Research Article Integration Analysis of MicroRNA and mRNA...

Research ArticleIntegration Analysis of MicroRNA andmRNA Expression Profiles in Human Peripheral BloodLymphocytes Cultured in Modeled Microgravity

C Girardi1 C De Pittagrave1 S Casara1 E Calura1 C Romualdi1 L Celotti12 and M Mognato1

1 Dipartimento di Biologia Universita degli Studi di Padova Via U Bassi 58B 35131 Padova Italy2 Laboratori Nazionali di Legnaro INFN Viale dellrsquoUniversita 2 Legnaro 35020 Padova Italy

Correspondence should be addressed to L Celotti luciacelottiunipdit and M Mognato maddalenamognatounipdit

Received 16 April 2014 Revised 22 May 2014 Accepted 22 May 2014 Published 23 June 2014

Academic Editor Mariano Bizzarri

Copyright copy 2014 C Girardi et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravitycondition simulated by a ground-based rotating wall vessel (RWV) bioreactor Our results show that 42miRNAswere differentiallyexpressed in MMG-incubated PBLs compared with 1 g incubated ones Among these miR-9-5p miR-9-3p miR-155-5p miR-150-3p and miR-378-3p were the most dysregulated To improve the detection of functional miRNA-mRNA pairs we performedgene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expressiondata The functional classification of miRNA-correlated genes evidenced significant enrichment in the biological processes ofimmuneinflammatory response signal transduction regulation of response to stress regulation of programmed cell death andregulation of cell proliferation We identified the correlation of miR-9-3p miR-155-5p miR-150-3p and miR-378-3p expressionwith that of genes involved in immuneinflammatory response (eg IFNG and IL17F) apoptosis (eg PDCD4 and PTEN) andcell proliferation (eg NKX3-1 and GADD45A) Experimental assays of cell viability and apoptosis induction validated the resultsobtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases thefrequency of apoptosis and decreases cell proliferation

1 Introduction

Exposure to spaceflight environment is known to cause inhumansmany adverse physiological changes including skele-tal muscle atrophy [1ndash3] cardiovascular and microvasculardisorders [4ndash6] bone deterioration [7 8] and impairedimmune system function [9 10] Immune systemdysfunctiondue to exposure to microgravity has been documented aswell in terms of reduced activationproliferation alteredcytokine production and altered signal transduction [11 12]Alterations in global gene expression patterns have beenalso observed in space-flown human cells involving mainlygenes of immune system activation [13 14] cytoskeleton [15]and cell cycle [16 17] However due to the difficulty andlimitations of performing experiments in the real micro-gravity of space many investigations have been conductedunder simulated microgravity conditions in which cells are

cultured in ground-based machines such as clinostats androtating wall vessel bioreactors that generate a residual 10minus3ndash10minus6 g force that approximates microgravity [5 18ndash24] Theresults indicate that similar to space microgravity simulatedmicrogravity affects both cell structure and function as wellas gene expression inmammalian cells [14 19 25] in bacteria[26 27] or in other living organisms [28ndash30]

Since molecular changes at the gene level may compro-mise cell function it is important to understand the cellularresponse to reduced gravity at the molecular level For thispurpose a class of noncoding RNAs called microRNAs(miRNAs) plays a key role miRNAs are a large family ofsmall RNAs of 18ndash24 nucleotides that are involved in post-transcriptional regulation of gene expression by interactingwith 31015840-untranslated regions (UTR) of target genes The reg-ulatory process is complex and occurs posttranscriptionallythrough miRNA interaction with a target site in the mRNA

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 296747 16 pageshttpdxdoiorg1011552014296747

2 BioMed Research International

that has partial or complete complementarity to the miRNAThe binding of miRNAs to complementary sequence of theirtarget mRNAs may repress translation or induce degradationofmRNAs [31] Recently the destabilization of targetmRNAsinstead of translational repression has been shown to be thepredominant mechanism for reduced protein output [32]Less often dsRNA formed by miRNA target complexes cantarget gene promoters and actually enhance transcription oftarget genes sometimes termed RNAa (RNA activation) [33]A single miRNA may have broad effects on gene expressionnetworks such as regulating cell lineage specificity cellularfunctions or stress response Besides a physiological roleof miRNAs in a variety of important biological processesincluding differentiation apoptosis [34] and fat metabolism[35] the miRNA-mediated gene regulation operates alsoduring viral infection [36] stress response pathway [37] andpathological processes such as tumorigenesis [38ndash42]

The present study is addressed to identify alterationsin miRNA profiles of human peripheral blood lympho-cytes (PBLs) incubated in modeled microgravity (MMG)with respect to those incubated in gravity 1 g To simulatemicrogravity we used a specialized bioreactor developed atthe NASA-Johnson Space Center (Houston TX USA) therotating wall vessel which represents a valid ground modelto simulate as far as possible a condition of reduced gravityTo identify miRNA-correlated genes whose expression levelwas significantly altered as a function ofMMGwe performedgene expression profiling on the same PBL samples assayedfor miRNA profiling and we integrated microRNAome andtranscriptome by using MAGIA2 [43] a web tool for theintegrative analysis of miRNA and genes expression dataincorporating transcriptional regulation A group ofmiRNA-mRNA pairs related to immunity cell proliferation andapoptosis was identified in PBLs incubated in MMG Thedifferences between MMG and 1 g on correlated miRNA-mRNA pairs involved in cell proliferation and apoptosiswere investigated by in vitro assays of clonogenic ability andapoptosis induction in PBLs incubated in MMGwith respectto those incubated in 1 g conditions

2 Materials and Methods

21 Lymphocytes Isolation and Microgravity SimulationHuman peripheral blood lymphocytes (PBLs) were obtainedfrom freshly collected ldquobuffy coatsrdquo from blood samples oftwelve healthy anonymous donors at the Blood Centre ofthe City Hospital of Padova (Italy) This study obtainedthe Ethics Approval from the Transfusion Medicine (TM)Ethics Committee of Blood Centre of the City Hospital ofPadova PBLs were isolated by separation on Biocoll densitygradient (BIOCHROM Berlin Germany) After isolationPBLs were preincubated overnight at a concentration of 3times 106mL in basal medium RPMI 1640 containing Gluta-MAX I (Invitrogen Life Technologies Carlsbad CA USA)124UmL penicillin 63120583gmL streptomycin sulfate and 10fetal bovine serum (FBS BIOCHROM Berlin Germany)After the overnight incubation PBLs consisting of peripheralmononuclear cells depleted of monocytes were suspended

at 1 times 106mL in basal medium and subjected to modeledmicrogravity simulated by the rotating wall vessel (RWV)bioreactor (Synthecon Cellon) placed inside a humidifiedincubator vertically rotating at 23 rpm [44] In the rotatingsystem the gravity is balanced by equal and oppositemechan-ical forces (centrifugal Coriolis and shear components)and the gravitational vector is reduced to about 10minus3 g Inthese conditions single cells are nearly always in suspensionrotating in quasi-stationary manner with the fluid in a low-shear culture environment [19 45] Ground-based (1 g) PBLswere kept at the same cell density in 75 cm2 flasks (FALCON)in the same medium After 24 48 or 72 h of incubationin MMG and 1 g PBLs were activated to enter cell cycle tomeasure cell proliferation by incubation in culture medium(CM) containing phytohaemagglutinin (PHA BIOCHROMBerlin Germany) and interleukin 2 (IL2 Chiron Siena Italy)as stimulating factors [46]

22 Total RNA Isolation Total RNA was isolated from107 PBLs at the end of 24 h incubation in MMG and 1 g byusing Trizol Reagent (Invitrogen Life Technologies CarlsbadCA USA) according to the manufacturerrsquos protocol TotalRNA was quantified using the ND-1000 spectrophotometer(Nanodrop Wilmington DE USA) and RNA integrity andthe content of miRNAs were assessed by capillary elec-trophoresis using the Agilent Bioanalyzer 2100 as previouslydescribed [47] Only total RNA samples with RNA integritynumber (RIN) values ge6 and with miRNA lt20 were usedfor microarray analysis

23 miRNA and Gene Expression Profiling MicroRNAs pro-filing was carried out in PBL samples incubated in MMGversus 1 g Analyses were performed by using the ldquoHumanmiRNA Microarray kit (V2)rdquo (Agilent Technologies) thatallows the detection of 723 known human (miRBase v101)and 76 human viral miRNAs Total RNA (200 ng) waslabeled with pCp Cy3 according to the Agilent protocoland unincorporated dyes were removed with MicroBioSpin6columns (BioRad) [48] Probes hybridization and slideswashing were performed as previously reported [47] AgilentFeature Extraction software version 10511 was used forimage analysis

Gene expression profiling was carried out in MMG-incubated PBLs versus 1 g incubated PBLs on total RNAextracted from the same PBL samples assayed for miRNAprofiling We used the ldquoWhole Human Genome Oligo Mi-croarrayrdquo (Agilent) consisting of sim41000 (60-mer) oligonu-cleotide probes which span conserved exons across thetranscripts of the targeted full-length genes 800 ng of totalRNAwas labeled with ldquoAgilent One-Color Microarray-BasedGene Expression protocolrdquo according to the manufacturerrsquosinstructions The method uses T7 RNA polymerase whichsimultaneously amplifies target material and incorporatescyanine 3-labeled CTP The Cy3-labeled cRNAs were puri-fied using Qiagenrsquos RNeasy mini spin columns (Qiagen)and quantified using the ND-1000 spectrophotometer (Nan-odrop Wilmington DE USA) Probes hybridization andslides washing were performed as previously reported [47]

BioMed Research International 3

Slides were scanned on an Agilent Microarray Scanner Sys-tem (model G2565CA) and Agilent Feature Extraction soft-ware version 10511 was used for image analysis Raw data areavailable on the Gene Expression Omnibus (GEO) website(httpwwwncbinlmnihgovgeo) using SuperSeries acces-sion number GSE57418 that groups microRNA (GSE57400)and mRNA expression profiles (GSE57408)

24 Statistical Analysis of miRNA and Gene Expression DataInterarray normalization of expression levels was performedwith cyclic Lowess formiRNA experiments and with quantilefor gene expression profiling [49] to correct possible exper-imental distortions Normalization function was applied toexpression data of all experiments and then values of spotreplicates within arrays were averagedThemodalities of spotquality measures and hybridization are reported previously[47] The identification of differentially expressed genes andmiRNAs was performedwith one- and two-class SignificanceAnalysis of Microarray (SAM) program [50] with defaultsettingsThe expression level of eachmiRNA andmRNAwascalculated as the log2 (MMG1 g) PBLs of the same donorPathway analysis on differentially expressed genes has beenperformed using Graphite web [51] hypergeometric test onReactome Pathways considering significant those categorieswith FDR lt 01

25 Identification of miRNA Target Genes and CorrelationAnalysis of miRNA and mRNA Expression Data To predictmiRNA targets we performed a computational analysis inte-grating mRNA and miRNA expression measurements fromthe same donor usingMAGIA2 web tool [43] freely availableat httpgencompbiounipditmagia2We used httpwwwmicrornaorg predictions and Pearson correlation (119903 gt 04)to estimate the degree of correlation between any putativepairs of miRNA and mRNA [52 53] To identify the bio-logical processes most involved in target prediction we haveperformed an enrichment analysis on Gene Ontology (GO)usingDAVIDweb tool v67 [54] considering significant thosecategories with FDR lt 02

Intraclass analyses have been performed consideringMMGand 1 g samples separately In order to have comparableresults the intraclass analysis has been performed usingMAGIA2 software with the same parameters predictors andcutoff described for the previous analysis Specific inter-actions for MMG and 1 g networks have been identifiedand a GO enrichment analysis (FDR lt 02) was performedseparately on the nodes belonging to the specific MMG and1 g networks using DAVID web tool

26 Quantitative Real-Time PCR (qRT-PCR) Assay In orderto verify the expression data generated by miRNA andmRNA microarrays we performed qRT-PCR experimentsfor miRNAs and genes which showed significant expressionchanges in MMG The following miRNAs were subjectedto the RT-qPCR validation miR-9-5p miR-378a miR-155-5p and miR-150-3p Reverse transcription of 10 ng of totalRNA with primers corresponding to each miRNA and toU48 small nuclear RNA (RNU48) as endogenous control

was performed as directed by the protocol of the two-stepTaqMan MicroRNA Assay kit (Applied Biosystems FosterCity CA USA) that incorporates a target-specific stem-loopreverse transcription primer to provide specificity for themature miRNA target For the PCR reaction 1 120583L of the RTreaction was combined with 05 120583L of TaqMan MicroRNAAssay 20x and 5 120583L of TaqMan Universal PCR Master Mixin a 10 120583L final volume The reactions were incubated ina Mastercycler EP gradient 119878 (Eppendorf) in 02mLPCRtubes for 30min at 16∘C and 30min at 42∘C followed by5min at 85∘C and then held at 4∘CThe resulting cDNA wasquantitatively amplified in 40 cycles on an ABI 7500 Real-Time PCR System using TaqMan Universal PCRMaster Mixand TaqMan MicroRNA Assays

For mRNA detection 1 120583g of total RNA was retro-transcribed with ImProm-II Reverse Transcription System(Promega) qRT-PCR was performed with the GoTaq qPCRMaster Mix (Promega) and gene-specific primers for IFNGIL17F TLR4 HLA-DRB1 and BCL6 genes and for GAPDHas reference qRT-PCR reactions were performed in quad-ruplicates in PBL samples from 6 to 8 donors Real-timePCR was performed using an Applied Biosystems 7500 FastReal-Time PCR System with cycling conditions of 95∘C for10min followed by 95∘C for 15 sec and 60∘C for 60 sec 45cycles in total The relative expression levels of miRNAsand mRNAs between samples were calculated using thecomparative delta CT (threshold cycle number) method(2minusΔΔCT) implemented in the 7500 Real-Time PCR Systemsoftware [55] Primersrsquo pairs used are as follows GAPDH(glyceraldehyde-3-phosphate dehydrogenase) fw 51015840-TCC-TCTGACTTCAACAGCGA-31015840 rev 51015840-GGGTCTTACTCC-TTGGAGGC-31015840 IFNG (interferon gamma) fw 51015840- GGC-ATTTTGAAGAATTGGAAAG-31015840 rev 51015840-TTTGGATGC-TCTGGTCATCTT-31015840 IL17F (interleukin 17) fw 51015840-GGC-ATCATCAATGAAAACCA-31015840 rev 51015840- TGGGGTCCCAAG-TGACAG-31015840 TLR4 (Toll-like receptor 4)fw 51015840-CCTGCG-TGAGACCAGAAAG-31015840 rev 51015840-TTCAGCTCCATGCAT-TGATAA-31015840 HLA-DRB1 (major histocompatibility com-plex class II DR beta 1) fw 51015840-ACAACTACGGGGTTG-TGGAG-31015840 rev 51015840-GCTGCCTGGATAGAAACCAC-31015840BCL6 (B-cell CLLlymphoma 6) fw 51015840-CGAATCCACACA-GGAGAGAAA-31015840 rev 51015840-ACGCGGTATTGCACCTTG-31015840

