Biochimica et Biophysica Acta 1861 (2016) 1671–1680

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Genes and miRNA expression signatures in peripheral bloodmononuclear cells in healthy subjects and patients with metabolicsyndrome after acute intake of extra virgin olive oil

Simona D'Amore a,b,c,1, Michele Vacca d,2, Marica Cariello a, Giusi Graziano b, Andria D'Orazio d, Roberto Salvia a,Rosa Cinzia Sasso b, Carlo Sabbà a, Giuseppe Palasciano c, Antonio Moschetta a,⁎a Department of Interdisciplinary Medicine, Clinica Medica “C. Frugoni”, Aldo Moro University of Bari, Italyb National Cancer Research Center, IRCCS Oncologico Giovanni Paolo II, Bari, Italyc Department of Biomedical Sciences and Human Oncology, Clinica Medica “A. Murri”, Aldo Moro University of Bari, Italyd Laboratory of Lipid Metabolism and Cancer, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy

Abbreviations: ACADM, acyl-CoA dehydrogenase, C-4acetyl-CoA acetyltransferase 1; AGO2, argonaute RISC cathydrocarbon receptor nuclear translocator like; CCNK, cyctor output cycles kaput; CRY2, cryptochrome 2 photolyase-lCXCR4, chemokine CXC motif chemokine receptor; EVOO,discovery rate; HIF1A, hypoxia inducible factor 1, alpha70 kDa protein 1A; IKKα, nuclear factor-κB kinase sureceptor-associated kinase 3; MeD, Mediterranean diet; miRsyndrome; MUFAs, monounsaturated fatty acids; NASHPBMCs, peripheral bloodmononuclear cells; PRDX3, peroxisor phosphatase and tensin homolog; RXRβ, retinoid sensosterol-C5-desaturase; TAB2, TGF-β-activated kinase1/MATGF-β-activated kinase1/MAP3K7 binding proteins 3.⁎ Corresponding author at: Clinica Medica “Cesa

Interdisciplinary Medicine, University of Bari “Aldo M70124 Bari, Italy.

E-mail address: antonio.moschetta@uniba.it (A. Mosch1 S. D'Amore is currently a Clinical Research Fellow a

University of Cambridge, UK.2 M. Vacca is currently Senior Clinician Scientist at theM

Nutrition Research (MRC-HNR), and Research Fellow at thUniversity of Cambridge, UK.

http://dx.doi.org/10.1016/j.bbalip.2016.07.0031388-1981/© 2016 Published by Elsevier B.V.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 January 2016Received in revised form 15 June 2016Accepted 9 July 2016Available online 12 July 2016

Extra virgin olive oil (EVOO) consumption has been associated with reduced cardiovascular risk but molecularmechanisms underlying its beneficial effects are not fully understood. Here we aimed to identify genes andmiRNAs expression changes mediated by acute high- and low-polyphenols EVOO intake. Pre- and post-challenge gene and miRNAs expression analysis was performed on the peripheral blood mononuclear cells(PBMCs) of 12 healthy subjects and 12 patients with metabolic syndrome (MS) by using microarray and RT-qPCR. In healthy subjects, acute intake of EVOO rich in polyphenols was able to ameliorate glycaemia and insulinsensitivity, and to modulate the transcription of genes and miRNAs involved in metabolism, inflammation andcancer, switching PBMCs to a less deleterious inflammatory phenotype;weaker effects were observed in patientswith MS as well as in healthy subjects following low-polyphenol EVOO challenge. Concluding, our study showsthat acute high-polyphenols EVOO intake is able to modify the transcriptome of PBMCs through the modulationof different pathways associated with the pathophysiology of cardio-metabolic disease and cancer. These bene-ficial effects are maximized in healthy subjects, and by the use of EVOO cultivars rich in polyphenols.Nutrigenomic changes induced by EVOO thus legitimate thewell-known beneficial effects of EVOO in promotinghuman health and, potentially, preventing the onset of cardiovascular disease and cancer.

© 2016 Published by Elsevier B.V.

Keywords:Extra virgin olive oilGene expressionmiRNAsPBMCs

to C-12 straight chain; ACAT1,alytic component 2; BMAL, aryllin K; CLOCK, circadian locomo-ike; CVD, cardiovascular disease;extra virgin olive oil; FDR, falsesubunit; HSPA1A, heat shock

bunit α; IRAK3, interleukin-1NAs, microRNAs; MS, metabolic, non-alcoholic steatohepatitis;redoxin 3; PTEN, tumor suppres-r retinoid X receptor beta; SCD5,P3K7 binding proteins 2; TAB3,

re Frugoni”, Department oforo”, Piazza Giulio Cesare 11,

etta).t the Department of Medicine,

edical Research Council Humane Institute ofMetabolic Science,

1. Introduction

Adherence to Mediterranean diet (MeD) has been shown to signifi-cantly protect against cardiovascular and cancer risks [1]. One ofthe main components of the Mediterranean diet is extra virginolive oil (EVOO), which is also considered a functional food. EVOO con-tains high levels of monounsaturated fatty acids (MUFAs; oleic acid:70–80%), and a number of biologically active minor components (e.g.phenolic compounds) playing a role in health promotion [2–4]. Severalstudies have shown that olive oil intake is effective in lowering bloodpressure in hypertensive patients, reducing lipid and DNA oxidation,ameliorating lipid profile and insulin-resistance, inflammation, andendothelial dysfunction, thus leading to protection against cardiovascu-lar disease (CVD) [3–5]. EVOO has also shown a favorable effect ontumorigenesis and cancer progression due to its ability to modify cellmembrane composition, signal transduction pathways, transcriptionfactors, and oncogenes [6]. Few studies have been conducted in humansto elucidate the nutrigenomics responses of inflammatory cells to the

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acute intake of super-physiological doses of EVOO; these studies werefocused mainly on mRNA changes thereby the adaptive modulation ofthe overall transcriptome (genome and miRNome) in response toEVOO is lacking so far [7]. Moreover, it is still unresolved whether ben-eficial changes in modulation of genes involved in proliferative, anti-oxidant, and inflammatory pathways are mediated by oleic acid orexecuted by minor components.

