possibly relevantaxes. Among disease subtypes.circRNA-dedi Subsequently,cated databases, the involvement CircInteractome of circRNAs [19], in specificCircAtlas pathological [20], and CSCD mechanisms [21] provide or biological information processes about shouldmiRNAs be potentially identified. sponged These chal- by lengescircRNAs. can beHowever, tackled bynone circRNA of them screening, allows the for analysis instance, of bynew massive expression circRNA data. silencing or overexpressionThe ceRNA studies, function and was with the experimental first described investigation for circRNAs of the [1,22] mechanisms and has important involved, onceimplications the circRNAs in cancer. whose We expression and other groups impacts used significant custom cell integrative features haveanalysis been of identified. circRNA, miRNA,Computational and gene expression predictions data, were with extensively systems used biology to identify methods, circRNA to infer correlation circRNA withregulatory diseases networks [18] and [23–26]. infer circRNABesides, the functions. Cerina tool Most for of predicting the efforts biological were put functions toward predictingof circRNAs the based miRNA-sponging on the ceRNA activity model of was circRNAs recently and made inferring available circRNA-miRNA-gene [27]. However, the regulatoryceRNA function axes. Among may not circRNA-dedicated apply to all circRNAs databases, [28,29]: CircInteractome systematic analyses [19], CircAtlas of circRNA [20], andsequences CSCD showed [21] provide that only information a minority about present miRNAs multiple potentially binding sites sponged for specific by circRNAs. miRNAs However,[30], and, in none general, of them circRNAs allows theare analysisnot bound of newto miRNA-loaded expression data. Argonaute proteins [31]. The ceRNA function was the first described for circRNAs [1,22] and has important implications in cancer. We and other groups