27 Cell Proliferation and Apoptosis Induction Cell viabilitywas determined by the T-cell cloning assay [44 46] at theend of 24 h 48 h and 72 h incubation in MMG and 1 gBriefly four 96-well U-bottomed microtiter plates with twoviable lymphocyteswell were seeded in medium CM in thepresence of 1 times 104 feeder cellswell (TK6 lymphoblastoidcells lethally irradiatedwith 40Gy of 120574-rays) Twoweeks laterthe plates were scored for growing colonies to calculate thecloning efficiency (CE) from the proportion of negative wellsassuming a Poisson distribution (CE = minusIn P

0

N where P0

isthe fraction of wells without cells andN is the number of cellsseeded into wells) [56]

Apoptotic index was determined in PBLs incubated for24 and 48 h in MMG and in parallel in 1 g For detectionof apoptotic morphology PBLs were fixed and stained with


hsa-miR-9-5phsa-miR-9-3p

hsa-miR-155-5phsa-miR-125a-5p

hsa-let-7e-5phsa-miR-376c-3phsa-miR-99b-5p

hsa-miR-29b-1-5phsa-miR-132-3p

hsa-let-7i-3phsa-miR-376a-3phsa-miR-193b-3p

hsa-let-7i-5phsa-miR-146b-5p

hsa-miR-221-5phsa-miR-505-5phsa-miR-10a-5p

hsa-miR-7-5phsa-miR-625-5phsa-miR-629-5p

hsa-miR-200a-3phsa-miR-342-5phsa-miR-192-5phsa-miR-7-1-3phsa-miR-185-5p

hsa-miR-107hsa-miR-103a-3p



hsa-miR-135a-3phsa-miR-378a-5p

hsa-miR-663ahsa-miR-940


hsa-miR-34b-5phsa-miR-150-3p

hsa-miR-378a-3p

Expression valueminus4 minus2 0 2 4 6

(a)

hsa-miR-376a-3phsa-miR-376c-3phsa-miR-192-5phsa-miR-7-1-3phsa-miR-625-5phsa-miR-342-5phsa-miR-10a-5phsa-miR-505-5phsa-miR-221-5phsa-miR-7i-5phsa-miR-7-5phsa-miR-629-3p5phsa-miR-146b-5phsa-miR-200a-3phsa-miR-145-5phsa-miR-193b-3phsa-miR-7i-5phsa-miR-29b-1-5phsa-miR-1323phsa-miR-99b-5phsa-miR-125a-5phsa-miR-7e-5phsa-miR-155-5phsa-miR-9-3phsa-miR-9-5phsa-miR-150-3phsa-miR-378a-3p5phsa-miR-378a-5phsa-miR-181a-3phsa-miR-34a-5phsa-miR-34b-5phsa-miR-362-5phsa-miR-532-5phsa-miR-423-3phsa-miR-103a-3phsa-miR-107hsa-miR-185-5phsa-miR-223-3phsa-miR-940

G D E F I C L A P B H Mminus371 00 307

(b)

Figure 1 Differentially expressed miRNAs in human PBLs incubated in MMG (a) The expression level of each miRNA indicated as foldchange is the mean of the expression values obtained from the transformed log2 ratio (MMG1 g) (b) Dendrogram of miRNAs differentiallyexpressed in MMG The range of expression value is from minus37 (green downregulation) to 307 (red upregulation) Grey boxes correspondto not available (NA) fluorescent signal from the microarray platform

2 120583gmL 46-diamino-2-phenylindol (DAPI Roche) in anantifade solution (Vectashield Vector Lab) as previouslydescribed [57] At least 2000 cells were scored for each time-point by fluorescence microscopy (1000x magnification)The activation of caspase-3 was measured by the caspase-3fluorescent assay kit (Clontech BD Biosciences) at the endof incubation in 1 g or MMG as previously described [58]The fluorescent emission at 505 nm (excitation at 400 nm)of cleaved 7-amino-4-trifluoromethyl coumarin (AFC) wasmeasured with a PerkinElmer LS-50 B spectrofluorimeter

3 Results

31 Identification of miRNAs Affected by MicrogravitymiRNA expression profiling was performed on total RNAextracted from PBLs of twelve healthy donors at the end of

24 h incubation time in MMG and in 1 g conditions By com-paring the expression profile of MMG-incubated versus 1 gincubated PBLs of the same donor we found 42 differentiallyexpressed miRNAs 25 upregulated and 17 downregulatedfor which raw data and means of miRNA expression valuesare available at Supplementary Table S1 (see SupplementaryTable S1 in the Supplementary Material available online athttpdxdoiorg1011552014296747) miR-9-5p miR-9-3pand miR-155-5p were the most upregulated (46- 35- and24-fold resp) whereas miR-378a and miR-150-3p were themost downregulated (sim2-fold) (Figure 1)

32 Effect of Microgravity on Gene Expression Profile Geneexpression analysis was performed in PBLs incubated for 24 hin MMG and in 1 g By comparing the expression profilesof MMG-incubated and 1 g incubated PBLs we identified1581 differentially expressed genes inMMG versus 1 g among


0

200

400

600

800

1000

Num

ber o

f diff

eren

tially

expr

esse

d ge

nes

1200

Upregulated Downregulated

(a)

57

1919

19 19

19

ImmunityHemostasisLipid metabolic processSignal transductionMetabolismCell migrationdevelopment

Cell surface interactionsDiseaseNeuronal systemTransmembrane transportof small moleculesMuscle contraction

283

188151

132

94

(b)

Figure 2 Results of gene expression analysis Differentially expressed genes (a) and pie chart of biological process () containing pathwayssignificantly enriched in PBLs incubated in MMG (b)

which 465 (29) genes were upregulated whereas 1116 (71)genes were downregulated (Figure 2(a) and SupplementaryTable S2) By selecting a 2-fold cut-off threshold we iden-tified 312 (197) genes in MMG 157 genes (10) showedalterations in expression level with a fold change greater than40 and among these 20 genes showed a fold change ge160(Supplementary Table S3) To identify sets of genes withexpression changes in MMG condition we used Graphite[51] a novel web tool for topological-based pathway analysesbased on high-throughput gene expression data analysesPathway analysis on differentially expressed genes has beenperformed by using hypergeometric test on Reactome Path-ways as implemented inGraphiteweb considering significantthose categories with FDR lt 01 We evidenced biologicalpathways significantly enriched in MMG 15 (283) wererelated to immunity 10 (188) to hemostasis 7 (132) tolipid metabolic process and signal transduction 5 (94) tometabolism 3 (57) to cell migrationdevelopment and 1(19) tocell surface interactions disease neuronal systemtransmembrane transport of small molecules and musclecontraction (Figure 2(b)) The list of pathways is reported inSupplementary Table S4

Among the immune pathways significantly enriched inMMG those of ldquoMHC class II antigen presentationrdquo ldquoTollReceptor Cascadesrdquo and ldquoDAP12 signalingrdquo showed the high-est number of differentially expressed genes (30 20 and 20genes resp) Also the ldquointerferon gamma signalingrdquo pathwaywas significantly enriched in MMG with 13 differentiallyexpressed genes Ten genes codifying for proteins of MHCclass II (HLA-DRA HLA-DRB1 HLA-DRB3 HLA-DRB4

HLA-DRB5 HLA-DQA1 HLA-DQA2 HLA-DPA1 HLA-DPB1 and HLA-DQB1) were common to nine pathways(Figure 3) CD4 codifying for a membrane glycoprotein of Tlymphocytes that interacts with the major histocompatibilitycomplex class II antigens was common to eight immunepathways RELA PTEN and PAK1 were included in threepathways whereas HLA-DOA andHLA-DMBwere includedin two pathways

33 Target Prediction and Integration Analysis of miRNAand mRNA Expression Profiles We examined the regulatoryeffects of miRNAs on global gene expression under modeledmicrogravity (MMG) condition in comparison with groundgravity (1 g) To predict the target genes of differentiallyexpressed miRNAs in MMG we performed a computationalanalysis using TargetScan tool which predicts biologicaltargets of miRNAs by searching for the presence of conserved8mer and 7mer sites that match the seed region of eachmiRNA [59] However all available software for target pre-diction is characterized by a large fraction of false positivethus the integration of target predictions with miRNA andgene target expression profiles has been proposed to refinemiRNA-mRNA interactions The correlation analyses on thedifferentially expressed miRNAs and mRNAs were carriedout with MAGIA2 software [43] by microRNA Pearson pre-diction analysis which allowed the identification of miRNA-mRNA interactions (Supplementary Table S5) To discoverfunctional relationships between miRNAs and the transcrip-tome and uncover the gene pathways that are regulated


Gene symbol description Fold change Toll

rece

ptor

casc

ade

HLA-DPA1 Major histocompatibility complex class II DP alpha 1 HLA-DPB1 Major histocompatibility complex class II DP beta 1 HLA-DQA1 Major histocompatibility complex class II DQ alpha 1HLA-DQA2 Major histocompatibility complex class II DQ alpha 2HLA-DQB1 Major histocompatibility complex class II DQ beta 1 HLA-DRA Major histocompatibility complex class II DR alphaHLA-DRB1 Major histocompatibility complex class II DR beta 1 HLA-DRB3 Major histocompatibility complex class II DR beta 3 HLA-DRB4 Major histocompatibility complex class II DR beta 4 HLA-DRB5 Major histocompatibility complex class II DR beta 5 HLA-DOA Major histocompatibility complex class II DO alphaHLA-DMB Major histocompatibility complex class II DM betaCD4 CD4 moleculeRELA v-rel reticuloendotheliosis viral oncogene homolog APTEN Phosphatase and tensin homologPAK1 p21 protein (Cdc42Rac)-activated kinase 1

minus178minus183minus151minus164minus14minus155minus161minus185minus174minus159minus095minus147minus078051

minus026minus132

DA

P12

signa

ling

Cos

timul

atio

n by

the C

D28

fam

ily

TCR

signa

ling

Dow

nstre

am T

CR si

gnal

ing

MH

C cla

ss II

antig

en p

rese

ntat

ion

Gen

erat

ion

of se

cond

mes

seng

er m

olec

ules

Tran

sloca

tion

of Z

AP-70

to im

mun

olog

ical

syna

pse

Phos

phor

ylat

ion

of C

D3

and

TCR

zeta

chai

ns

Inte

rfero

n ga

mm

a sig

nalin

g

PD-1

signa

ling

Figure 3 Differentially expressed genes common to immune-related pathways identified by Reactome database in PBLs incubated inMMGThe expression value of each gene indicated as fold change is the mean of expression levels calculated as the log2 ratio (MMG1 g) on PBLsamples (see Supplementary Table S2)

by miRNAs in MMG we performed Gene Ontology (GO)analysis using DAVID [54]

In our analysis we used high classification stringency andconsidered only GO terms that have 119875 lt 01 after permu-tation corrections (Benjamini) (Table 1) Several GO termsbelonged to immune system function (ie ldquoinnate immuneresponserdquo ldquoinflammatory responserdquo ldquoregulation of cytokineproductionrdquo ldquopositive regulation of immune system processrdquoand ldquoresponse to bacteriumrdquo) in accordance with the resultsof pathway analysis on transcriptome (see SupplementaryTable S4) GO terms of ldquocell developmentrdquo ldquoregulation ofcell differentiationrdquo ldquoregulation of cell communicationrdquo ldquocellmotilityrdquo and ldquocell migrationrdquo were significantly enriched inMMG together with the category ldquoorgan developmentrdquo Inaddition the biological categories of ldquoregulation of signaltransductionrdquo ldquoregulation of response to stressrdquo ldquoregulationof cell deathrdquo and ldquoregulation of cell proliferationrdquo wereenriched in PBLs incubated in MMG

To determine whether different miRNAs within a GOcategory interact with the same target genes we performednetwork analysis using MAGIA2 [43] a software platformfor the visualization of complex miRNA-mRNA interactionsWe focused on miRNAs that correlated both positively andnegatively with the GO categories of immuneinflammatory

response regulation of programmed cell death and reg-ulation of cell proliferation As shown in Figure 4 mosttranscripts are associated with more than one miRNA as inthe case of TLR4 transcript correlated with eight differentmiRNAs (miR-10a-5p miR-7-5p miR-135a-3p miR-103a-3pmiR-7-1-3p miR-107 miR-629-5p and miR-362-5p)

By using Cytoscape [60] we visualized the func-tional interactions between miRNAs whose expression levelschanged the most in PBLs incubated in MMG such as miR-9-5p miR-9-3p miR-155-5p miR-150-3p and miR-378a-3pand correlated target genes involved in GO categories ofimmuneinflammatory response regulation of programmedcell death and regulation of cell proliferation (Figure 5)

Our results show that miR-155-5p correlates with IFNGIL17F BCL6 and RELA involved in immuneinflammatoryresponse with PTEN BNIP3L APAF1 and PDCD4 involvedin regulation of programmed cell death and with NKX3-1 involved in regulation of cell proliferation miR-150-3pcorrelates with immune-related genes (IFNG IL1A andHLA-DRB1) and with proapoptotic gene PDCD4 miR-9-3p correlates with genes regulating cell proliferation (NKX3-1 GADD45A and TP53BP1) apoptosis (APAF1 BNIP3L)and immunity (CCL7 CXCL5 and BCL6) Among genesenriched within the three functional categories miR-9-5p


Table 1 Selected GO terms of biological processes significantly affected by microgravity The complete list of GO terms can be found inSupplementary Table S6