MicroRNAs (miRNAs) are small 19–23 nucleotide RNA moleculesthat act as regulators of RNA degradation and/or blocking protein trans-lation of specific sets of target proteins [8], thus acting as master trans-duction regulators of genetic networks, cellular, and physiologicalprocesses. As it occurs for genes, miRNA regulation is tightlymodulated,and miRNA dysregulation has been documented in models of disease(e.g. metabolic syndrome,MS; atherosclerosis; cancer) [9]. Additionally,miRNA have been proposed to be secreted by certain cells (e.g. macro-phages and platelets) exerting regulatory effects on other cell types inan “endocrine” or “paracrine” fashion [10]. However, this is just thesunrise of a proper understanding of all the functions regulated bythese small RNA sequences as well as for their use as effective targetsfor treatment.

Here we performed a study aimed to characterize the effects ofEVOO on the miRNome and transcriptome of circulating inflammatorycells (peripheral blood mononuclear cells, PBMCs) that could underlinethe molecular mechanisms exerted by EVOO in the prevention of thecardio-metabolic risk. PBMCs have been recognized as carriers of sys-temic signals, and the adaptive changes of their transcriptome havebeen proposed either as biomarkers of awide range of pathological con-ditions (e.g. inflammatory disease, MS, and cancer) or tools to investi-gate response to medication, and nutritional changes [11,12].

We aimed to identify genes andmiRNAs expression changesmediat-ed by acute EVOO intake expression patterns in the PBMCs of healthysubjects, and patients with MS. Our results show that acute intake ofEVOO rich in polyphenols is able to ameliorate glycaemia and insulinsensitivity, and to promote the transcription of genes and miRNAsinvolved in the anti-inflammatory, and anti-cancer responses as wellas in the energy homeostasis. Interestingly, these effects are partiallylost in patients with MS as well as by the administration of low-polyphenols EVOO thus pointing to the importance of the phenolic frac-tion and to the health status of the organism that receives EVOO in orderto achieve the best transcriptional response of PBMCs.

2. Methods

2.1. Chemical profile of extra virgin olive oil

The two EVOOs selected for thepresent studywere both produced inApulia, and similar in fatty acid composition (Supplementary Table 1);differing mainly for the content in polyphenols (the “Coratina” cultivarhad higher content in polyphenols when compared to the “Peranzana”cultivar). We used the same stocks of EVOO to treat all the participantsincluded in each treatment arm.

2.2. Study population

Patient recruitment was performed in the Clinic of Nutrition (Head:Prof. Antonio Moschetta) of the Clinica Medica “Augusto Murri”, AldoMoro University of Bari, Italy (Director: Prof. Giuseppe Palasciano).Twelve healthy subjects (6M:6F; mean age 29± 2 yrs.), and 12 patientsat the first diagnosis of MS (6M:6F; mean age 35± 3 yrs.) were recruit-ed for this study (baseline characteristics are shown in SupplementaryTable 2). The diagnosis of MS was assessed in accordance to the AdultTreatment Panel III (presence of three or more criteria) [13]. The pres-ence of MS complications (CVD, cerebrovascular diseases) as well as ofother diseases were the exclusion criteria. None of the subjects was asmoker and/or underwent pharmacological therapy (except for anti-hypertensive drugs in MS patients). Subjects with a daily consumption

of alcohol over 25 g/day, and intake of anti-oxidant supplements duringthe last 2 months prior to study enrollment were excluded. In all sub-jects, background information was collected, and physical examinationwas performed. Cardiovascular risk was assessed using the scoring sys-tem of the Progetto Cuore [14]. The study protocol was approved by theEthical Committee of the Azienda Ospedaliero-Universitaria Policlinico diBari, Italy.

2.3. Study design, dietary control and virgin olive oil administration

Prior to both dietary interventions (ingestion of high-polyphenolsand low-polyphenols EVOO), patients followed a 1-weekwashout peri-od in which no olive oil consumption was allowed. During the first4 days subjects were asked to control anti-oxidant intake, while duringthe 3 days prior to the intervention they followed a strict low-phenoliccompound diet. Female volunteers were scheduled to start the inter-vention at the beginning of their follicular phases.

After 12 h fasting, subjects underwent EVOO intake [50mL (44 g)] at8 am in a single dose. All the subjects consumed the total amount ofEVOO administered. During the following 4 h, subjects abstained fromfood and drinks (with the exception of water that was allowed insmall quantities), and were instructed not to perform physical exercise(Supplementary Fig. 1).

2.4. Sample collection, biochemical measurements, and PBMC isolation

After overnight fasting and 4 h after EVOO administration, samplesfor serum biochemistry and whole blood (18 mL) for PBMC isolationwere collected. PBMCs were isolated immediately after collection ofblood using a standard, previously validated protocol [11]. Cells werethen stored at−80 °C until RNA extraction.

2.5. RNA sample preparation

RNA was extracted from the PBMCs or cell subpopulations pelletusing QIAzol® Lysis Reagent (Qiagen, Hilden, Germany), according tothe manufacturer's instructions. To avoid possible DNA contamination,the RNA was treated with DNAase-1 (Ambion, Foster City, CA). RNApurity (A260/A280 N 1.75), and the concentrationwas checked by spec-trophotometer, while the RNA integrity was assessed by Bio-RadExperion™ (Bio-Rad, Hercules, CA). Only samples with Relative QualityIndex (RQI) N8 were used for microarray analysis while samples withRQI N 6.5were used for quantitative RTqPCR. Samples were stored in al-iquot at−80 °C prior to use.

