miR-34a-5p and miR-34a-3p contribute to thesignaling pathway of p53 by targeting

overlapping sets of genes

Abderrahim Chafik

Ministere de l’Emploi et des Affaires Sociales, Rue AlJommayz, Hay Riad , Rabat, Morocco

The Abdus Salam International Centre for Theoretical Physics(ICTP), Strada Costiera, 11 I - 34151 Trieste Italy

August 11, 2016

Abstract

In contrary to the common belief that only one strand of the pre-miRNA is active (usually the 5p one that is the more abundant) whilethe second one (miRNA*) is discarded, functional 5p and 3p have beenobserved for many miRNAs. Among those miRNAs is miR-34a whichis a target gene of the tumor suppressor p53. In this paper we havere-examined the role of miR-34a-5p and miR-34a-3p in the signalingpathway of p53. We found that they target overlapping sets of genes(MDM2 and THBS1). By a GO enrichment analysis we found thatTHBS1 is involved in cancer and metastasis relevant processes. Wehave also deduce that p53, MDM2 and miR-34a are linked by a type1 incoherent FFL that represent a novel mechanism for acceleratingthe response of p53 to external stress signals.

1 Introduction

p53 has been dubbed ”guardian of the genome” [1] or gatekeeper [2]due to its central role in maintaining the genomic stability and tu-mor suppression [3, 4, 5]. It has been found to be mutated in about

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half of the human cancers [6, 7]. Since its discovery p53 has beensubject to a tremendous amount of work making it one of the mostextensively studied gene. Its tumor suppressive role consists in induc-ing anti-proliferative cellular responses to a variety of stress signals,namely a cell-cycle arrest, senescence or an apoptosis. p53 can thenbe activated in response to DNA damage, hypoxia, or aberrant growthsignals resulting from deregulated expression of oncogenes [8, 9, 10].Under normal conditions p53 is expressed at low level as a result of theaction of the ubiquitin-protein ligase MDM2, that repress p53 throughubiquitin-mediated degradation [11, 12]. In response to a stress sig-nal as DNA damage p53 undergoes post-transcriptional modifications(phosphorylation and acetylation) by various kinases, including ATM,ATR, DNA-PK, Chk1, and Chk2 [13]. These modifications activatep53 by inhibiting MDM2 from binding to its N-terminal and allow itto carry out its major function as a transcription factor that bindsto specific DNA sequences and activate the transcription of adjacentgenes. By inducing the expression of its target genes p53 inducescell-cycle arrest or apoptosis.

MicroRNAs (miRNAs) are small (20 25 nucleotides) non-codingRNAs that negatively regulate gene expression post transcriptionallyby base-pairing to the 3’UTR of target mRNAs leading to repression ofprotein production or mRNA degradation (For a recent introductionsee for example [14]). a single miRNA can target hundreds of differentmRNAs, and a single mRNA can be coordinately suppressed by multi-ple different miRNAs, They are involved in many biological processes,in particular cancer-relevant processes such as proliferation, cell cy-cle control, apoptosis, differentiation, migration and metabolism. AmiRNA can have either oncogenic or tumor suppressive function. Ex-amples of tumor suppressor miRNAs are let-7 family, miR-29, miR-34and miR -15, and of oncogene miRNAs are miR-17-92, miR-155 andmiR-221.

The biogenesis of miRNAs can be summarized as follows: a pri-mary transcript (pri-miRNA) is first generated by RNA polymeraseII as separate transcriptional units or embedded within the introns ofprotein coding genes. Then the primary transcript is processed by themicroprocessor complex containing the RNase III enzyme Drosha toan approximate 70-nucleotide (nt) pre-miRNA hairpin (the precursor-miRNA) [15, 16, 17]. Pre-miRNAs are subsequently exported to thecytoplasm by exportin 5 (XPO5) [18, 19] where their terminal loopsare excised by the RNase III Dicer to give rise to a double-stranded 22

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nt stem composed of 5’ and 3’ strands representing 5p and 3p respec-tively. While one of the two strands (the passenger strand miRNA*)is discarded, the other one (the guide) miRNA is then embeded intothe RISC (RNA Induced Silencing Complex) to complementary targetmRNA for post-transcriptional gene silencing [20].