GOID Term Count 119875 value Fold enrichment FDRGO0045087 Innate immune response 25 242 times 10minus5 2566978193 0040412626GO0009966 Regulation of signal transduction 88 167 times 10minus4 1471577879 027973741GO0048468 Cell development 62 171 times 10minus4 1610338849 0285253218GO0045595 Regulation of cell differentiation 54 268 times 10minus4 1648 0448057857GO0006954 Inflammatory response 41 323 times 10minus4 1787513228 0539044451GO0080134 Regulation of response to stress 37 543 times 10minus4 1806696296 090478009GO0010646 Regulation of cell communication 95 822 times 10minus4 1380599647 1366092587GO0048513 Organ development 139 822 times 10minus4 1290910116 1366713032GO0042060 Wound healing 26 838 times 10minus4 2025910165 1393334889GO0007165 Signal transduction 198 0001398603 1211224944 2313943814GO0001817 Regulation of cytokine production 27 0001406003 1926233766 2326052387GO0048514 Blood vessel morphogenesis 26 0001709187 193009009 2820918727GO0001817 Regulation of cytokine production 15 0001406003 1926233766 2326052387GO0007399 Nervous system development 88 0002097026 1359812471 3450524099GO0022603 Regulation of anatomical structure morphogenesis 28 0002408369 1831111111 3953172097GO0048870 Cell motility 33 0002838826 1710188679 464407324GO0048522 Positive regulation of cellular process 159 0003551259 1221594406 5777298149GO0002684 Positive regulation of immune system process 31 0003855111 1711490787 6256755641GO0050865 Regulation of cell activation 26 0003880839 1819447983 6297247375GO0051174 Regulation of phosphorus metabolic process 51 0003964604 1486259947 6428964883GO0001944 Vasculature development 28 0003981783 1767969349 6455956993GO0043066 Negative regulation of apoptosis 41 0004040803 156952381 6548633436GO0010941 Regulation of cell death 83 0004115909 1341019608 6666445839GO0009617 Response to bacterium 22 0004991824 19032021 8030143455GO0043067 Regulation of programmed cell death 82 000552374 1328770895 8849108723GO0008285 Negative regulation of cell proliferation 40 000576007 154741784 9210770389GO0010557 Positive regulation of macromolecule biosynthetic process 62 0006543793 139014966 104004889GO0016477 Cell migration 30 0006706505 1664646465 1064564555GO0042127 Regulation of cell proliferation 71 0009848433 1331149033 152576907GO0048523 Negative regulation of cellular process 139 0012514364 119870225 189945349

correlates with BCL6 miR-378a-3p is correlated with HLA-DRB1 GPNMB and NKX3-1 the same transcripts togetherwith IL17F are correlated also with miR-378a-5p

The microarray data from miRNA and gene expressionprofiling were validated by real-time qPCR experiments forfour miRNAs (miR-9-5p miR-155-5p miR-378a and miR-150-3p) and five mRNAs (IFNG IL17F BCL6 HLA-DRB1and TLR4) whose expression level was significantly alteredby MMG incubation (Figure 6) miR-9-5p and miR-155-5p together with IFNG IL17F and BCL6 transcripts wereupregulated in MMG whereas miR-378a and miR-150-3ptogether with HLA-DRB1 and TLR4 transcripts were down-regulated in MMG

34 Intraclass Integrated Analysis Recently Censi and col-leagues [61] observed a significant increase in the numberand strength of genes correlation under stress conditionssuch as disease and environmental or physiological changesTo evaluate whether the stress induced by MMG increasesthe amount of correlation of the system with respect to

1 g control condition we integrated mRNAs and miRNAsdata separately for MMG and 1 g using MAGIA2 By com-paring the two regulatory networks we observed a similarnumber of interactions between MMG (190 interactions)and 1 g (218 interactions) (Figure S1) indicating that therewas no significant connectivity enrichment under modeledmicrogravity By contrast GeneOntology analysis performedon MMG- and 1 g-specific interactions reported 50 GOcategories significantly enriched in only MMG condition(119875 lt 02 after Benjamini corrections Supplementary TableS7) 10 out of these were previously described in Table 1 Inparticular the GO categories ldquoregulation of cellular processrdquoand ldquocell differentiationrdquo were significantly affected byMMGWith intraclass analysis the GO categories of immunity andcell death were not enriched in MMG probably because inour study the number of PBL samples available for suchanalysis was relatively small On the whole the intraclassanalysis shows that modeled microgravity does not increasethe general connectivity of miRNA-gene interaction networkbut rather increases the transcriptome plasticity with respect


hsa-let-7i-3p

hsa-miR-200a-3p

TP73

CXCL10

hsa-miR-34a-5p

IRAK2

hsa-miR-663aIL18RAP

CCL19

hsa-miR-150-3p

BMP6

IL6

CXCL11CD180

CCR7

hsa-miR-378a-5p

FOS

FCN2

hsa-miR-125a-5p

hsa-miR-132-3pPDPN

hsa-miR-505-5phsa-miR-34b-5p

hsa-miR-155-5p

hsa-miR-9-3p PRDX2

ATRN

FN1

SPP1HMOX1

RELAhsa-miR-7-5p

hsa-let-7i-5p

LYZ

CLEC7AVSIG4

hsa-miR-10a-5p

hsa-miR-940

hsa-miR-362-5p

hsa-miR-532-5pCD55

hsa-miR-9-5p

hsa-miR-146b-5p

IL1RAPhsa-miR-7-1-3p

hsa-miR-103a-3p

CXCL1

CIITA

TLR4

TLR7

hsa-miR-629-5p

hsa-miR-107

MEFV

hsa-miR-376a-3p

hsa-miR-376c-3p

IL17F

IL1A

hsa-let-7e-5phsa-miR-221-5p

hsa-miR-185-5p

CCL7

hsa-miR-135a-3p

SIGLEC1 TLR5

LIPA AIF1

CFP


hsa-miR-1225-5p

C1QCF2R

C5

C1QB

hsa-miR-378a-3p

(a) Immuneinflammatory response

GIMAP5

DDAH2 HMOX1

SOX4PRDX1

hsa-miR-505-5p

hsa-miR-223-3p

SNCA hsa-miR-221-5p

hsa-miR-192-5p

UNC13B

PTPRFRNF7

TXNIPAPAF1

RUNX3

MGMTPTENNOTCH2IFNG

MPOETS1

NRG1

PRDX2

ANXA4

TNFSF13

NEFL GPX1NAIP

BNIP3L hsa-miR-185-5p

STK17ATLR4


hsa-miR-135a-3p

hsa-miR-99b-5p

IL12A

ITGA1CHD8

RAG1

UACASH3RF1

IL1A

HGF

KCNMA1

HTATIP2

SMOCAMK1D

IL6NOL3

AVEN

APOE

hsa-miR-663a

hsa-miR-200a-3p

hsa-miR-378a-5phsa-let-7i-5phsa-miR-532-5p

hsa-miR-376c-3phas-miR-362-5p

TAF9B

LTB

RASGRF2

LGALS1

TRIO

MEF2CBCL6

NQO1

ERBB3

AIFM3

TRAF1

FURIN

NR4A2

SERPINB2

FGD2

PIM2

NR4A1

TNFSF14

TIMP3

CEBPG

FGD4CARD6

RAB27A

ARHGEF3NF1

PPT1

F2RRELA

VAV2

SMAD6

ESR1

BMF ITSN1

MUC2

FOXO3

hsa-miR-132-3p

hsa-miR-423-3p

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-378a-3p

hsa-miR-629-5p

SLC25A4

hsa-let-7e-5p

MAP3K5

hsa-miR-181a-3p

TP73hsa-let-7i-3p

PPP1R13Bhsa-miR-9-3p

hsa-miR-940

hsa-miR-7-1-3p

hsa-miR-625-5p


hsa-miR-342-5p

hsa-miR-7-5p

hsa-miR-155-5p

hsa-miR-29b-1-5p

hsa-miR-10a-5p

(b) Regulation of programmed cell deathLTB

VSIG4

hsa-a-423-3p

SMO

hsa-let-7i-5p

hsa-miR-629-3p

hsa-let-7e-5p

VEGFB

NCK2

hsa-miR-505-5pHMOX1

IL12RB2 IL6CEBPA

CD86

CYP27B1

hsa-miR-7-5p

RARRES1

hsa-miR-940

hsa-miR-629-5p

hsa-miR-146b-5p

KLF11

RAC2

APOEPDCD1LG2

IFI30

F2R

hsa-miR-342-5p

hsa-miR-625-5pIL1A

SYKKLF4

HHEX

BCL6

AIF1

PDGFRB

IL12A

IFNGCTTNBP2

GPNMB

EREG

HES1

ETS1

hsa-miR-378a-5p

hsa-miR-155-5p

hsa-miR-378a-3p

NKX3-1

PPARG

CD33

NOTCH4

hsa-miR-221-5p

hsa-miR-135a-3p

ERBB3

CXCL10

COMTTNFRS13B

hsa-miR-34a-5p

hsa-miR-376c-3p

TNFSF13

hsa-miR-1225-5p

GPX1

hsa-miR-132-3p

hsa-miR-185-5p CXCL5CXCL1

KLF5FIGF

SOX2

KIFAP3 DLG3

hsa-miR-532-5p

hsa-miR-192-5p

hsa-miR-193b-3pPLAU

PTGS1PTEN

PDGFBIRS1

hsa-miR-10a-5p

SOX4NDN

hsa-miR-223-3p

hsa-miR-29b-1-5p

NOTCH2

hsa-miR-7-1-3pTXNIP

hsa-miR-107

CD9

NF1

NRG1

SLAMF1

RUNX3

PTPRF

MUC2

hsa-miR-125a-5p

hsa-miR-103a-3p

CHRNA10

RELA

PDGFCIL13RA1

hsa-miR-9-3p

hsa-miR-34b-5p hsa-miR-663a

GRN

hsa-miR-9-5p

hsa-miR-362-5pTIMP2

(c) Regulation of cell proliferation

Figure 4 Network analysis on correlated miRNA-mRNA pairs in PBLs incubated in MMG Network analyses were performed by MAGIA2software using miRNAs correlating both positively and negatively with transcripts involved in immuneinflammatory response (a) inregulation of programmed cell death (b) and in regulation of cell proliferation (c) Circles represent transcripts and triangles representmiRNAs

to 1 g gravity condition as evidenced by the enriched GOcategories

35 In Vitro Validation of GO Analysis Effects of MMG onCell Proliferation and Apoptosis Experimental assays wereperformed to validate the results obtained by bioinformaticsanalyses on the GO categories ldquoregulation of cell prolifera-tionrdquo and ldquoregulation of programmed cell deathrdquo associatedwith variations in miRNA expression under MMG To mea-sure cell proliferation quiescent (119866

0

) PBLs from the samedonorwere incubated for different times (24 h 48 h and 72 h)in 1 g and inMMGAt the end of incubation times the colony

forming ability has been determined by the T-cell cloningassay [44] in which cells were incubated in medium contain-ing mitogen factors (ie PHA and IL2) to trigger their cellcycle entry Our results showed that cloning efficiency (CE)decreased with time in both gravity conditions howeverMMG incubation affected the ability of PBLs to form colonies(119875 lt 005 at 24 h Figure 7(a)) To investigate whether MMGincubation increased the frequency of apoptotic cells PBLswere scored for the presence of apoptotic bodies Apoptoticindex was very similar at 24 and 48 h and significantlyhigher in PBLs incubated in MMG than in 1 g (Figure 7(b)119875 lt 005) In the same PBL samples caspase-3 activationassayed by the cleavage of the peptide substrate DEVD-AFC


Expression value

Regu

latio

n of

cell

prol

ifera

tion

Regu

latio

n of

pro

gram

med

cell

deat

hIm

mun

ein

flam

mat

ory

resp

onse

hsa-miR-155-5pIFNG

hsa-miR-125a-5p hsa-miR-940

hsa-miR-150-3p

hsa-miR-150-3p

hsa-miR-9-3p

CCL7hsa-let-7e-5p hsa-miR-7-5p

hsa-miR-135a-3p

hsa-miR-378a-5p

hsa-miR-135a-3p

IL17F

hsa-miR-155-5p

hsa-miR-9-3p

CXCL5

IL1A

hsa-miR-7-5pRELA

hsa-miR-155-5p hsa-miR-155-5p

hsa-miR-378a-5p hsa-miR-150-3p

HLA-DRB1hsa-miR-378a-3p hsa-miR-629-5p

hsa-miR-9-3p hsa-miR-9-5pBCL6

hsa-miR-629-5p

hsa-miR-34a-5p

hsa-miR-185-5p

hsa-miR-223-3p

hsa-miR-107

hsa-miR-185-5pPTEN

hsa-miR-10a-5p hsa-miR-7-1-3p

hsa-miR-29b-1-5p


hsa-miR-7-5p

hsa-miR-9-3p

hsa-miR-7-1-3p

hsa-miR-29b-1-5p

hsa-let-7e-5p

hsa-miR-505-5pAPAF1


hsa-miR-221-5p

hsa-miR-376a-3p

hsa-miR-9-3pTP53BP1

hsa-miR-9-3p

hsa-miR-29b-1-5p

GADD45A


hsa-miR-9-3p

hsa-miR-378a-3p

hsa-miR-378a-3p

hsa-miR-155-5p

hsa-miR-1225-5p

hsa-miR-185-5phsa-miR-185-5pNKX3-1

hsa-miR-107GPNMB


PDCD4

hsa-miR-200a-3p

hsa-miR-135a-3p

hsa-miR-532-5p

hsa-miR-629-5p

hsa-miR-362-5p

minus51 0 75

hsa-miR-378a-5p

BNIP3Lhsa-miR-155-5p

hsa-let-7i-5p

Figure 5 Cytoscape visualization of miRNA-mRNA correlations in PBLs incubated 24 h in MMG Relationships between miRNAs andcorrelated target genes involved in ldquoimmuneinflammatory responserdquo (IFNG CCL7 IL17F HLA-DRB1 IL1A CXCL5 RELA and BCL6)ldquoregulation of programmed cell deathrdquo (PTEN BNIP3L APAF1 and PDCD4) and ldquoregulation of cell proliferationrdquo (GPNMB NKX3-1TP53BP1 and GADD45A) Circles represent transcripts and triangles represent miRNAs the expression levels of each feature are representedas color scale


0

05

1

15

2

25

3

35

4

45

miR-9-5p miR-155-5p

MMG

Relat

ive e

xpre

ssio

n

lowastlowastlowast

lowastlowast

1g

0

02

04

06

08

1

12

miR-378a miR-150-3p

Relat

ive e

xpre

ssio

n

lowastlowastlowast lowastlowastlowast

MMG1g

(a)

0

5

10

15

20

25

IFNG IL17F BCL6

80

120

140

Relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

MMG1g

0

02

04

06

08

1

12

TLR4 HLA-DRB1

Relat

ive e

xpre

ssio

nlowastlowastlowast lowastlowastlowast

MMG1g

(b)

Figure 6 Microarray data validation by quantitative real-time PCR (qRT-PCR) Validation of microarray data by qRT-PCR in MMG-incubated versus 1 g incubated PBLs The results are consistent with the cumulative microarray data of miRNAs (a) and mRNAs (b) Values(fold change dark grey bars) are means plusmn SE of expression levels calculated as the log2 (MMG1 g) on PBL samples from 4 to 6 differentdonors The value ldquo1rdquo of control 1 g PBLs (light grey bars) is arbitrarily given when no change is observed ( lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001 andlowast