2.6. Microarray analysis of gene and miRNA expression profiles

Microarray gene and miRNAs expression analysis was conductedon RNA extracted from PBMCs. For GE expression: whole RNA (400 ng)was used for cRNA synthesis using the Illumina Total Prep RNA Amplifi-cation kit (Ambion, Austin, TX, US) following the manufacturer'sinstructions, and randomly hybridized to the array. Genome-wide ex-pression profile analysis of PBMCs was performed using the Illuminawhole genome direct hybridization assay (HumanHT-12 v.4 ExpressionBeadChip Kit). All the bead chips (GE) were read on the same micro-array platform at one time (Illumina iScan System, San Diego, CA,USA). For miRNA expression: whole RNA (400 ng) was studied usingthe Illumina Universal MicroRNA Expression Profiling (Human V2MicroRNA Expression profiling kit & Illumina Universal Bead Chips).All the bead chips (GE/miR)were read on the samemicroarray platform(Illumina iScan System, SanDiego, CA, USA). Upon themanufacturer in-structions, data were processed using the Illumina Genome StudioSoftware through specific algorithms of filtration and cleaning of thesignal. All the items with detection p value N0.001 were excluded.Data from PBMCs were normalized together with the quantile method[15]. Background was subtracted. Final output consisted of normalized

Table 1Changes in metabolic parameters in healthy subjects after high-polyphenols and low-polyphenols EVOO intake.

High-polyphenols EVOO Low-polyphenols EVOO

T0 T1 p T0 T1 p

N 12 12 – 12 12 –M:F 6/6 6/6 – 6/6 6/6 –Total cholesterol (mg/dL) 182.6 ± 31.6 186.8 ± 31.6 NS 180.7 ± 32.2 182.5 ± 35.3 NSHDL-c (mg/dL) 62.6 ± 12.6 64.8 ± 12.2 NS 58.3 ± 12.0 60.1 ± 14.9 NSLDL-c (mg/dL) 102.3 ± 27.9 104.4 ± 28.7 NS 103.0 ± 28.1 106.6 ± 31.4 NSTG (mg/dL) 58.6 ± 25.0 56.7 ± 18.2 NS 64.5 ± 37.2 73.5 ± 31.3 NSGlucose (mg/dL) 85.7 ± 5.2 78.7 ± 5.7 b0.01 84.3 ± 6.9 80.6 ± 5.3 NSInsulin (microUI/mL) 8.3 ± 2.3 6.3 ± 3.1 0.09 8.0 ± 2.5 7.7 ± 3.5 NSHOMA-IR 1.7 ± 0.4 1.2 ± 0.6 b0.05 1.7 ± 0.5 1.5 ± 0.7 NS

Data are presented as mean ± SD. Statistical significant values (p-value ≤ 0.05) are highlighted in bold. Abbreviations: high-density lipoprotein cholesterol, HDL-c; homeostatic modelassessment for insulin resistance, HOMA-IR; low-density lipoprotein cholesterol, LDL-c; non-significant, NS; triglyceride, TG; T0, baseline values; T1, 4 h after EVOO administration values.

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fluorescence intensity of each probe (AVG signal), representing the ex-pression levels of each gene. This initial screening allowed us to identify8708 genes to be exported from Genome Studio Software on which,after transformation log2-transformed data,we thus performed the sta-tistical analysis. The identification of up- or down-regulated genes byhigh-polyphenols EVOO ingestion was performed by comparing geneexpression in PBMCs at baseline and after 4 h of EVOO intake, in bothhealthy subjects and MS patients.

2.7. Reverse-transcription and quantitative RTqPCR

For genes: according to the manufacturer's instructions, 190 ng ofRNA were reverse-transcribed in a volume of 20 μL using the HighCapacity RNA-to-cDNA Kit (Applied Biosystem, Foster City, CA). Real-time quantification was done using previously-validated TaqMan®Assays (Applied Biosystems, Foster City, CA), according to manufac-turer instructions. Quantitative normalization of cDNA in eachsample was performed using glyceraldehyde-3-phosphate dehydroge-nase (GAPDH), and cyclophilin as internal controls. FormiRNAs: quanti-fication of miRNA was done using TaqMan miRNA Assays (AppliedBiosystems). Briefly, 10 ng of RNA were reverse-transcribed and real-time quantification was done using previously-validated TaqMan®Assays (Applied Biosystems, Foster City, CA), according to themanufac-turer instructions. Quantitative normalization was performed usingU6snRNA and RNU48 as internal controls.

For all experiments the PCR assayswere performed in 96well opticalreaction plates using the ABI 7500HT system (Applied Biosystems, Fos-ter City, CA). All reactions were run in triplicate. Relative quantificationwas performed using the ΔΔCT method.

2.8. Statistical analysis

The datasets underwent a restrictive screening for excluding thepresence of outliers, before starting the analyses. In order to minimize

Table 2Changes in metabolic parameters in MS subjects after high-polyphenols EVOO intake.