Since it was believed that according to the thermodynamic stabilityof the pre-miRNA cells preferentially select the less stable one of thetwo strands (the guide) and destroy the other one (the miRNA*),early works miRNAs have focused on the guide strand (which wasusually considered as the 5p one because it was found to be moreabundant than its counterpart miRNA* in humans [21]). However,even though miRNA* are less abundant they are often present andremain functional because they conserve their seed sequences and havebeen isolated from RISC [22, 23] . It has been shown by profilinganalyses that both strands could be co-accumulated in some tissueswhile being subjected to strand selection in others [24, 25, 26]. Theinterplay between the 5p and 3p strands from the same precursorhas been shown either in arm-switching where the dominant miRNAis switched from one arm of the precursor to the other [27] or bytargeting overlapping sets of genes when the abundances of the twostrands are similar [28]. Functional 5p and 3p have been characterizedfor many miRNAs. Examples include miR-9 [29], miR-17 [30], miR-19[23], miR-28 [31], miR-30c [32], miR-125a [33], miR-142 [34], miR-155[35], miR-199 [36, 37], miR-223 [38], miR-342 [39], miR-2015 [40],miR-18a [41], and miR-582 [42]. In some cases the two arms functionin opposing ways (e.g. miR-28 [31] and miR-125 [33]), while in othersthey function in joint fashion (e.g. miR-199 [37] and miR-155 [35]).

miRNAs have been shown to be important components in the p53network Their interactions with p53 have been demonstrated throughthe identification of several miRNAs as direct target genes of p53.By inducing the expression of specific miRNAs that have a tumorsuppressive function a novel mechanism for tumor suppression for p53has been then revealed. In particular the role of the miR-34 familyhas been reported in several studies [43].

In mammalians miR-34 family consists of miR-34a, miR-34b andmiR-34c, that are encoded by two different genes. miR-34a is en-coded by an individual transcript in chromosome 1 and expressed in amajority of tissues, while miR-34b and miR-34c share a common pri-mary transcript in chromosome 11 and are mainly expressed in lungtissues. Several studies have reported that the members of miR-34

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family were direct target genes of p53 and their upregulation inducesapoptosis and cell-cycle arrest. [44, 45, 46, 47, 48, 49, 50]. Indeed,Ectopic expression of miR-34 induces cell cycle arrest in both primaryand tumour-derived cell lines [50]. Inactivation of miR-34a stronglyattenuates p53-mediated apoptosis in cells exposed to genotoxic stress[46]. mir-34b/mir-34c were also down-regulated in p53-null humanovarian carcinoma cells and both cooperate in suppressing prolifera-tion of neoplastic epithelial ovarian cells [45]. Since cell-cycle arrestand apoptosis are the responses of p53 to the stress signals, these factsimply that miR-34 mediate the tumor suppressive function of p53. Onthe other hand the members of the miR-34 family can have decreasedexpression in cancer because of the inactivating mutations of p53 orthe expression of viral inhibitors of p53, but also as a consequence oftheir own mutational or epigenetic inactivation.

Since 30% of all genes and the majrity of the genetic pathways areregulated by miRNAs [51, 52, 53], we can expect some miRNAs to reg-ulate p53 and its pathway. This hypothesis has been verified and somemiRNAs have been identified as regulators of p53. miR-504 can nega-tively regulate p53 expression through its binding to two binding sitesin human p53 3-UTR [54]. It has also been reported that miR-125bis another miRNA targeting p53 [55]. Another regulator is miR-29that was identified as a positive regulator of p53 that up-regulate p53protein levels and induce p53-mediated apoptosis through repressionof p85 [56]. Furthermore, miR-34a, which is a transcription target ofthe p53 protein, was also found to positively regulate p53 activity andfunction in apoptosis through its direct negative regulation of SIRT1[57]. SIRT1 is a negative regulator of p53, which physically interactswith p53 and deacetylates Lys382 of p53 [58].

The purpose of this paper is the study of the role of both miR-34a-5p and miR-34a-3p in the signaling pathway of the p53.

2 Materiels and methods

We begin by using DIANA-miRPath v3. 0 which is a miRNA pathwayanalysis web-server, providing accurate statistics utilizing predicted orexperimentally validated miRNA interactions derived from DIANA-TarBase [59]. We first perform a KEGG reverse search for miRNAsin the p53 signaling pathway (—hsa04115) by choosing the TarBase v7.0 for method. In the same web-server we obtain the target genes of

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the different miRNAs that we found. The distribution of the targetgenes in the pathway can also be displayed.

Then we use the cytoscape 3.4.0 software to visualize the interac-tions where the relevant genes are involved. We take the intAct 1 .to be our source of interactions. We filter by taxonomy identifier torestrict the obtained network to the human case. In order to formclusters of genes that share a similar function we use the clusterMakera Cytoscape application with the GLay Community Clustering algo-rithm. the BiNGO a Cytoscape Plugin [60] gives the Gene Ontology(GO) terms that are significantly overrepresented in each cluster.