119875 lt 005 t-test)

increased significantly in PBLs incubated 48 h in MMG withrespect to those in 1 g (Figure 7(c) 119875 lt 005)

4 Discussion

In the present study we evaluated the effects of modeledmicrogravity (MMG) on human PBLs by analyzing miRNAand gene expression profiles in comparison with PBLs cul-tured in Earth gravity condition (1 g) Our results reported 42differentially expressed miRNAs in PBLs cultured for 24 h inMMGwith respect to 1 g of which 14 (miR-34a-5p miR-34b-5p miR-663a miR-135a-3p miR-1225-5p miR-940 miR-221-5p miR-29b-1-5p miR-10a-5p let-7i-3p miR-200a-3p miR-7-5p miR-7-1-3p and miR-505-5p) were found altered also

by 120574-irradiation as assessed in our previous study [47] Themost dysregulatedmiRNAs identified in the present work arethe upregulated miR-9-5p miR-9-3p and miR-155-5p andathe downregulated ones are miR-150-3p and miR-378a-3pSuch miRNAs have been found altered in human tumors inparticular miR-9 is an oncogenic miRNA overexpressed inmixed lineage leukemia- (MLL-) rearranged acute myeloidleukemia [62] in muscle-invasive bladder cancer [63] andin osteosarcoma cell lines [64] miR-155 is commonly upreg-ulated in hematological malignancies [65 66] and has beenlinked to the development of breast lung and stomachtumors [67ndash70] miR-150 is significantly downregulated inmost cases of acute myeloid leukemia [71] and colorectalcancer [72] in addition miR-150 has an important role in


25

20

15

10

5

0

lowast

24 48 72

Incubation time (hrs)

Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)

Figure 7 Cell proliferation and apoptosis induction in human PBLs incubated in MMG and in 1 g (a) T-cell cloning assay performed atthe end of 24 h 48 h and 72 h of incubation in the two gravity conditions Data are means plusmn SE from thirteen independent experiments(b) Apoptotic index at the end of incubation for 24 h and 48 h in MMG and 1 g determined by nuclear chromatin condensation with DAPIstaining (c) Caspase-3 activation at the end of 24 h and 48 h incubation in MMG and 1 g assessed by fluorimetric assay (au arbitrary units)Data in (b) and (c) are means plusmn SE from 3-4 independent experiments (lowast119875 lt 005 t-test)

normal hematopoiesis and its aberrant downregulation is asensitivemarker indicative of lymphocyte depletion and bonemarrow damage [73] miR-378 (actually annotated as miR-378a) is significantly downregulated in colorectal cancer [74]in cutaneous squamous cell carcinoma [75] and in renalcell carcinoma [76] The effects of microgravity on miRNAexpression profile are currently reported in only one studycarried out in human lymphoblastoid TK6 cells incubatedunder simulated microgravity for 72 h [77] Among thedysregulated miRNAs only two were common to our datamiR-150 and miR-34a although the direction and intensityof fold change were different demonstrating the cell typespecific signature of miRNA profile

miRNAs modulate gene expression by interacting withthe 31015840UTR of target genes and since a single miRNA couldhave hundreds to thousands of predicted target genes [25]it is difficult to determine the true target regulated by themiRNA which affects a biological function Moreover bind-ing of multiple miRNAs to one target could further increasethe complexity of target prediction The identification ofmiRNA target genes is usually performed by bioinformaticprediction algorithms based on (i) sequence similarity searchpossibly considering target site evolutionary conservationand (ii) thermodynamic stability However it is known thatthe results of target prediction algorithms are characterizedby very low specificity [78] For this purpose the integrationof target predictions with miRNA and gene expression pro-files has been recently proposed to improve the detection offunctional miRNA target relationships [79 80] Therefore toidentify the most likely target genes of miRNAs differentiallyexpressed in MMG we defined gene expression signatureon the same samples of PBLs assayed for miRNA profilingthen we integrated expression profiles from both miRNAsand mRNAs with in silico target predictions to reducethe number of false positives and increase the number of

biologically relevant targets [81ndash83] Our results of geneexpression profiling reported the downregulation of multiplegenes in MMG (71) in accordance with previous findingsin activated human T lymphocytes incubated for 24 h insimulated microgravity [21] Moreover we found that about20 of genes responded to MMG by more than 2-foldchange in expression level and twenty genes showed a ge16-fold change in expression Most of these top dysregulatedgenes were immune-related such as those codifying forinflammatory cytokines (CCL1 CCL7 CXCL5 CXCL11 andIL1A) and for proteins with a role in immunoregulatoryfunctions (IFNG TNIP3 TREM1 APOC1 FCN1 FCN2and CPVL) (Supplementary Table S3) Biological pathwaysenriched in PBLs exposed to MMG were mainly involved inimmunity (Figure 2(b)) including adaptive immune systemresponse (ie PD-1 signaling phosphorylation of CD3 andTCR zeta chains translocation of ZAP-70 to immunologicalsynapse MHC class II antigen presentation TCR signalingand costimulation by the CD28 family) innate immunesystem response (ie Toll Receptor Cascades) and cytokinesignaling in immune system (ie interferon gamma signal-ing) (Supplementary Table S4) All these pathways includedten downregulated genes codifying for MHCmolecules classII (HLA-DPA1 HLA-DPB1 HLA-DQA1 HLA-DQA2 HLA-DQB1 HLA-DRA HLA-DRB1 HLA-DRB3 HLA-DRB4and HLA-DRB5) which are expressed in antigen presentingcells (APC) and play a central role in the immune system bypresenting peptides derived from extracellular proteins [84]Therefore the downregulation of these genes suggests that thedisplay of antigens at the cell surface of APCmay be disturbedby gravity reduction affecting the efficiency of immuneresponse as observed in astronauts during spaceflight andimmediately afterwards [85ndash87] Moreover our data are inaccordance with the inhibition of immediate early genesin T-cell activation observed in space microgravity [14]


and with alterations of gene expression in human activatedT-cells incubated in modeled microgravity including thedownregulation of HLA-DRA gene [21]

By integrating the transcriptome and microRNAomewe detected significant miRNA-mRNA relationships underMMG Since miRNAs act prevalently through target degra-dation expression profiles of miRNAs and target genesare generally expected to be inversely correlated Neverthe-less since miRNA activity is part of complex regulatorynetworks and gene expression profiles are the result ofdifferent levels of regulation also positive correlation (ieupregulated miRNAupregulated mRNA or downregulatedmiRNAdownregulated mRNA) is expected Indeed in an invivo mouse model the activated expression of miRNAs hasbeen shown to correlate with activated expression of mRNAsrather thanwithmRNAdownregulation [88] GeneOntology(GO) analysis conducted on the significantly correlatedmiRNA-mRNA pairs evidenced the biological categoriessignificantly overrepresented in MMG (Table 1) Many GOterms of immune response were enriched such as ldquoinnateimmune responserdquo ldquoinflammatory responserdquo ldquoregulation ofcytokine productionrdquo ldquopositive regulation of immune systemprocessrdquo and ldquoresponse to bacteriumrdquo Notably the mostdysregulated miRNAs detected in the present study miR-378a-3p miR-150-3p miR-155-5p miR-9-3p and miR-9-5pare significantly correlated with immune-related genes Inparticular miR-378a-3p is positively correlated with tran-scripts of MHC molecules class II such as HLA-DRB1(Figure 5) and HLA-DOA HLA-DRB5 and HLA-DQA2(not shown) miR-150-3p is negatively correlated with HLA-DRB1 IFNG and IL1Atranscripts whereas miR-155-5p ispositively correlated with IFNG and IL17F and negativelycorrelated with RELA and BCL6 (Figure 5) IL1A IL17Fand IFN-120574 are proinflammatory cytokines acting during theimmune response IFN-120574 is a soluble cytokine having broaderroles in activation of immune responses in part throughupregulating transcription of genes involved in antigen pro-cessingpresentation in cell cycle regulation and apoptosisand its correlation with miR-155 has been recently validated[89] BCL6 which encodes a nuclear transcriptional repres-sor has a role not only in regulation of lymphocyte functionbut also in cell survival and differentiation Similarly thepleiotropic transcription factor RELA has a role in immunebiological process and it is also involved in cell growth andapoptosis Besides miR-155-5p BCL6 is correlated with fourmiRNAs including miR-9 (3p and 5p) in addition miR-9-3p is positively correlated with CCL7 and CXCL5 (Figure 5)Recent evidences show that miR-9 is highly involved inimmunity and inflammatory diseases [90ndash92] by enhancingIFN-120574 production in activated human CD4(+) T-cells [91]Moreover Gao et al [90] have shown that miR-9 disturbsthe display of antigens at the cell surface by suppressing theexpression of MHC class I gene transcription

Together with the categories of immune response GOanalysis reported that also the categories of regulation of cellproliferation and regulation of programmed cell death weresignificantly enriched in MMG as previously reported in 120574-irradiated PBLs [47] Interestingly such categories were notenriched from pathway analysis conducted on transcriptome

and the reason could be that integrated analysis of miRNAsand mRNAs expression profiles evidences the posttran-scriptional effect mediated by miRNAs on gene expressionAmong genes involved in cell proliferation the transcriptionfactor NKX3-1 (24-fold upregulated) which mediates non-cell autonomous regulation of gene expression and inhibitscell proliferation is correlated with miR-9-3p miR-155-5pmiR-378a-3p and miR-378-5p (Figure 5) TP53BP1 (14-foldupregulated) encoding for a chromatin-associated factorinvolved in cell cycle checkpoint and growth is correlatedwith miR-9-3p GADD45A (15-fold upregulated) regulatingcell cycle arrest DNA repair cell survival senescence andapoptosis is also correlated with miR-9-3p GPNMB (32-fold downregulated) expressed in a wide array of normaltissues such as bone hematopoietic system and skin whereit influences cell proliferation adhesion differentiation andsynthesis of extracellular matrix proteins [93] is targetedby five miRNAs including miR-378a-3p Among miRNA-correlated genes involved in apoptosis we identified PDCD4(proapoptotic 14-fold upregulated) and found out that it iscorrelated with seven miRNAs including miR-155-5p andmiR-150-3p BNIP3L (proapoptotic 15-fold downregulated)also correlated with seven miRNAs including miR-155-5pandmiR-9-3p APAF1 (proapoptotic 13-fold downregulated)correlated with four miRNAs including miR-155-5p andmiR-9-3p and PTEN (proapoptotic) correlated with sevenmiRNAs including miR-155-5p Notably many transcripts(ie BCL6 PTEN BNIP3L PDCD4 NKX3-1 and GPNMB)are targeted by multiple miRNAs indicating a pleiotropiceffect in gene regulation by coexpressed endogenousmiRNAsin MMG Moreover our results suggest that under MMGcondition a small group of miRNAs regulates transcriptomeby modulating the same transcripts within one pathway

To validate the results of Gene Ontology analysis weevaluated whether the GO categories ldquoregulation of cell pro-liferationrdquo and ldquoregulation of programmed cell deathrdquo wereaffected byMMG incubation by performing biological assaysof T-cell cloning and apoptosis induction Our results showthat cloning ability of PBLs was lower after 24 h incubationin MMG than in 1 g in accordance with the suppression ofproliferative response of human lymphocytes to mitogenicstimulation in microgravity [94 95] The ability to originateclones in PBLs incubated for 48h and 72 h decreased in bothgravity conditions probably because the longer119866

0

-phase con-dition experienced by PBLs affected their responsiveness toenter into cell cycleThe antiproliferative effect of micrograv-ity has been recently reported also in human thyroid cancercells [96] and in human lung adenocarcinoma cells [97]Our results of apoptosis induction demonstrated that boththe formation of apoptotic bodies activation and caspase-3activation increased significantly in PBLs incubated inMMGthan in 1 g The activation of apoptotic process seems relatedto the overexpression of PDCD4 and RELA rather than tothe underexpression of BNIP3L and APAF1 indicating theexistence of complex regulatory networks between miRNAsand mRNAs that occur at different levels of regulation IFN-120574 besides having an important role in activating innateand adaptive immune responses plays important roles in


inhibiting cell proliferation and inducing apoptosis Its over-expression in MMG mediated by miR-9-3p and miR-155-5p could thus mediate the antiproliferative effect and theapoptosis induction In addition the correlation betweenmiR-9-3p and TP53BP1 could explain the clonogenicitydecrease and apoptosis increase in PBLs incubated in MMGIndeed overexpression of TP53BP1 has been demonstrated todecrease the clonogenicity and induce apoptosis in ovariancancer cells [98]

5 Conclusions

Our results show that MMG leads to changes in expressionlevel of a considerable fraction of microRNAome and tran-scriptome in human PBLs miRNAs differentially expressedin MMG are correlated with immuneinflammatory-relatedgenes as IFNG accordingly with the important role ofmiRNAs in immune function regulation Since inflammationworks as a tumor-promoting agent the abnormal expressionof such miRNAs under microgravity condition could influ-ence the carcinogenic process by affecting cancer cell immuneescape Moreover miRNAs mostly dysregulated in MMGsuch as miR-9 miR-155 and miR-150 are oncogenic sug-gesting that their abnormal expression can influence the car-cinogenic process The results of miRNA-mRNA integrationanalysis demonstrate that MMG increases the transcriptomeplasticity compared with 1 g condition and that categories ofregulation of cell proliferation and programmed cell deathare affected by MMG as confirmed by in vitro experimentalvalidation Taken togetherour results of high-throughputexpression analysis and miRNA-mRNA integration analysisgive new insight into the complex genetic mechanisms of cellresponse to stress environment under reduced gravity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

M Mognato and C De Pitta conceived and designed theexperiments C Girardi S Casara and M Mognato per-formed the experiments C De Pitta C Romualdi E CaluraL Celotti and M Mognato analyzed the data M Mognatoand L Celotti wrote the paper Cristina Girardi and CristianoDe Pitta contributed equally to this work

Acknowledgments

The authors gratefully acknowledge M De Bernard forcritical discussion and R Mazzaro for graphical supportThis work was done with the support of the Italian SpaceAgency (ASI XMAB fromMolecules to Man 1014060) toL Celotti and of the University of Padova (CPDA061783) toMMognatoThe authors also apologize to the authors whosework could not be cited due to space limitations

References

[1] R H Fitts D R Riley and J J Widrick ldquoFunctional and struc-tural adaptations of skeletal muscle to microgravityrdquo Journal ofExperimental Biology vol 204 no 18 pp 3201ndash3208 2001