T0 T1 p

N 12 12 –M:F 6/6 6/6 –Total cholesterol (mg/dL) 183.2 ± 33.0 187.5 ± 34.4 NSHDL-c (mg/dL) 40.2 ± 10.3 39.8 ± 9.9 NSLDL-c (mg/dL) 124.8 ± 27.8 126.9 ± 27.9 NSTG (mg/dL) 144.4 ± 45.4 173.5 ± 57.1 NSGlucose (mg/dL) 90.1 ± 9.7 88.4 ± 5.3 NSInsulin (microUI/mL) 14.3 ± 10.5 11.9 ± 6.6 NSHOMA-IR 3.3 ± 2.5 2.6 ± 1.5 NS

Data are presented as mean ± SD. Abbreviations: high-density lipoprotein cholesterol,HDL-c; homeostatic model assessment for insulin resistance, HOMA-IR; low-density lipo-protein cholesterol, LDL-c; non-significant, NS; triglyceride, TG; T0, baseline values; T1, 4 hafter EVOO administration values.

the effect of variation caused by non-biological factors, all the genetic(GE) values were normalized directly in Illumina Genome Studio Soft-ware with the quantile method, and then were analyzed with classicalstatistical approaches to evaluate differences among groups and corre-lations between clinical and prognostic variables and levels of expres-sion of genes. In particular, for continuous variables the differencebetween paired samples was assessed with Wilcoxon signed-rank teston the Log2 normalized data. Comparison of the effects of EVOOamong the two study populations (CTRL vs. MS) was performed byusing Mann-Whitney test on fold changes. The raw p-values werethen adjusted by the Benjamini-Hochberg procedure to control thefalse discovery rate (FDR), the expected proportion of incorrectlyrejected null hypotheses (“false discoveries”) [16,17]. Data are shownas means ± SD (Supplementary Table 3A). All the analyses were per-formed using SAS (Release 9.2, 2008) and R (version 2.12.2). Thesedatasets are publically available on GEO (Ref: GSE75027; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE75027).

2.9. Bio-informatics functional analyses

After statistical analysis, differentially expressed genes withingroupswere studied using the Core Analysis Function of Ingenuity Path-way Analysis (Ingenuity System Inc., USA). IPA (Ingenuity System, USA,www.ingenuity.com) was used to determine the functional annotationand the significant Biological Functions related to the differentiallyexpressed genes, and to compare the pathway enrichment amonggroups [18]. Networks showing relationships and interactions betweendifferentially expressed genes and others that functionally interact withthem, were generated and ranked in terms of significance of genesparticipating to the pathway (p b 0.01). Only genes significant atMann-Whitney (or FDR), and gene-class testing (pathway analysis)were considered biologically relevant [17], and selected for furtherqPCR confirmations. To integrate mRNA and miRNA expression data,we analyzed putative miRNA:mRNA pairs using another feature of IPA(that interrogates Target Scan Human database) regarding their foldchange, where the expression of the miRNA is the opposite of itsmRNA target (i.e. mRNA down-regulated vs. miRNA up-regulated orgenes up-regulated vs. miRNA down-regulated; see SupplementaryTable 4).

3. Results

3.1. Metabolic effects of EVOO in healthy subjects and patients with MS

We recruited 12 healthy controls who underwent oral adminis-tration of a single dose of 50-mL of high-polyphenols and low-polyphenols EVOO preceded by a 7-days washout period. The laborato-ry findings before and after high- and low-polyphenols EVOOs intakeare summarized in Table 1. Four hours after high-polyphenols EVOOadministration, we found a decrease in serum glucose, insulin and

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=SE75027http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=SE75027http://www.ingenuity.com

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HOMA-IR (Table 1). No significant differences were observed in theother assessed metabolic variables (serum total cholesterol, LDL-c,HDL-c, and TGs). On the other hand, low-phenols EVOO did notinduce significant changes in serum biochemistry in the same sub-jects (Table 1). In order to check if the metabolic effects of high-polyphenols EVOOaremaintained also inmetabolic disease, we recruit-ed 12 patients with MS undergoing the same dietary intervention. Theeffects on glucose metabolism induced by high-polyphenols EVOOwere not confirmed in patients with MS (Table 2).

3.2. Gene expression profiling in PBMCs after high-polyphenols EVOO intakeand validation by RTqPCR

In order to study the effects induced by high-polyphenols EVOO onPBMCs at transcriptional level, we performed gene expression microar-rays in both healthy controls and patients withMS. At the paired analy-sis (T0, baseline values, vs. T1, 4 h after EVOO administration), high-polyphenols EVOO induced the modulation of 2438 annotated genes(1376 up-regulated and 1062 down-regulated - out of total 2447 differ-entially modulated; p b 0.05) in the PBMCs of healthy subjects, and of954 annotated genes (403 up-regulated and 551 down-regulated - outof total 963 differentially modulated; p b 0.05) in the PBMCs of patientswith MS. Of these hits, 389 annotated genes (195 over-expressed and194 under-expressed) were consistently changed in both groups(CTRL and MS). The complete list of the genes differentially modulatedeither in the CTRL group or in theMS group is shown in SupplementaryTable 3A; these results are summarized in Figs. 1A and 2A.

In order to provide a framework for interpretation of our results, wefunctionally clustered significant (p b 0.01) biological pathways usingthe Core Function of the IPA Software [19] (Figs. 1B and 2B, and detailsin Supplementary Table 3B and C for pathways up-regulated and down-regulated, respectively). High-polyphenols EVOO intake induced dra-matic changes in the mRNA abundance of a number of genes involvedin the modulation of PBMCs functions. The pathway enrichment (Figs.1B and 2B) highlights the modulation of the immune responses (e.g.up-regulation of CD28 Signaling in T Helper Cells, and Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes; suppressionof the NFAT in the regulation of the immune response and of PI3K Sig-naling in B Lymphocytes), and of different inflammatory signals (sup-pression of both B Cell and T Cell Receptors, NF-κB, IL1/3/8, RANK andthrombin signaling cascades as well as of the LPS-stimulated MAPK ac-tivation, and of the NRF2-mediated Oxidative Stress Responses), a neg-ative modulation of cell proliferation and cancer (up-regulation of theDNA Damage Response systems, suppression of different pathways in-volved in cell and cancer proliferation such as ERK/MAPK, CXCR4,HGF/EGF, HIF1α signaling cascades). Also pathways involved in metab-olism and cardio-metabolic risk appear to be modulated by high-polyphenols EVOO intake, as shown by predicted suppression of ProteinKinase A, PI3K/AKT signaling, CREB, mTOR, cholesterol biosynthesis, aswell as of mechanisms involved in Cardiac Hypertrophy, Adipogenesis,and Circadian Rhythms; on the other hand glucose metabolism (bothgluconeogenesis and glycolysis as well as the biosynthesis of Acetyl-CoA by the Pyruvate Dehydrogenase Complex), and pathways involvedin the metabolism of different amino acids (Aspartate, Cysteine, Isoleu-cine, Leucine, Methionine, Valine, Tryptophan) appeared to be up-regulated. Also a set of transcripts coding for proteins participating tothe transcriptional machinery of different nuclear receptors appearedto bemodulated by high-polyphenols EVOO (e.g. both androgen and es-trogen receptors, glucocorticoid receptor, and retinoid acid receptors).Intriguingly, the fatty acid sensor peroxisome proliferator-activated