3 Results and discussion

The result of the KEGG reverse search was a list containing 660 differ-ent miRNAs including hsa-miR-34a-5p (p-value = 1.840530e-100) andhsa-miR-34a-3p (p-value = 4.325757e-5). The respective p-values canbe interpreted as expressing the fact that 5p is more abundant than3p. The list of target genes contains 30 genes for miR-34a-5p andtwo for miR-34a-3p (MDM2 and THBS1) that are also target genes ofthe miR-34a-5p. The involvement of these genes in the p53 signalingpathway is shown in figure 1.

THBS1

By loading the list of the 30 target genes of miR-34a-5p (containingalso the two targets of miR-34a-3p) in Cytoscape 3.4.0 and searchingthe interaction in IntAct we obtain a network composed of 2492 nodesand 6493 edges. After clustering this network by the clusterMakerapplication we obtain the result shown in figure 2 where the clustercontaining THBS1 was highlighted, This cluster is shown in figure 3.

In order to find the pathways in which the THBS1 gene is involvedwe use the BINGO application of Cytoscape, and the relevant GOterms that are overexpressed in the corresponding cluster are given intable 1

1IntAct is one of the largest available repositories for curated molecular interactionsdata, storing PPIs as well as interactions involving other molecules. It is hosted by theEuropean Bioinformatics Institute. IntAct has evolved into a multisource curation plat-form and many other databases curate into IntAct and make their data available throughit [23].

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Figure 1: The signaling pathway of p53 with the target genes of miR-34-5p/3p highlighted

Figure 2: The result of the clustering of the initial network representingdifferent interactions of the target genes of miR-34a-5p/3p

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Figure 3: The cluster representing THBS1 and its interactions

GO terms overexpressed p-valueRegulation of response to external stimulus 2.34 × 10−6

Regulation of response to stress 3.09 × 10−6

Regulation of cell adhesion 6.29 × 10−4

Positive regulation of apoptosis 5.48 × 10−3

Negative regulation of cell-matrix adhesion 3.07 × 10−3

Induction of apoptosis 1.42 × 10−3

Negative regulation of angiogenesis 6.48 × 10−3

Table 1: GO term enrichment for the THBS1

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p53

miR-34a

MDM2

Figure 4: Type 1 incoherent FFL composed of p53, miR-34a and MDM2

According to this analysis we can deduce that the THBS1 gene isinvolved in regulating tumor suppression processes. But it is also clearthat it contributes to some anti metastatic pathways like angiogenesisand cell adhesion.

MDM2

As was mentioned in the introduction MDM2 and p53 are linked bya negative feedback loop. However, since MDM2 is a target gene ofmiR-34a which means that it is negatively regulated by this miRNA,we can represent the interaction between the three molecules (p53,MDM2 and miR-34a) as a Feed Forward Loop (FFL). According tothe signs of the three interactions (activation/repression) the presentloop is a type 1 incoherent FFL (figure 4).

The dynamics of this FFL can be described as follows: when p53begins to accumulate as a response to stress signals MDM2 first risessince it is positively regulated by this gene. miR-34a is one of thedirect target genes of p53, thus, at some level of p53 the expressionof miR-34a begins also to increase, when it reaches some cellular levelMDM2 begins to decrease. It has been demonstrated that the responsetime of the type 1 incoherent FFL is smaller than the one of a singleregulation system [61].

Thus in cases where speedy responses are needed this type of reg-ulatory loop is more advantageous than the simple one. In our caseMDM2 is the principal cell antagonist of p53 that limits its anti growthfunction in normal cells. Indeed, in unstressed cells p53 is present atvery low levels due to the continuous degradation by ubiquitination

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which is mediated by MDM2. In stressed cells, in order for p53 to ac-cumulate in response to stress signals it has to escape the degradationby MDM2. This is achieved by some protein kinases that phospho-rylate its amino-terminal sites which are close to the MDM2 bindingregions of the protein, inhibiting the interaction between the two pro-teins. on the other hand MDM2 itself is the product of p53-induciblegene. Thus the two genes are linked by an autoregulatory negativefeedback loop ([62]and references therein). Thus the FFL constitutean other mechanism that accelerates the stabilization of p53 in re-sponse to external stimilus.

Acknowledgment

I wish to thank the Abdus Salam ictp for hospitality.

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