[2] M Narici B Kayser P Barattini and P Cerretelli ldquoEffects of 17-day spaceflight on electrically evoked torque and cross-sectionalarea of the human triceps suraerdquo European Journal of AppliedPhysiology vol 90 no 3-4 pp 275ndash282 2003

[3] S Trappe D Costill P Gallagher et al ldquoExercise in spacehuman skeletal muscle after 6 months aboard the InternationalSpace Stationrdquo Journal of Applied Physiology vol 106 no 4 pp1159ndash1168 2009

[4] S I M Carlsson M T S Bertilaccio E Ballabio and J AMMaier ldquoEndothelial stress by gravitational unloading effectson cell growth and cytoskeletal organizationrdquo Biochimica etBiophysica Acta vol 1642 no 3 pp 173ndash179 2003

[5] M Infanger P Kossmehl M Shakibaei et al ldquoInductionof three-dimensional assembly and increase in apoptosis ofhuman endothelial cells by simulated microgravity impact ofvascular endothelial growth factorrdquo Apoptosis vol 11 no 5 pp749ndash764 2006

[6] R M Baevsky V M Baranov I I Funtova et al ldquoAuto-nomic cardiovascular and respiratory control during prolongedspaceflights aboard the International Space Stationrdquo Journal ofApplied Physiology vol 103 no 1 pp 156ndash161 2007

[7] J D Sibonga H J Evans H G Sung et al ldquoRecovery ofspaceflight-induced bone loss bone mineral density after long-duration missions as fitted with an exponential functionrdquo Bonevol 41 no 6 pp 973ndash978 2007

[8] J H Keyak A K Koyama A LeBlanc Y Lu and T F LangldquoReduction in proximal femoral strength due to long-durationspaceflightrdquo Bone vol 44 no 3 pp 449ndash453 2009

[9] O Ullrich K Huber and K Lang ldquoSignal transduction in cellsof the immune system in microgravityrdquo Cell Communicationand Signaling vol 6 article 9 2008

[10] B E Crucian R P Stowe D L Pierson and C F SamsldquoImmune system dysregulation following short- vs long-duration spaceflightrdquo Aviation Space and Environmental Medi-cine vol 79 no 9 pp 835ndash843 2008

[11] G Sonnenfeld J S Butel and W T Shearer ldquoEffects of thespace flight environment on the immune systemrdquo Reviews onEnvironmental Health vol 18 no 1 pp 1ndash17 2003

[12] G Sonnenfeld ldquoEditorial space flight modifies T cellactivationmdashrole of microgravityrdquo Journal of Leukocyte Biologyvol 92 no 6 pp 1125ndash1126 2012

[13] A Semov N Semova C Lacelle et al ldquoAlterations in TNF-and IL-related gene expression in space-flown WI38 humanfibroblastsrdquoTheFASEB Journal vol 16 no 8 pp 899ndash901 2002

[14] T T Chang I Walther C-F Li et al ldquoThe RelNF-120581B pathwayand transcription of immediate early genes in T cell activationare inhibited bymicrogravityrdquo Journal of Leukocyte Biology vol92 no 6 pp 1133ndash1145 2012

[15] M L Lewis L A Cubano B Zhao et al ldquocDNA microarrayreveals altered cytoskeletal gene expression in space-flownleukemic T lymphocytes (Jurkat)rdquo The FASEB Journal vol 15no 10 pp 1783ndash1785 2001

[16] S J PardoM J PatelMC Sykes et al ldquoSimulatedmicrogravityusing the Random Positioning Machine inhibits differentiationand alters gene expression profiles of 2T3 preosteoblastsrdquoAmerican Journal of Physiology vol 288 no 6 pp C1211ndashC12212005


[17] M Monticone Y Liu N Pujic and R Cancedda ldquoActivationof nervous system development genes in bone marrow derivedmesenchymal stem cells following spaceflight exposurerdquo Jour-nal of Cellular Biochemistry vol 111 no 2 pp 442ndash452 2010

[18] D Grimm J Bauer P Kossmehl et al ldquoSimulated microgravityalters differentiation and increases apoptosis in human follicu-lar thyroid carcinoma cellsrdquo The FASEB Journal vol 16 no 6pp 604ndash606 2002

[19] M Maccarrone N Battista M Meloni et al ldquoCreating con-ditions similar to those that occur during exposure of cellsto microgravity induces apoptosis in human lymphocytesby 5-lipoxygenase-mediated mitochondrial uncoupling andcytochrome c releaserdquo Journal of Leukocyte Biology vol 73 no4 pp 472ndash481 2003

[20] S J Crawford-Young ldquoEffects of microgravity on cell cytoskele-ton and embryogenesisrdquo International Journal of DevelopmentalBiology vol 50 no 2-3 pp 183ndash191 2006

[21] N E Ward N R Pellis S A Risin and D Risin ldquoGeneexpression alterations in activated human T-cells induced bymodeledmicrogravityrdquo Journal of Cellular Biochemistry vol 99no 4 pp 1187ndash1202 2006

[22] J Q Clement S M Lacy and B L Wilson ldquoGene expres-sion profiling of human epidermal keratinocytes in simulatedmicrogravity and recovery culturesrdquo Genomics Proteomics andBioinformatics vol 6 no 1 pp 8ndash28 2008

[23] R Kumari K P Singh and J W DuMond Jr ldquoSimulatedmicrogravity decreases DNA repair capacity and induces DNAdamage in human lymphocytesrdquo Journal of Cellular Biochem-istry vol 107 no 4 pp 723ndash731 2009

[24] C-Y Kang L Zou M Yuan et al ldquoImpact of simulated micro-gravity on microvascular endothelial cell apoptosisrdquo EuropeanJournal of Applied Physiology vol 111 no 9 pp 2131ndash2138 2011

[25] J Krutzfeldt M N Poy andM Stoffel ldquoStrategies to determinethe biological function of microRNAsrdquoNature Genetics vol 38no 1 pp S14ndashS19 2006

[26] C A Nickerson C M Ott J W Wilson et al ldquoLow-shearmodeled microgravity a global environmental regulatory sig-nal affecting bacterial gene expression physiology and patho-genesisrdquo Journal of Microbiological Methods vol 54 no 1 pp1ndash11 2003

[27] K Arunasri M Adil K Venu Charan C Suvro S HimabinduReddy and S Shivaji ldquoEffect of simulated microgravity on Ecoli K12 MG1655 growth and gene expressionrdquo PLoS ONE vol8 no 3 Article ID e57860 2013

[28] O Marcu M P Lera M E Sanchez et al ldquoInnate immuneresponses of Drosophila melanogaster are altered by space-flightrdquo PLoS ONE vol 6 no 1 Article ID e15361 2011

[29] Y Honda A Higashibata Y Matsunaga et al ldquoGenes down-regulated in spaceflight are involved in the control of longevityin Caenorhabditis elegansrdquo Scientific Reports vol 2 article 4872012

[30] A I Manzano J J W A van Loon P C M Christianen J MGonzalez-Rubio F J Medina and R Herranz ldquoGravitationaland magnetic field variations synergize to cause subtle varia-tions in the global transcriptional state of Arabidopsis in vitrocallus culturesrdquo BMC Genomics vol 13 no 1 article 105 2012

[31] D P Bartel ldquoMicroRNAs target recognition and regulatoryfunctionsrdquo Cell vol 136 no 2 pp 215ndash233 2009

[32] H Guo N T Ingolia J S Weissman and D P BartelldquoMammalian microRNAs predominantly act to decrease targetmRNA levelsrdquo Nature vol 466 no 7308 pp 835ndash840 2010

[33] V Huang Y Qin J Wang et al ldquoRNAa is conserved inmammalian cellsrdquo PLoS ONE vol 5 no 1 Article ID e88482010

[34] L B Frankel N R Christoffersen A Jacobsen M Lindow AKrogh and A H Lund ldquoProgrammed cell death 4 (PDCD4)is an important functional target of the microRNA miR-21 inbreast cancer cellsrdquoThe Journal of Biological Chemistry vol 283no 2 pp 1026ndash1033 2008

[35] M N Poy M Spranger and M Stoffel ldquomicroRNAs and theregulation of glucose and lipid metabolismrdquo Diabetes Obesityand Metabolism vol 9 no 2 pp 67ndash73 2007

[36] N Stern-Ginossar N Elefant A Zimmermann et al ldquoHostimmune system gene targeting by a viral miRNArdquo Science vol317 no 5836 pp 376ndash381 2007

[37] C J Marsit K Eddy and K T Kelsey ldquoMicroRNA responsesto cellular stressrdquo Cancer Research vol 66 no 22 pp 10843ndash10848 2006

[38] P M Voorhoeve C le Sage M Schrier et al ldquoA geneticscreen implicates miRNA-372 and miRNA-373 as oncogenes intesticular germ cell tumorsrdquo Cell vol 124 no 6 pp 1169ndash11812006

[39] E A C Wiemer ldquoThe role of microRNAs in cancer no smallmatterrdquo European Journal of Cancer vol 43 no 10 pp 1529ndash1544 2007

[40] W C S Cho ldquoOncomiRs the discovery and progress ofmicroRNAs in cancersrdquo Molecular Cancer vol 6 article 602007

[41] S Mi J Lu M Sun et al ldquoMicroRNA expression signaturesaccurately discriminate acute lymphoblastic leukemia fromacute myeloid leukemiardquo Proceedings of the National Academyof Sciences of the United States of America vol 104 no 50 pp19971ndash19976 2007

[42] S M Hammond ldquoMicroRNAs as tumor suppressorsrdquo NatureGenetics vol 39 no 5 pp 582ndash583 2007

[43] A Bisognin G Sales A Coppe S Bortoluzzi andC RomualdildquoMAGIA2 from miRNA and genes expression data integrativeanalysis to microRNA-transcription factor mixed regulatorycircuits (2012 update)rdquoNucleic Acids Research vol 40 no 1 ppW13ndashW21 2012

[44] M Mognato and L Celotti ldquoModeled microgravity affects cellsurvival and HPRT mutant frequency but not the expressionof DNA repair genes in human lymphocytes irradiated withionising radiationrdquoMutation Research vol 578 no 1-2 pp 417ndash429 2005

[45] B R Unsworth and P I Lelkes ldquoGrowing tissues in micrograv-ityrdquo Nature Medicine vol 4 no 8 pp 901ndash907 1998

[46] S-M Hou F J Van Dam F De Zwart et al ldquoValidation ofthe human T-lymphocyte cloning assaymdashring test report fromthe EU concerted action on HPRT mutation (EUCAHM)rdquoMutation Research vol 431 no 2 pp 211ndash221 1999

[47] C Girardi C de Pitta S Casara et al ldquoAnalysis of miRNAandmRNA expression profiles highlights alterations in ionizingradiation response of human lymphocytes under modeledmicrogravityrdquo PLoS ONE vol 7 no 2 Article ID e31293 2012

[48] H Wang R A Ach and B O Curry ldquoDirect and sensitivemiRNA profiling from low-input total RNArdquo RNA vol 13 no1 pp 151ndash159 2007

[49] B M Bolstad R A Irizarry M Astrand and T P Speed ldquoAcomparison of normalizationmethods for high density oligonu-cleotide array data based on variance and biasrdquo Bioinformaticsvol 19 no 2 pp 185ndash193 2003


[50] V G Tusher R Tibshirani and G Chu ldquoDiagnosis of multiplecancer types by shrunken centroids of gene expressionrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 98 pp 5116ndash5121 2001

[51] G Sales E Calura P Martini and C Romualdi ldquoGraphite webweb tool for gene set analysis exploiting pathway topologyrdquoNucleic Acids Research vol 41 pp 89ndash97 2013

[52] F Xin M Li C Balch et al ldquoComputational analysis ofmicroRNA profiles and their target genes suggests significantinvolvement in breast cancer antiestrogen resistancerdquo Bioinfor-matics vol 25 no 4 pp 430ndash434 2009

[53] HWang andW-H Li ldquoIncreasingMicroRNA target predictionconfidence by the relative R2 methodrdquo Journal of TheoreticalBiology vol 259 no 4 pp 793ndash798 2009

[54] D W Huang B T Sherman and R A Lempicki ldquoSystematicand integrative analysis of large gene lists using DAVID bioin-formatics resourcesrdquo Nature Protocols vol 4 no 1 pp 44ndash572009

[55] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862T methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[56] R J Albertini K L Castle and W R Borcherding ldquoT-cellcloning to detect the mutant 6-thioguanine-resistant lympho-cytes present in human peripheral bloodrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 79 no 21 I pp 6617ndash6621 1982

[57] MMognato CGirardi S Fabris and L Celotti ldquoDNA repair inmodeledmicrogravity double strand break rejoining activity inhuman lymphocytes irradiated with 120574-raysrdquoMutation Researchvol 663 no 1-2 pp 32ndash39 2009

[58] S Canova F Fiorasi M Mognato et al ldquoldquoModeled micrograv-ityrdquo affects cell response to ionizing radiation and increasesgenomic damagerdquo Radiation Research vol 163 no 2 pp 191ndash199 2005

[59] B P Lewis C B Burge and D P Bartel ldquoConserved seedpairing often flanked by adenosines indicates that thousandsof human genes are microRNA targetsrdquo Cell vol 120 no 1 pp15ndash20 2005

[60] M S Cline M Smoot E Cerami et al ldquoIntegration ofbiological networks and gene expression data using CytoscaperdquoNature Protocols vol 2 no 10 pp 2366ndash2382 2007

[61] F Censi A Giuliani P Bartolini and G Calcagnini ldquoA multi-scale graph theoretical approach to gene regulation networks acase study in atrial fibrillationrdquo IEEETransactions onBiomedicalEngineering vol 58 no 10 pp 2943ndash2946 2011

[62] P Chen C Price Z Li et al ldquomiR-9 is an essential onco-genic microRNA specifically overexpressed in mixed lineageleukemia-rearranged leukemiardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no28 pp 11511ndash11516 2013

[63] G Pignot G Cizeron-Clairac S Vacher et al ldquoMicroRNAexpression profile in a large series of bladder tumors identifi-cation of a 3-miRNA signature associated with aggressivenessof muscle-invasive bladder cancerrdquo International Journal ofCancer vol 132 no 11 pp 2479ndash2491 2013

[64] H M Namloslashs L A Meza-Zepeda T Baroslashy et al ldquoModulationof the osteosarcoma expression phenotype by microRNAsrdquoPLoS ONE vol 7 no 10 Article ID e48086 2012

[65] P S Eis W Tam L Sun et al ldquoAccumulation of miR-155and BIC RNA in human B cell lymphomasrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 10 pp 3627ndash3632 2005

[66] Y Pan M Meng G Zhang H Han and Q Zhou ldquoOncogenicmicroRNAs in the genesis of leukemia and lymphomardquoCurrentPharmaceutical Design 2014