Fig. 1. Pathway enrichment analysis of genes differentiallymodulated by high-polyphenols EVOhigh-polyphenols intake (T0, baseline values vs. T1, 4 h after EVOO intake), 1181were up-regucommon pattern of expression in both groups. (B) Pathways significantly enriched (p b 0.01) inbox represents the number of genes of the associated pathway after the nutritional interventionresults are provided in Supplementary Table 3B.

receptor alpha (PPARα), and its coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α or PPARGC1A),although not modulated at mRNA level, are predicted as activated bothby the pathway analysis and by IPA “upstreamregulator”prediction tool(that predicts the status of activation of a specificmodulator on the basisof the differential modulation of its known targets; Supplementary Fig.2); this is also paralleled by an up-regulation of the transcripts encodingthe retinoid X receptors (RXRα and RXRβ, see Supplementary Table 3A)forming heterodimers with PPARs. Interestingly, we previously de-scribed that PPARs and RXRα are suppressed in the PBMCs of patientswithMS [11]; this could explain, at least in part, the lack of reactivenessto EVOO challenge in the PBMCs of MS patients. In fact, PBMCs of pa-tients with MS showed a reduced number of genes significantly modu-lated by high-polyphenols EVOO (Figs. 1A and 2A) and reduced (vs.CTRL) changes in the genes being significantly regulated by EVOO (Sup-plementary Table 3A); as a consequence of these weaker changes, alsomost of the pathways differentially modulated in the CTRL group weremuch less enriched in the MS patients.

Overall, we depicted here the gene expression adaptive changesinduced by an acute oral administration of phenol-rich EVOO in PBMC.This modulation of the transcriptome is more evident in healthysubjects and parallels the beneficial effects in glucose metabolism ob-served in their biochemistry following acute EVOO intake.

3.3. RTqPCR confirmation of the gene expression changes induced by highpolyphenols EVOO in PBMCs of healthy subjects and patients with MS

In order to confirm by RTqPCR the most important hits modulatedby high-polyphenols EVOO, we considered “biologically relevant” onlythose genes “statistically significant” (p b 0.05, fold change N1.3) andenriched in “significantly modulated” (p b 0.01) pathways; we then se-lected hits for confirmation by RTqPCR on the basis of their biologicalrelevance (Fig. 3). We confirmed the up-regulation of: A) acyl-CoA de-hydrogenase, C-4 to C-12 straight chain (ACADM) transcript (acyl-CoAdehydrogenase which catalyzes the first reaction of fatty acid β-oxidation [20]); B) and of acetyl-CoA acetyltransferase 1 (ACAT1;involved in the glutaryl-CoA degradation pathway, and in the mainte-nance of lipid homoeostasis promoting cholesterol esterification) [21];C) retinoid X receptor beta (RXRβ; RXRs mediate the function of alarge number of other nuclear hormone receptors and are involved indifferent metabolic pathways, and exert anti-inflammatory, and anti-cancer actions [22]); D) heat shock 70 kDa protein 1A (HSPA1A playsan essential role in the energy production, folding/unfolding of proteins,cell-cycle control and signaling, and the protection of cells againststress/apoptosis [23]); E) peroxiredoxin 3 (PRDX3 plays an importantrole in the antioxidant defense system, and homeostasis within mito-chondria [24]). On the other hand, we confirmed the suppression of:F) sterol-C5-desaturase (SCD5 participates to cholesterol biosynthesispathway, and cell proliferation networks [25]); G) interleukin-1receptor-associated kinase 3 (IRAK3 is involved in the regulation of hu-moral immune response and in the NF-κB, and IL-8 signaling [26]); H)chemokine CXC motif chemokine receptor (CXCR4 is a chemokine re-ceptor implicated in the regulation of lymphocyte migration, autoim-munity, and cancer progression [27]); I) cyclin K (CCNK controls cellcycle, and division [28]); J) hypoxia inducible factor 1, alpha subunit(HIF1A is the master modulator of the cellular responses to hypoxia,but is also involved in inflammation, and cancer cell survival [29]); K)cryptochrome 2 photolyase-like (CRY2 acts as a negative feedbackloop component of the circadian clock [30]; L) argonaute RISC catalyticcomponent 2 (AGO2 is involved in miRNA biogenesis thus being a

O in the PBMCs of healthy subjects andMS patients. (A) Of the 1389 genes up-regulated bylated in healthy subjects, 208 were up-regulated inMS patients while 195 genes showed athe PBMCs of healthy subjects andMS patients after high-polyphenols EVOO intake. Eachthat are either up-regulated in healthy subjects (white), or inMSpatients (black). Detailed

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centralmodulator of RNApost-transcriptionalmodification, and proteinsynthesis [31]). As shown by microarray analysis, most of the changesinduced by high-polyphenols EVOO in healthy subjects were less pro-nounced or absent in the PBMCs of patients with MS.

In order to investigatewhether themodulation on genes involved inproliferative, anti-oxidant, and inflammatory pathways observed in thePBMCs of healthy subjects were mediated by the minor components(polyphenols) of EVOO or by its mayor components (mono- and poly-unsaturated fatty acids), we compared changes in the expression ofthe selected candidate genes after acute intake of phenol-rich EVOO tophenol-poor EVOO that did not show any evident metabolic benefit.Most of the genes previously validated by RTqPCR (and discussedabove) were not changed after low-polyphenol EVOO challenge (datanot shown); on the other hand, in linewith the results obtained follow-ing high-polyphenol EVOO, a significant increase of HSPA1A and RXRβ,and a decrease of CXCR4 were observed (Supplementary Fig. 3), thuspointing to this genes as potentially modulated by fatty acids ratherthan other components of EVOO.

Overall, we confirmed here that EVOO is able to induce changes inthe PBMC transcriptome in key genes for lipid metabolism, inflamma-tion, proliferation, and cancer. The effects are maximized in healthysubjects (when compared to patients with MS), and by the use ofEVOO cultivars rich in polyphenols.

3.4. miRNAs expression profiling in PBMCs after EVOO intake and validationby RTqPCR

Since one of the genes modulated by EVOO is AGO2, which is in-volved inmiRNA processing, we also studiedmiRNA expression profilesusing microarrays (containing 1.146 oligonucleotide probes comple-mentary to mature forms of miRNAs and annotated on miRBase). Ofthe 14miRNA (6 up-regulated and 8 down-regulated) potentiallymod-ulated according to the miRNA arrays (Fig. 4A), only 8 miRNAs wereexpressed above the sensitivity limit of the TaqMan miRNA assays(RTqPCR, Fig. 4B–I). In agreement with AGO2 suppression, most of themiRNA confirmed were suppressed in our database. In details, we con-firmed the suppression of: 1) miR-146b-5p (over-expressed in humanatherosclerotic plaques [32], and up-regulated in human monocytes/macrophage stimulated with oxidized-LDL [33]); 2) the oncogenic[34]miR-19a-3p; 3)miR-181b-5p (involved in themodulation of inflam-mation and cancer [35]); 4) miR-107 (known to be suppressed by fattyacids, and associated to obesity, insulin resistance and fatty liver [36]);5) miR-769-5p; 6) andmiR-192-5p (that has been associated to the im-pairment in glucose metabolism, fatty liver, and acute myocardial in-farction [37,38]). Among up-regulated miRNAs, we were able toconfirm the over-expression of only two miRNAs, namely the anti-inflammatory [39] miR-23b-3p and the tumor-suppressor [40,41] miR-519b-3p (Fig. 4H and 4I). The other miRNAs had expression levelsbelow the sensitivity limit of the TaqMan probes. Intriguingly, aswe ob-served for gene expression, and in line with the lack of modulation ofAGO2, most of these miRNAs (with the exception of miR-19a-3p)were not significantly modulated in the PBMCs of patients with MS.

In conclusion, our results point to EVOO-induced changes in themiRNome which actually support the modulation of EVOO-associatedmodulation of AGO2, and further support the idea that acute high-polyphenols EVOO intake may exert transcriptional effects potentiallybeneficial on insulin resistance (i.e. miR-107), inflammation (i.e. miR-181b-5p, miR-23b-3p), and cancer (miR-19a-3p, miR-519b-3p). In-triguingly, most of these beneficial effects of EVOO were not confirmedin MS patients undergoing the same treatment. Different possible

Fig. 2. Pathway enrichment analysis of genes differentiallymodulated byhigh-polyphenols EVOby high-polyphenols intake (T0, baseline values vs. T1, 4 h after EVOO intake), 868 were downshowed a common pattern of expression in both groups. (B) Pathways significantly enriched (intake. Each box represents the number of genes of the associated pathway after the nutritipatients (black). Detailed results are provided in Supplementary Table 3C.

explanations could justify these findings, including the lack of a meta-bolic adaptation in insulin signaling following EVOO in this cohort orof responsiveness of these cells at gene level (e.g. the absence of modu-lation of AGO2 that is involved in miRNA processing); more research isneeded to further address these hypotheses.

4. Discussion

Nutritional interventions are considered efficient approaches toprevent the onset of a wide range of chronic diseases including type 2diabetes, obesity, and MS, due to their potential to ameliorate plasmaglucose and lipids, as well as blood pressure, thus reducing the risk ofCVD [1]. Considering the epidemic of obesity and MS, and the fact thatMeD has been proven to be beneficial for cardio-metabolic risk, effortsshould thus bemade to better understand the relative role of each com-ponent of MeD to these benefits. Several cross-sectional, epidemiologi-cal studies, randomized intervention trials on human or research inanimals have pointed to a candidate beneficial role of EVOO in metabo-lism, and inflammation [2–5].Moving from these evidences, we used anintegrative approach bridging metabolic characterization of healthysubjects and MS patients with the study of adaptive changes in thegenome and miRNome following acute EVOO intake to dissect the mo-lecularmechanisms underlying the beneficial effects of EVOO suggestedby previous epidemiological studies. The application of gene/miRNA ex-pression analysis in studying humanphysiology and disease is relativelynew and rapidly evolving. Different gene expression studies in PBMCsclearly show that these cells are suitable not only for providing themolecular signatures of disease but also to identify tools for predictingresponsiveness of patients to treatments [12]. Our results confirm thishypothesis. We were able to prove that a single dose of EVOO is ableto transiently ameliorate insulin sensitivity in healthy subjects. Thiswas paralleled by transcriptomic changes both atmRNA andmiRNA ex-pression level in the PBMCs; on the other hand, EVOOdid not showben-efits in the biochemistry of patients with MS, and this was paralleledalso by weaker transcriptomic changes. Part of these effects may be at-tributed to the “healthy” fatty acids (MUFAs/PUFAs) abundant in EVOO,and part can be due to the minor contents of EVOO.