[67] M V Iorio M Ferracin C-G Liu et al ldquoMicroRNA geneexpression deregulation in human breast cancerrdquo CancerResearch vol 65 no 16 pp 7065ndash7070 2005

[68] Z Lu Y Ye D Jiao J Qiao S Cui and Z Liu ldquoMiR-155 andmiR-31 are differentially expressed in breast cancer patientsand are correlated with the estrogen receptor and progesteronereceptor statusrdquo Oncology Letters vol 4 no 5 pp 1027ndash10322012

[69] F Gao J Chang H Wang and G Zhang ldquoPotential diagnosticvalue ofmiR-155 in serum from lung adenocarcinoma patientsrdquoOncology Reports vol 31 no 1 pp 351ndash357 2014

[70] G Higgs and F Slack ldquoThe multiple roles of microRNA-155 inoncogenesisrdquo Journal of Clinical Bioinformatics vol 3 no 1 p17 2013

[71] H Fayyad-Kazan N BitarM Najar et al ldquoCirculatingmiR-150andmiR-342 in plasma are novel potential biomarkers for acutemyeloid leukemiardquo Journal of TranslationalMedicine vol 11 no1 article 31 2013

[72] M Yanlei P Zhang F Wang et al ldquomiR-150 as a potentialbiomarker associated with prognosis and therapeutic outcomein colorectal cancerrdquo Gut vol 61 no 10 pp 1447ndash1453 2012

[73] N K Jacob J V Cooley T N Yee et al ldquoIdentification of Sensi-tive SerummicroRNABiomarkers for Radiation BiodosimetryrdquoPLoS ONE vol 8 no 2 Article ID e57603 2013

[74] G J Zhang H Zhou H X Xiao Y Li and T Zhou ldquoMiR-378 isan independent prognostic factor and inhibits cell growth andinvasion in colorectal cancerrdquo BMC Cancer vol 14 no 1 p 1092014

[75] M Sand M Skrygan D Georgas et al ldquoMicroarray analysis ofmicroRNA expression in cutaneous squamous cell carcinomardquoJournal of Dermatological Science vol 68 no 3 pp 119ndash1262012

[76] S Hauser L M Wulfken S Holdenrieder et al ldquoAnalysisof serum microRNAs (miR-26a-2lowast miR-191 miR-337-3p andmiR-378) as potential biomarkers in renal cell carcinomardquoCancer Epidemiology vol 36 no 4 pp 391ndash394 2012

[77] L S Mangala Y Zhang Z He et al ldquoEffects of simulatedmicrogravity on expression profile of microRNA in humanlymphoblastoid cellsrdquo The Journal of Biological Chemistry vol286 no 37 pp 32483ndash32490 2011

[78] P Alexiou MMaragkakis G L Papadopoulos M Reczko andA G Hatzigeorgiou ldquoLost in translation an assessment andperspective for computational microrna target identificationrdquoBioinformatics vol 25 no 23 pp 3049ndash3055 2009

[79] J Nunez-Iglesias C-C Liu T E Morgan C E Finch and XJ Zhou ldquoJoint genome-wide profiling of miRNA and mRNAexpression in Alzheimers disease cortex reveals alteredmiRNAregulationrdquo PLoS ONE vol 5 no 2 Article ID e8898 2010

[80] LMa YHuangWZhu et al ldquoAn integrated analysis ofmiRNAand mRNA expressions in non-small cell lung cancersrdquo PLoSONE vol 6 no 10 Article ID e26502 2011

[81] J C Engelmann and R Spang ldquoA least angle regression modelfor the prediction of canonical and non-canonical miRNA-mRNA interactionsrdquo PLoS ONE vol 7 no 7 Article ID e406342012

[82] N Bossel Ben-Moshe R Avraham M Kedmi et al ldquoContext-specific microRNA analysis identification of functionalmicroRNAs and their mRNA targetsrdquo Nucleic Acids Researchvol 40 no 21 pp 10614ndash10627 2012


[83] S Artmann K Jung A Bleckmann and T Beiszligbarth ldquoDetec-tion of simultaneous group effects inmicroRNA expression andrelated target gene setsrdquo PLoS ONE vol 7 no 6 Article IDe38365 2012

[84] R N Germain ldquoMHC-dependent antigen processing andpeptide presentation providing ligands for T lymphocyte acti-vationrdquo Cell vol 76 no 2 pp 287ndash299 1994

[85] I V Konstantinova E N Antropova V I Legenkov and VD Zazhirey ldquoStudy of the reactivity of blood lymphoid cells increw members of Soyuz 6 7 and 8 before and after space flightrdquoKosmicheskaia Biologiia iMeditsina vol 7 no 6 pp 35ndash40 1973(Russian)

[86] A Cogoli and A Tschopp ldquoLymphocyte reactivity duringspaceflightrdquo Immunology Today vol 6 no 1 pp 1ndash4 1985

[87] N Gueguinou C Huin-Schohn M Bascove et al ldquoCouldspaceflight-associated immune system weakening preclude theexpansion of human presence beyond Earths orbitrdquo Journal ofLeukocyte Biology vol 86 no 5 pp 1027ndash1038 2009

[88] Y O Nunez J M Truitt G Gorini et al ldquoPositively correlatedmiRNA-mRNA regulatory networks in mouse frontal cortexduring early stages of alcohol dependencerdquo BMCGenomics vol14 p 725 2013

[89] R P Sullivan L A Fogel J W Leong et al ldquoMicroRNA-155 tunes both the threshold and extent of NK cell activationvia targeting of multiple signaling pathwaysrdquo The Journal ofImmunology vol 191 no 12 pp 5904ndash5913 2013

[90] F Gao Z-L Zhao W-T Zhao et al ldquoMiR-9 modulates theexpression of interferon-regulated genes and MHC class Imolecules in humannasopharyngeal carcinoma cellsrdquoBiochem-ical and Biophysical Research Communications vol 431 no 3pp 610ndash616 2013

[91] F Bazzoni M Rossato M Fabbri et al ldquoInduction andregulatory function of miR-9 in human monocytes and neu-trophils exposed to proinflammatory signalsrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol106 no 13 pp 5282ndash5287 2009

[92] S Thiele J Wittmann H-M Jack and A Pahl ldquomiR-9enhances IL-2 production in activated human CD4+ T cells byrepressing Blimp-1rdquo European Journal of Immunology vol 42no 8 pp 2100ndash2108 2012

[93] M Singh F Del carpio-Cano J Y Belcher et al ldquoFunctionalroles of osteoactivin in normal and disease processesrdquo CriticalReviews in Eukaryotic Gene Expression vol 20 no 4 pp 341ndash357 2010

[94] M Cogoli-Greuter M A Meloni L Sciola et al ldquoMovementsand interactions of leukocytes in microgravityrdquo Journal ofBiotechnology vol 47 no 2-3 pp 279ndash287 1996

[95] I Walther P Pippia M A Meloni F Turrini F Mannu and ACogoli ldquoSimulated microgravity inhibits the genetic expressionof interleukin-2 and its receptor in mitogen-activated T lym-phocytesrdquo FEBS Letters vol 436 no 1 pp 115ndash118 1998

[96] X Ma J Pietsch M Wehland et al ldquoDifferential gene expres-sion profile and altered cytokine secretion of thyroid cancer cellsin spacerdquoThe FASEB Journal vol 28 no 2 pp 813ndash835 2014

[97] D Chang H Xu Y Guo et al ldquoSimulated microgravity altersthe metastatic potential of a human lung adenocarcinoma celllinerdquo In Vitro Cellular and Developmental BiologymdashAnimal vol49 no 3 pp 170ndash177 2013

[98] S Hong X Li Y ZhaoQ Yang and B Kong ldquo53BP1 suppressestumor growth and promotes susceptibility to apoptosis ofovarian cancer cells through modulation of the Akt pathwayrdquoOncology Reports vol 27 no 4 pp 1251ndash1257 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


that has partial or complete complementarity to the miRNAThe binding of miRNAs to complementary sequence of theirtarget mRNAs may repress translation or induce degradationofmRNAs [31] Recently the destabilization of targetmRNAsinstead of translational repression has been shown to be thepredominant mechanism for reduced protein output [32]Less often dsRNA formed by miRNA target complexes cantarget gene promoters and actually enhance transcription oftarget genes sometimes termed RNAa (RNA activation) [33]A single miRNA may have broad effects on gene expressionnetworks such as regulating cell lineage specificity cellularfunctions or stress response Besides a physiological roleof miRNAs in a variety of important biological processesincluding differentiation apoptosis [34] and fat metabolism[35] the miRNA-mediated gene regulation operates alsoduring viral infection [36] stress response pathway [37] andpathological processes such as tumorigenesis [38ndash42]

The present study is addressed to identify alterationsin miRNA profiles of human peripheral blood lympho-cytes (PBLs) incubated in modeled microgravity (MMG)with respect to those incubated in gravity 1 g To simulatemicrogravity we used a specialized bioreactor developed atthe NASA-Johnson Space Center (Houston TX USA) therotating wall vessel which represents a valid ground modelto simulate as far as possible a condition of reduced gravityTo identify miRNA-correlated genes whose expression levelwas significantly altered as a function ofMMGwe performedgene expression profiling on the same PBL samples assayedfor miRNA profiling and we integrated microRNAome andtranscriptome by using MAGIA2 [43] a web tool for theintegrative analysis of miRNA and genes expression dataincorporating transcriptional regulation A group ofmiRNA-mRNA pairs related to immunity cell proliferation andapoptosis was identified in PBLs incubated in MMG Thedifferences between MMG and 1 g on correlated miRNA-mRNA pairs involved in cell proliferation and apoptosiswere investigated by in vitro assays of clonogenic ability andapoptosis induction in PBLs incubated in MMGwith respectto those incubated in 1 g conditions

2 Materials and Methods

21 Lymphocytes Isolation and Microgravity SimulationHuman peripheral blood lymphocytes (PBLs) were obtainedfrom freshly collected ldquobuffy coatsrdquo from blood samples oftwelve healthy anonymous donors at the Blood Centre ofthe City Hospital of Padova (Italy) This study obtainedthe Ethics Approval from the Transfusion Medicine (TM)Ethics Committee of Blood Centre of the City Hospital ofPadova PBLs were isolated by separation on Biocoll densitygradient (BIOCHROM Berlin Germany) After isolationPBLs were preincubated overnight at a concentration of 3times 106mL in basal medium RPMI 1640 containing Gluta-MAX I (Invitrogen Life Technologies Carlsbad CA USA)124UmL penicillin 63120583gmL streptomycin sulfate and 10fetal bovine serum (FBS BIOCHROM Berlin Germany)After the overnight incubation PBLs consisting of peripheralmononuclear cells depleted of monocytes were suspended

at 1 times 106mL in basal medium and subjected to modeledmicrogravity simulated by the rotating wall vessel (RWV)bioreactor (Synthecon Cellon) placed inside a humidifiedincubator vertically rotating at 23 rpm [44] In the rotatingsystem the gravity is balanced by equal and oppositemechan-ical forces (centrifugal Coriolis and shear components)and the gravitational vector is reduced to about 10minus3 g Inthese conditions single cells are nearly always in suspensionrotating in quasi-stationary manner with the fluid in a low-shear culture environment [19 45] Ground-based (1 g) PBLswere kept at the same cell density in 75 cm2 flasks (FALCON)in the same medium After 24 48 or 72 h of incubationin MMG and 1 g PBLs were activated to enter cell cycle tomeasure cell proliferation by incubation in culture medium(CM) containing phytohaemagglutinin (PHA BIOCHROMBerlin Germany) and interleukin 2 (IL2 Chiron Siena Italy)as stimulating factors [46]

22 Total RNA Isolation Total RNA was isolated from107 PBLs at the end of 24 h incubation in MMG and 1 g byusing Trizol Reagent (Invitrogen Life Technologies CarlsbadCA USA) according to the manufacturerrsquos protocol TotalRNA was quantified using the ND-1000 spectrophotometer(Nanodrop Wilmington DE USA) and RNA integrity andthe content of miRNAs were assessed by capillary elec-trophoresis using the Agilent Bioanalyzer 2100 as previouslydescribed [47] Only total RNA samples with RNA integritynumber (RIN) values ge6 and with miRNA lt20 were usedfor microarray analysis

23 miRNA and Gene Expression Profiling MicroRNAs pro-filing was carried out in PBL samples incubated in MMGversus 1 g Analyses were performed by using the ldquoHumanmiRNA Microarray kit (V2)rdquo (Agilent Technologies) thatallows the detection of 723 known human (miRBase v101)and 76 human viral miRNAs Total RNA (200 ng) waslabeled with pCp Cy3 according to the Agilent protocoland unincorporated dyes were removed with MicroBioSpin6columns (BioRad) [48] Probes hybridization and slideswashing were performed as previously reported [47] AgilentFeature Extraction software version 10511 was used forimage analysis

Gene expression profiling was carried out in MMG-incubated PBLs versus 1 g incubated PBLs on total RNAextracted from the same PBL samples assayed for miRNAprofiling We used the ldquoWhole Human Genome Oligo Mi-croarrayrdquo (Agilent) consisting of sim41000 (60-mer) oligonu-cleotide probes which span conserved exons across thetranscripts of the targeted full-length genes 800 ng of totalRNAwas labeled with ldquoAgilent One-Color Microarray-BasedGene Expression protocolrdquo according to the manufacturerrsquosinstructions The method uses T7 RNA polymerase whichsimultaneously amplifies target material and incorporatescyanine 3-labeled CTP The Cy3-labeled cRNAs were puri-fied using Qiagenrsquos RNeasy mini spin columns (Qiagen)and quantified using the ND-1000 spectrophotometer (Nan-odrop Wilmington DE USA) Probes hybridization andslides washing were performed as previously reported [47]










0

N where P0




















hsa-miR-378a-3p


(a)



(b)



3 Results





0

200

400

600

800

1000

Num

ber o

f diff

eren

tially

expr

esse

d ge

nes

1200


(a)

57

1919

19 19

19



283

188151

132

94

(b)








rece

ptor

casc

ade



minus026minus132

DA

P12

signa

ling

Cos

timul

atio

n by

the C

D28

fam

ily

TCR

signa

ling

Dow

nstre

am T

CR si

gnal

ing

MH

C cla

ss II

antig

en p

rese

ntat

ion

Gen

erat

ion

of se

cond

mes

seng

er m

olec

ules

Tran

sloca

tion

of Z

AP-70

to im

mun

olog

ical

syna

pse

Phos

phor

ylat

ion

of C

D3

and

TCR

zeta

chai

ns

Inte

rfero

n ga

mm

a sig

nalin

g

PD-1

signa

ling
















hsa-let-7i-3p

hsa-miR-200a-3p

TP73

CXCL10

hsa-miR-34a-5p

IRAK2

hsa-miR-663aIL18RAP

CCL19

hsa-miR-150-3p

BMP6

IL6

CXCL11CD180

CCR7

hsa-miR-378a-5p

FOS

FCN2

hsa-miR-125a-5p

hsa-miR-132-3pPDPN


hsa-miR-155-5p

hsa-miR-9-3p PRDX2

ATRN

FN1

SPP1HMOX1

RELAhsa-miR-7-5p

hsa-let-7i-5p

LYZ

CLEC7AVSIG4

hsa-miR-10a-5p

hsa-miR-940

hsa-miR-362-5p

hsa-miR-532-5pCD55

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-103a-3p

CXCL1

CIITA

TLR4

TLR7

hsa-miR-629-5p

hsa-miR-107

MEFV

hsa-miR-376a-3p

hsa-miR-376c-3p

IL17F

IL1A


hsa-miR-185-5p

CCL7

hsa-miR-135a-3p

SIGLEC1 TLR5

LIPA AIF1

CFP


hsa-miR-1225-5p

C1QCF2R

C5

C1QB

hsa-miR-378a-3p


GIMAP5

DDAH2 HMOX1

SOX4PRDX1

hsa-miR-505-5p

hsa-miR-223-3p

SNCA hsa-miR-221-5p

hsa-miR-192-5p

UNC13B

PTPRFRNF7

TXNIPAPAF1

RUNX3

MGMTPTENNOTCH2IFNG

MPOETS1

NRG1

PRDX2

ANXA4

TNFSF13

NEFL GPX1NAIP


STK17ATLR4


hsa-miR-135a-3p

hsa-miR-99b-5p

IL12A

ITGA1CHD8

RAG1

UACASH3RF1

IL1A

HGF

KCNMA1

HTATIP2

SMOCAMK1D

IL6NOL3

AVEN

APOE

hsa-miR-663a

hsa-miR-200a-3p



TAF9B

LTB

RASGRF2

LGALS1

TRIO

MEF2CBCL6

NQO1

ERBB3

AIFM3

TRAF1

FURIN

NR4A2

SERPINB2

FGD2

PIM2

NR4A1

TNFSF14

TIMP3

CEBPG

FGD4CARD6

RAB27A

ARHGEF3NF1

PPT1

F2RRELA

VAV2

SMAD6

ESR1

BMF ITSN1

MUC2

FOXO3

hsa-miR-132-3p

hsa-miR-423-3p

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-378a-3p

hsa-miR-629-5p

SLC25A4

hsa-let-7e-5p

MAP3K5

hsa-miR-181a-3p

TP73hsa-let-7i-3p


hsa-miR-940

hsa-miR-7-1-3p

hsa-miR-625-5p


hsa-miR-342-5p

hsa-miR-7-5p

hsa-miR-155-5p

hsa-miR-29b-1-5p

hsa-miR-10a-5p


VSIG4

hsa-a-423-3p

SMO

hsa-let-7i-5p

hsa-miR-629-3p

hsa-let-7e-5p

VEGFB

NCK2

hsa-miR-505-5pHMOX1

IL12RB2 IL6CEBPA

CD86

CYP27B1

hsa-miR-7-5p

RARRES1

hsa-miR-940

hsa-miR-629-5p

hsa-miR-146b-5p

KLF11

RAC2

APOEPDCD1LG2

IFI30

F2R

hsa-miR-342-5p

hsa-miR-625-5pIL1A

SYKKLF4

HHEX

BCL6

AIF1

PDGFRB

IL12A

IFNGCTTNBP2

GPNMB

EREG

HES1

ETS1

hsa-miR-378a-5p

hsa-miR-155-5p

hsa-miR-378a-3p

NKX3-1

PPARG

CD33

NOTCH4

hsa-miR-221-5p

hsa-miR-135a-3p

ERBB3

CXCL10

COMTTNFRS13B

hsa-miR-34a-5p

hsa-miR-376c-3p

TNFSF13

hsa-miR-1225-5p

GPX1

hsa-miR-132-3p


KLF5FIGF

SOX2

KIFAP3 DLG3

hsa-miR-532-5p

hsa-miR-192-5p

hsa-miR-193b-3pPLAU

PTGS1PTEN

PDGFBIRS1

hsa-miR-10a-5p

SOX4NDN

hsa-miR-223-3p

hsa-miR-29b-1-5p

NOTCH2

hsa-miR-7-1-3pTXNIP

hsa-miR-107

CD9

NF1

NRG1

SLAMF1

RUNX3

PTPRF

MUC2

hsa-miR-125a-5p

hsa-miR-103a-3p

CHRNA10

RELA

PDGFCIL13RA1

hsa-miR-9-3p


GRN

hsa-miR-9-5p

hsa-miR-362-5pTIMP2





0




Expression value

Regu

latio

n of

cell

prol

ifera

tion

Regu

latio

n of

pro

gram

med

cell

deat

hIm

mun

ein

flam

mat

ory

resp

onse

hsa-miR-155-5pIFNG


hsa-miR-150-3p

hsa-miR-150-3p

hsa-miR-9-3p


hsa-miR-135a-3p

hsa-miR-378a-5p

hsa-miR-135a-3p

IL17F

hsa-miR-155-5p

hsa-miR-9-3p

CXCL5

IL1A

hsa-miR-7-5pRELA





hsa-miR-629-5p

hsa-miR-34a-5p

hsa-miR-185-5p

hsa-miR-223-3p

hsa-miR-107

hsa-miR-185-5pPTEN


hsa-miR-29b-1-5p


hsa-miR-7-5p

hsa-miR-9-3p

hsa-miR-7-1-3p

hsa-miR-29b-1-5p

hsa-let-7e-5p

hsa-miR-505-5pAPAF1


hsa-miR-221-5p

hsa-miR-376a-3p

hsa-miR-9-3pTP53BP1

hsa-miR-9-3p

hsa-miR-29b-1-5p

GADD45A


hsa-miR-9-3p

hsa-miR-378a-3p

hsa-miR-378a-3p

hsa-miR-155-5p

hsa-miR-1225-5p


hsa-miR-107GPNMB


PDCD4

hsa-miR-200a-3p

hsa-miR-135a-3p

hsa-miR-532-5p

hsa-miR-629-5p

hsa-miR-362-5p

minus51 0 75

hsa-miR-378a-5p


hsa-let-7i-5p



0

05

1

15

2

25

3

35

4

45

miR-9-5p miR-155-5p

MMG

Relat

ive e

xpre

ssio

n

lowastlowastlowast

lowastlowast

1g

0

02

04

06

08

1

12

miR-378a miR-150-3p

Relat

ive e

xpre

ssio

n


MMG1g

(a)

0

5

10

15

20

25

IFNG IL17F BCL6

80

120

140

Relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

MMG1g

0

02

04

06

08

1

12

TLR4 HLA-DRB1

Relat

ive e

xpre

ssio


MMG1g

(b)


119875 lt 005 t-test)


4 Discussion




25

20

15

10

5

0

lowast

24 48 72


Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)











0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










0

N where P0




















hsa-miR-378a-3p


(a)



(b)



3 Results





0

200

400

600

800

1000

Num

ber o

f diff

eren

tially

expr

esse

d ge

nes

1200


(a)

57

1919

19 19

19



283

188151

132

94

(b)








rece

ptor

casc

ade



minus026minus132

DA

P12

signa

ling

Cos

timul

atio

n by

the C

D28

fam

ily

TCR

signa

ling

Dow

nstre

am T

CR si

gnal

ing

MH

C cla

ss II

antig

en p

rese

ntat

ion

Gen

erat

ion

of se

cond

mes

seng

er m

olec

ules

Tran

sloca

tion

of Z

AP-70

to im

mun

olog

ical

syna

pse

Phos

phor

ylat

ion

of C

D3

and

TCR

zeta

chai

ns

Inte

rfero

n ga

mm

a sig

nalin

g

PD-1

signa

ling
















hsa-let-7i-3p

hsa-miR-200a-3p

TP73

CXCL10

hsa-miR-34a-5p

IRAK2

hsa-miR-663aIL18RAP

CCL19

hsa-miR-150-3p

BMP6

IL6

CXCL11CD180

CCR7

hsa-miR-378a-5p

FOS

FCN2

hsa-miR-125a-5p

hsa-miR-132-3pPDPN


hsa-miR-155-5p

hsa-miR-9-3p PRDX2

ATRN

FN1

SPP1HMOX1

RELAhsa-miR-7-5p

hsa-let-7i-5p

LYZ

CLEC7AVSIG4

hsa-miR-10a-5p

hsa-miR-940

hsa-miR-362-5p

hsa-miR-532-5pCD55

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-103a-3p

CXCL1

CIITA

TLR4

TLR7

hsa-miR-629-5p

hsa-miR-107

MEFV

hsa-miR-376a-3p

hsa-miR-376c-3p

IL17F

IL1A


hsa-miR-185-5p

CCL7

hsa-miR-135a-3p

SIGLEC1 TLR5

LIPA AIF1

CFP


hsa-miR-1225-5p

C1QCF2R

C5

C1QB

hsa-miR-378a-3p


GIMAP5

DDAH2 HMOX1

SOX4PRDX1

hsa-miR-505-5p

hsa-miR-223-3p

SNCA hsa-miR-221-5p

hsa-miR-192-5p

UNC13B

PTPRFRNF7

TXNIPAPAF1

RUNX3

MGMTPTENNOTCH2IFNG

MPOETS1

NRG1

PRDX2

ANXA4

TNFSF13

NEFL GPX1NAIP


STK17ATLR4


hsa-miR-135a-3p

hsa-miR-99b-5p

IL12A

ITGA1CHD8

RAG1

UACASH3RF1

IL1A

HGF

KCNMA1

HTATIP2

SMOCAMK1D

IL6NOL3

AVEN

APOE

hsa-miR-663a

hsa-miR-200a-3p



TAF9B

LTB

RASGRF2

LGALS1

TRIO

MEF2CBCL6

NQO1

ERBB3

AIFM3

TRAF1

FURIN

NR4A2

SERPINB2

FGD2

PIM2

NR4A1

TNFSF14

TIMP3

CEBPG

FGD4CARD6

RAB27A

ARHGEF3NF1

PPT1

F2RRELA

VAV2

SMAD6

ESR1

BMF ITSN1

MUC2

FOXO3

hsa-miR-132-3p

hsa-miR-423-3p

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-378a-3p

hsa-miR-629-5p

SLC25A4

hsa-let-7e-5p

MAP3K5

hsa-miR-181a-3p

TP73hsa-let-7i-3p


hsa-miR-940

hsa-miR-7-1-3p

hsa-miR-625-5p


hsa-miR-342-5p

hsa-miR-7-5p

hsa-miR-155-5p

hsa-miR-29b-1-5p

hsa-miR-10a-5p


VSIG4

hsa-a-423-3p

SMO

hsa-let-7i-5p

hsa-miR-629-3p

hsa-let-7e-5p

VEGFB

NCK2

hsa-miR-505-5pHMOX1

IL12RB2 IL6CEBPA

CD86

CYP27B1

hsa-miR-7-5p

RARRES1

hsa-miR-940

hsa-miR-629-5p

hsa-miR-146b-5p

KLF11

RAC2

APOEPDCD1LG2

IFI30

F2R

hsa-miR-342-5p

hsa-miR-625-5pIL1A

SYKKLF4

HHEX

BCL6

AIF1

PDGFRB

IL12A

IFNGCTTNBP2

GPNMB

EREG

HES1

ETS1

hsa-miR-378a-5p

hsa-miR-155-5p

hsa-miR-378a-3p

NKX3-1

PPARG

CD33

NOTCH4

hsa-miR-221-5p

hsa-miR-135a-3p

ERBB3

CXCL10

COMTTNFRS13B

hsa-miR-34a-5p

hsa-miR-376c-3p

TNFSF13

hsa-miR-1225-5p

GPX1

hsa-miR-132-3p


KLF5FIGF

SOX2

KIFAP3 DLG3

hsa-miR-532-5p

hsa-miR-192-5p

hsa-miR-193b-3pPLAU

PTGS1PTEN

PDGFBIRS1

hsa-miR-10a-5p

SOX4NDN

hsa-miR-223-3p

hsa-miR-29b-1-5p

NOTCH2

hsa-miR-7-1-3pTXNIP

hsa-miR-107

CD9

NF1

NRG1

SLAMF1

RUNX3

PTPRF

MUC2

hsa-miR-125a-5p

hsa-miR-103a-3p

CHRNA10

RELA

PDGFCIL13RA1

hsa-miR-9-3p


GRN

hsa-miR-9-5p

hsa-miR-362-5pTIMP2





0




Expression value

Regu

latio

n of

cell

prol

ifera

tion

Regu

latio

n of

pro

gram

med

cell

deat

hIm

mun

ein

flam

mat

ory

resp

onse

hsa-miR-155-5pIFNG


hsa-miR-150-3p

hsa-miR-150-3p

hsa-miR-9-3p


hsa-miR-135a-3p

hsa-miR-378a-5p

hsa-miR-135a-3p

IL17F

hsa-miR-155-5p

hsa-miR-9-3p

CXCL5

IL1A

hsa-miR-7-5pRELA





hsa-miR-629-5p

hsa-miR-34a-5p

hsa-miR-185-5p

hsa-miR-223-3p

hsa-miR-107

hsa-miR-185-5pPTEN


hsa-miR-29b-1-5p


hsa-miR-7-5p

hsa-miR-9-3p

hsa-miR-7-1-3p

hsa-miR-29b-1-5p

hsa-let-7e-5p

hsa-miR-505-5pAPAF1


hsa-miR-221-5p

hsa-miR-376a-3p

hsa-miR-9-3pTP53BP1

hsa-miR-9-3p

hsa-miR-29b-1-5p

GADD45A


hsa-miR-9-3p

hsa-miR-378a-3p

hsa-miR-378a-3p

hsa-miR-155-5p

hsa-miR-1225-5p


hsa-miR-107GPNMB


PDCD4

hsa-miR-200a-3p

hsa-miR-135a-3p

hsa-miR-532-5p

hsa-miR-629-5p

hsa-miR-362-5p

minus51 0 75

hsa-miR-378a-5p


hsa-let-7i-5p



0

05

1

15

2

25

3

35

4

45

miR-9-5p miR-155-5p

MMG

Relat

ive e

xpre

ssio

n

lowastlowastlowast

lowastlowast

1g

0

02

04

06

08

1

12

miR-378a miR-150-3p

Relat

ive e

xpre

ssio

n


MMG1g

(a)