It is not surprising that different pathways involved in lipid and glu-cosemetabolism and cardio-metabolic risk aremodulated by EVOO.Wefound of particular interest the modulation of the transcriptional ma-chinery of different nuclear receptors (i.e. glucocorticoid, androgenand estrogen receptors; retinoid acid receptors; PPARα); these tran-scription factors act as master transcriptional regulators of differentcell functions (e.g. metabolism, inflammation, cell proliferation, differ-entiation and apoptosis, circadian rhythm, etc.). We found particularlyintriguing the predicted activation of the fatty acid sensor PPARα, andof its coactivator PGC-1α; this is paralleled by the up-regulation ofRXRs transcripts (both α and β) that form obligate heterodimers withPPARs. The fatty acids contained in EVOO are known to activate PPARs[42], and different polyphenols have been shown to be able to activatethe AMPK/SIRT1/PGC1 cascade [43] thus the activation of PPARα/PGC1α is consistent with EVOO challenge. PPARα/RXR heterodimersexert different beneficial effects in the cells, promoting the transcriptionof genes involved in fatty acid oxidation & mitochondrial function (inour dataset we observe the up-regulation of ACAT1, and ACADM), glu-coneogenesis and glycolysis (both promoted in our dataset), and sup-pressing inflammation [44,45]. Interestingly, we previously describeda suppression of both PPARα and RXRα in patients with MS [11] andthis could explain, at least in part, the lack of transcriptional responsive-ness to EVOO of the PBMCs isolated from MS patients.

O in the PBMCs of healthy subjects andMSpatients. (A)Of the 1225genes down-regulated-regulated in healthy subjects, 357 were down-regulated in MS patients while 194 genesp b 0.01) in the PBMCs of healthy subjects and MS patients after high-polyphenols EVOOonal intervention that are either down-regulated in healthy subjects (white), or in MS

Fig. 3. RT-qPCR validation of genes differentially modulated by high-polyphenols EVOO in the PBMCs of healthy subjects and patients with MS. Relative mRNA expression of genesmodulated by high-polyphenols EVOO intake (T0, baseline values vs. T1, 4 h after EVOO intake): up-regulated in both groups (A, B) ACADM and ACAT1; up-regulated in healthysubjects and unchanged in MS patients (C, D, E) RXRβ, HSPA1A, and PRDX3; down-regulated in both groups (H and J) CXCR4 and HIF1A; down-regulated in healthy subjects andunchanged in MS patients (F, G, I, K, L) SCD5, IRAK3, CCNK, CRY2, and AGO2. The data were normalized on the geometrical mean ("best-keeper" gene) of GAPDH and CYCLOPHILINmRNA levels, presented as relative expression values, and plotted as means ± SD (* = p ≤ 0.05). Statistical significance (p-value ≤0.05) was assessed by Wilcox Signed Rank test.

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The most important transcriptional changes induced by high-polyphenols EVOO were documented in the activation status of path-ways involved in PBMCs functions, including themodulation of immuneresponses and the suppression of pro-inflammatory signals. The anti-inflammatory effects of EVOO are well documented [4,46], and our re-sults support these evidences due to the modulation of a number ofpathways and genes involved in the inflammatory response. Indeed,we observed the up-regulation of Fcγ receptor-mediated phagocytosisin macrophages and monocytes pathway (which is essential for the im-mune response), and the suppression of several genes involved in theregulation of inflammatory response such as IRAK3, which is involvedin the regulation of humoral immune response and in NF-κB and IL-8 sig-naling pathways (it also plays an important role in tumorigenesis throughthe modulation of the pro-inflammatory microenvironment promotingcancer [26]), and of CXCR4, a chemokine receptor implicated in the regu-lation of lymphocyte migration, autoimmunity, and cancer [27].

EVOO consumption has been also proposed to be protective for can-cer [4]; in linewith this, we observed an EVOO-induced amodulation ofgenes involved in cell proliferation and survival, as well as in differentkey processes of tumorigenesis: up-regulation of the DNA DamageResponse systems (protective for cancer), suppression of different path-ways involved in cell proliferation (e.g. ERK/MAPK, CXCR4, HGF/EGF,HIF1α signaling cascades), the induction of HSPA1A, which plays anessential role in the energy production, folding/unfolding of proteins,

cell-cycle control and signaling, and the protection of cells againststress/apoptosis [23]. Also p53 pathway (essential for cell growth regu-lation and apoptosis) appears to be modulated by EVOO; this modula-tion featured the suppression of CCNK mRNA (that controls cell cycleand division) [28], and of hypoxia inducible factor 1, alpha subunit(HIF1A) which is involved in various cellular functions, including hyp-oxia, inflammation, and cancer cell survival [29]. Although still present,the previously-mentioned effects of EVOOwere partially lost in patientswith MS suggesting an impairment of the adaptive response in thePBMCs of these subjects. This goes in parallel with the lack of metabolic(glucose metabolism) benefits of EVOO in the same cohort of MSpatients, and needs to be further studied to understand the underlyingmolecular mechanisms.

Of note, EVOO also down-regulated AGO2, a key player in miRNAprocessing (pre-miRNA cleavage and mature miRNA release) [31] thuspointing to miRNA as possible indirect targets modulated by EVOO.The second part of our work was thus studying the adaptive changesof the miRNome of PBMCs following acute EVOO intake. miRNAs haveemerged as key regulators of physiological processes, including inflam-mation, proliferation, and glucose/lipid metabolism [9], and theirdysregulation has been shown to contribute to multiple disease, thusmaking them attractive therapeutic targets. We have proven that acuteEVOO intake induces different effects also at the level of themiRNome, in-cluding the promotion of miRNAs exerting anti-inflammatory (i.e. the

Fig. 4. miRNA profiling in the PBMCs of healthy subjects and patients with MS after high-polyphenols EVOO intake. (A) Changes in miRNA expression patterns in the PBMCs of healthysubjects after high-polyphenols EVOO intake are shown as lateral bars representing fold changes among groups (detailed results are provided in Supplementary Table 4A and B). RelativemRNA expression levels ofmiRNAsmodulated by high-polyphenols EVOO (T0, baseline values vs. T1, 4 h after EVOO intake): up-regulated in both groups (C)miR-19a-3p; up-regulated inhealthy subjects and unaffected inMS patients (H and I) miR-23b-3p andmiR-519b-3p; down-regulated (B, D, E, F, G), miR-146p-5p, miR-181b-5p, miR-107, miR-769-5p, andmiR-192-5p, in PBMCs of healthy subjects after EVOO intake. The data were normalized on the geometrical mean ("best-keeper" gene) of U6snRNA and RNU48 levels, presented as relativeexpression values, and plotted as means ± SD (* = p ≤ 0.05). Statistical significance (p-value ≤0.05) was assessed by Wilcox Signed Rank test.

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miR-23b-3p [39]), and tumor-suppressor (i.e. miR-519b-3p [40,41])properties, and the suppression of miRNA promoting inflammation (i.e.miR-181b-5p), cancer (miR-19a-3p) and insulin resistance (i.e. miR-107). Interestingly, miR-107 has been already shown to the modulatedby lipids in vitro (human enterocytes) by linoleic acid [36], and its over-expression has been associated to insulin resistance (miR-107 controlsthe post-transcriptional regulation of calveolin-1 [47], a key mediator ofinsulin pathway [47]) in obesity, metabolic disorders, and fatty liver;themodulation ofmiR-107 is thus of potential biologic interest in this co-hort, since insulin sensitivity appears to be transiently ameliorated inhealthy subjects following EVOO intake and, on the contrary, no changesin miR-107 are observed in MS subjects that have also no changes inmarkers of insulin sensitivity. Nevertheless, the paralogues miR-107/miR-103 have been also involved in the regulation of innate immunity,inflammatory responses, and circadian clock, but the biological meaningof their function is yet to be properly understood [36,48–51]. Interesting-ly, in parallel of the lack of responsiveness of glucose metabolism andtranscriptome (including AGO2), also miRNA showed a reduced respon-siveness to EVOO in PBMCs extracted from patients with MS.

Overall, our message at a glance is that the intake of EVOO contain-ing high phenols improves insulin sensitivity and modulates differentpathways in inflammatory cells of healthy subjects; most of thesechanges point to a beneficial role of EVOO in promoting health towardits anti-inflammatory, anti-cancer, and anti-oxidant properties. Thiscould be of particular relevance since circulating PBMCs are involvedin immune functions and also participate in the pathophysiologicalevents promoting atherosclerosis, and cancer. Our study thus providesnew insights to decode the molecular mechanisms legitimating thebeneficial effects of high phenols EVOO in promoting human health,and preventing onset of different disease.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbalip.2016.07.003.

Sources of funding

The work was funded by Apulian Region - Italy (POR StrategicProjects, CIP PS_101 to G.P.), Programma Operativo Nazionale(PON01_01958 PIVOLIO to A.M.) and Italian Association for Cancer Re-search (AIRC, Milan, Italy, IG 10410 to A.M.). M. Vacca is supported bythe Fondazione Umberto Veronesi (fellowship), and by theMedical Re-search Council (MRC-HNR; MC_PC_13030).

Conflict of interest

The authors declare that they have no conflict of interest.

Transparency document

The Transparency document associated with this article can befound, in online version.

Acknowledgments

We thank physicians and nurses of the Clinica Medica “AugustoMurri”, Aldo Moro University of Bari (Italy), Dr. A. Morgano andmembers of the laboratory at the Consorzio Mario Negri Sud, SantaMaria Imbaro, Chieti (Italy), and Prof. Villani of the Department ofBiochemistry University of Bari for their help and support duringthe study. We are also grateful to Dr. A. Sgaramella for EVOO chemi-cal analyses and to A. Cassanelli for supplying EVOO for this study.This study is dedicated to Franco Catapano*, Confederazione ItalianaAgricoltori (CIA).

doi:10.1016/j.bbalip.2016.07.003doi:10.1016/j.bbalip.2016.07.003http://doi.dx.org/10.1016/j.bbalip.2016.07.003

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Genes and miRNA expression signatures in peripheral blood mononuclear cells in healthy subjects and patients with metabolic...1. Introduction2. Methods2.1. Chemical profile of extra virgin olive oil2.2. Study population2.3. Study design, dietary control and virgin olive oil administration2.4. Sample collection, biochemical measurements, and PBMC isolation2.5. RNA sample preparation2.6. Microarray analysis of gene and miRNA expression profiles2.7. Reverse-transcription and quantitative RTqPCR2.8. Statistical analysis2.9. Bio-informatics functional analyses

3. Results3.1. Metabolic effects of EVOO in healthy subjects and patients with MS3.2. Gene expression profiling in PBMCs after high-polyphenols EVOO intake and validation by RTqPCR3.3. RTqPCR confirmation of the gene expression changes induced by high polyphenols EVOO in PBMCs of healthy subjects and p...3.4. miRNAs expression profiling in PBMCs after EVOO intake and validation by RTqPCR

4. DiscussionSources of fundingConflict of interestTransparency documentAcknowledgmentsReferences