0

5

10

15

20

25

IFNG IL17F BCL6

80

120

140

Relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

MMG1g

0

02

04

06

08

1

12

TLR4 HLA-DRB1

Relat

ive e

xpre

ssio


MMG1g

(b)


119875 lt 005 t-test)


4 Discussion




25

20

15

10

5

0

lowast

24 48 72


Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)











0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















hsa-miR-378a-3p


(a)



(b)



3 Results





0

200

400

600

800

1000

Num

ber o

f diff

eren

tially

expr

esse

d ge

nes

1200


(a)

57

1919

19 19

19



283

188151

132

94

(b)








rece

ptor

casc

ade



minus026minus132

DA

P12

signa

ling

Cos

timul

atio

n by

the C

D28

fam

ily

TCR

signa

ling

Dow

nstre

am T

CR si

gnal

ing

MH

C cla

ss II

antig

en p

rese

ntat

ion

Gen

erat

ion

of se

cond

mes

seng

er m

olec

ules

Tran

sloca

tion

of Z

AP-70

to im

mun

olog

ical

syna

pse

Phos

phor

ylat

ion

of C

D3

and

TCR

zeta

chai

ns

Inte

rfero

n ga

mm

a sig

nalin

g

PD-1

signa

ling
















hsa-let-7i-3p

hsa-miR-200a-3p

TP73

CXCL10

hsa-miR-34a-5p

IRAK2

hsa-miR-663aIL18RAP

CCL19

hsa-miR-150-3p

BMP6

IL6

CXCL11CD180

CCR7

hsa-miR-378a-5p

FOS

FCN2

hsa-miR-125a-5p

hsa-miR-132-3pPDPN


hsa-miR-155-5p

hsa-miR-9-3p PRDX2

ATRN

FN1

SPP1HMOX1

RELAhsa-miR-7-5p

hsa-let-7i-5p

LYZ

CLEC7AVSIG4

hsa-miR-10a-5p

hsa-miR-940

hsa-miR-362-5p

hsa-miR-532-5pCD55

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-103a-3p

CXCL1

CIITA

TLR4

TLR7

hsa-miR-629-5p

hsa-miR-107

MEFV

hsa-miR-376a-3p

hsa-miR-376c-3p

IL17F

IL1A


hsa-miR-185-5p

CCL7

hsa-miR-135a-3p

SIGLEC1 TLR5

LIPA AIF1

CFP


hsa-miR-1225-5p

C1QCF2R

C5

C1QB

hsa-miR-378a-3p


GIMAP5

DDAH2 HMOX1

SOX4PRDX1

hsa-miR-505-5p

hsa-miR-223-3p

SNCA hsa-miR-221-5p

hsa-miR-192-5p

UNC13B

PTPRFRNF7

TXNIPAPAF1

RUNX3

MGMTPTENNOTCH2IFNG

MPOETS1

NRG1

PRDX2

ANXA4

TNFSF13

NEFL GPX1NAIP


STK17ATLR4


hsa-miR-135a-3p

hsa-miR-99b-5p

IL12A

ITGA1CHD8

RAG1

UACASH3RF1

IL1A

HGF

KCNMA1

HTATIP2

SMOCAMK1D

IL6NOL3

AVEN

APOE

hsa-miR-663a

hsa-miR-200a-3p



TAF9B

LTB

RASGRF2

LGALS1

TRIO

MEF2CBCL6

NQO1

ERBB3

AIFM3

TRAF1

FURIN

NR4A2

SERPINB2

FGD2

PIM2

NR4A1

TNFSF14

TIMP3

CEBPG

FGD4CARD6

RAB27A

ARHGEF3NF1

PPT1

F2RRELA

VAV2

SMAD6

ESR1

BMF ITSN1

MUC2

FOXO3

hsa-miR-132-3p

hsa-miR-423-3p

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-378a-3p

hsa-miR-629-5p

SLC25A4

hsa-let-7e-5p

MAP3K5

hsa-miR-181a-3p

TP73hsa-let-7i-3p


hsa-miR-940

hsa-miR-7-1-3p

hsa-miR-625-5p


hsa-miR-342-5p

hsa-miR-7-5p

hsa-miR-155-5p

hsa-miR-29b-1-5p

hsa-miR-10a-5p


VSIG4

hsa-a-423-3p

SMO

hsa-let-7i-5p

hsa-miR-629-3p

hsa-let-7e-5p

VEGFB

NCK2

hsa-miR-505-5pHMOX1

IL12RB2 IL6CEBPA

CD86

CYP27B1

hsa-miR-7-5p

RARRES1

hsa-miR-940

hsa-miR-629-5p

hsa-miR-146b-5p

KLF11

RAC2

APOEPDCD1LG2

IFI30

F2R

hsa-miR-342-5p

hsa-miR-625-5pIL1A

SYKKLF4

HHEX

BCL6

AIF1

PDGFRB

IL12A

IFNGCTTNBP2

GPNMB

EREG

HES1

ETS1

hsa-miR-378a-5p

hsa-miR-155-5p

hsa-miR-378a-3p

NKX3-1

PPARG

CD33

NOTCH4

hsa-miR-221-5p

hsa-miR-135a-3p

ERBB3

CXCL10

COMTTNFRS13B

hsa-miR-34a-5p

hsa-miR-376c-3p

TNFSF13

hsa-miR-1225-5p

GPX1

hsa-miR-132-3p


KLF5FIGF

SOX2

KIFAP3 DLG3

hsa-miR-532-5p

hsa-miR-192-5p

hsa-miR-193b-3pPLAU

PTGS1PTEN

PDGFBIRS1

hsa-miR-10a-5p

SOX4NDN

hsa-miR-223-3p

hsa-miR-29b-1-5p

NOTCH2

hsa-miR-7-1-3pTXNIP

hsa-miR-107

CD9

NF1

NRG1

SLAMF1

RUNX3

PTPRF

MUC2

hsa-miR-125a-5p

hsa-miR-103a-3p

CHRNA10

RELA

PDGFCIL13RA1

hsa-miR-9-3p


GRN

hsa-miR-9-5p

hsa-miR-362-5pTIMP2





0




Expression value

Regu

latio

n of

cell

prol

ifera

tion

Regu

latio

n of

pro

gram

med

cell

deat

hIm

mun

ein

flam

mat

ory

resp

onse

hsa-miR-155-5pIFNG


hsa-miR-150-3p

hsa-miR-150-3p

hsa-miR-9-3p


hsa-miR-135a-3p

hsa-miR-378a-5p

hsa-miR-135a-3p

IL17F

hsa-miR-155-5p

hsa-miR-9-3p

CXCL5

IL1A

hsa-miR-7-5pRELA





hsa-miR-629-5p

hsa-miR-34a-5p

hsa-miR-185-5p

hsa-miR-223-3p

hsa-miR-107

hsa-miR-185-5pPTEN


hsa-miR-29b-1-5p


hsa-miR-7-5p

hsa-miR-9-3p

hsa-miR-7-1-3p

hsa-miR-29b-1-5p

hsa-let-7e-5p

hsa-miR-505-5pAPAF1


hsa-miR-221-5p

hsa-miR-376a-3p

hsa-miR-9-3pTP53BP1

hsa-miR-9-3p

hsa-miR-29b-1-5p

GADD45A


hsa-miR-9-3p

hsa-miR-378a-3p

hsa-miR-378a-3p

hsa-miR-155-5p

hsa-miR-1225-5p


hsa-miR-107GPNMB


PDCD4

hsa-miR-200a-3p

hsa-miR-135a-3p

hsa-miR-532-5p

hsa-miR-629-5p

hsa-miR-362-5p

minus51 0 75

hsa-miR-378a-5p


hsa-let-7i-5p



0

05

1

15

2

25

3

35

4

45

miR-9-5p miR-155-5p

MMG

Relat

ive e

xpre

ssio

n

lowastlowastlowast

lowastlowast

1g

0

02

04

06

08

1

12

miR-378a miR-150-3p

Relat

ive e

xpre

ssio

n


MMG1g

(a)

0

5

10

15

20

25

IFNG IL17F BCL6

80

120

140

Relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

MMG1g

0

02

04

06

08

1

12

TLR4 HLA-DRB1

Relat

ive e

xpre

ssio


MMG1g

(b)


119875 lt 005 t-test)


4 Discussion




25

20

15

10

5

0

lowast

24 48 72


Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)











0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

200

400

600

800

1000

Num

ber o

f diff

eren

tially

expr

esse

d ge

nes

1200


(a)

57

1919

19 19

19



283

188151

132

94

(b)








rece

ptor

casc

ade



minus026minus132

DA

P12

signa

ling

Cos

timul

atio

n by

the C

D28

fam

ily

TCR

signa

ling

Dow

nstre

am T

CR si

gnal

ing

MH

C cla

ss II

antig

en p

rese

ntat

ion

Gen

erat

ion

of se

cond

mes

seng

er m

olec

ules

Tran

sloca

tion

of Z

AP-70

to im

mun

olog

ical

syna

pse

Phos

phor

ylat

ion

of C

D3

and

TCR

zeta

chai

ns

Inte

rfero

n ga

mm

a sig

nalin

g

PD-1

signa

ling
















hsa-let-7i-3p

hsa-miR-200a-3p

TP73

CXCL10

hsa-miR-34a-5p

IRAK2

hsa-miR-663aIL18RAP

CCL19

hsa-miR-150-3p

BMP6

IL6

CXCL11CD180

CCR7

hsa-miR-378a-5p

FOS

FCN2

hsa-miR-125a-5p

hsa-miR-132-3pPDPN


hsa-miR-155-5p

hsa-miR-9-3p PRDX2

ATRN

FN1

SPP1HMOX1

RELAhsa-miR-7-5p

hsa-let-7i-5p

LYZ

CLEC7AVSIG4

hsa-miR-10a-5p

hsa-miR-940

hsa-miR-362-5p

hsa-miR-532-5pCD55

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-103a-3p

CXCL1

CIITA

TLR4

TLR7

hsa-miR-629-5p

hsa-miR-107

MEFV

hsa-miR-376a-3p

hsa-miR-376c-3p

IL17F

IL1A


hsa-miR-185-5p

CCL7

hsa-miR-135a-3p

SIGLEC1 TLR5

LIPA AIF1

CFP


hsa-miR-1225-5p

C1QCF2R

C5

C1QB

hsa-miR-378a-3p


GIMAP5

DDAH2 HMOX1

SOX4PRDX1

hsa-miR-505-5p

hsa-miR-223-3p

SNCA hsa-miR-221-5p

hsa-miR-192-5p

UNC13B

PTPRFRNF7

TXNIPAPAF1

RUNX3

MGMTPTENNOTCH2IFNG

MPOETS1

NRG1

PRDX2

ANXA4

TNFSF13

NEFL GPX1NAIP


STK17ATLR4


hsa-miR-135a-3p

hsa-miR-99b-5p

IL12A

ITGA1CHD8

RAG1

UACASH3RF1

IL1A

HGF

KCNMA1

HTATIP2

SMOCAMK1D

IL6NOL3

AVEN

APOE

hsa-miR-663a

hsa-miR-200a-3p



TAF9B

LTB

RASGRF2

LGALS1

TRIO

MEF2CBCL6

NQO1

ERBB3

AIFM3

TRAF1

FURIN

NR4A2

SERPINB2

FGD2

PIM2

NR4A1

TNFSF14

TIMP3

CEBPG

FGD4CARD6

RAB27A

ARHGEF3NF1

PPT1

F2RRELA

VAV2

SMAD6

ESR1

BMF ITSN1

MUC2

FOXO3

hsa-miR-132-3p

hsa-miR-423-3p

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-378a-3p

hsa-miR-629-5p

SLC25A4

hsa-let-7e-5p

MAP3K5

hsa-miR-181a-3p

TP73hsa-let-7i-3p


hsa-miR-940

hsa-miR-7-1-3p

hsa-miR-625-5p


hsa-miR-342-5p

hsa-miR-7-5p

hsa-miR-155-5p

hsa-miR-29b-1-5p

hsa-miR-10a-5p


VSIG4

hsa-a-423-3p

SMO

hsa-let-7i-5p

hsa-miR-629-3p

hsa-let-7e-5p

VEGFB

NCK2

hsa-miR-505-5pHMOX1

IL12RB2 IL6CEBPA

CD86

CYP27B1

hsa-miR-7-5p

RARRES1

hsa-miR-940

hsa-miR-629-5p

hsa-miR-146b-5p

KLF11

RAC2

APOEPDCD1LG2

IFI30

F2R

hsa-miR-342-5p

hsa-miR-625-5pIL1A

SYKKLF4

HHEX

BCL6

AIF1

PDGFRB

IL12A

IFNGCTTNBP2

GPNMB

EREG

HES1

ETS1

hsa-miR-378a-5p

hsa-miR-155-5p

hsa-miR-378a-3p

NKX3-1

PPARG

CD33

NOTCH4

hsa-miR-221-5p

hsa-miR-135a-3p

ERBB3

CXCL10

COMTTNFRS13B

hsa-miR-34a-5p

hsa-miR-376c-3p

TNFSF13

hsa-miR-1225-5p

GPX1

hsa-miR-132-3p


KLF5FIGF

SOX2

KIFAP3 DLG3

hsa-miR-532-5p

hsa-miR-192-5p

hsa-miR-193b-3pPLAU

PTGS1PTEN

PDGFBIRS1

hsa-miR-10a-5p

SOX4NDN

hsa-miR-223-3p

hsa-miR-29b-1-5p

NOTCH2

hsa-miR-7-1-3pTXNIP

hsa-miR-107

CD9

NF1

NRG1

SLAMF1

RUNX3

PTPRF

MUC2

hsa-miR-125a-5p

hsa-miR-103a-3p

CHRNA10

RELA

PDGFCIL13RA1

hsa-miR-9-3p


GRN

hsa-miR-9-5p

hsa-miR-362-5pTIMP2





0




Expression value

Regu

latio

n of

cell

prol

ifera

tion

Regu

latio

n of

pro

gram

med

cell

deat

hIm

mun

ein

flam

mat

ory

resp

onse

hsa-miR-155-5pIFNG


hsa-miR-150-3p

hsa-miR-150-3p

hsa-miR-9-3p


hsa-miR-135a-3p

hsa-miR-378a-5p

hsa-miR-135a-3p

IL17F

hsa-miR-155-5p

hsa-miR-9-3p

CXCL5

IL1A

hsa-miR-7-5pRELA





hsa-miR-629-5p

hsa-miR-34a-5p

hsa-miR-185-5p

hsa-miR-223-3p

hsa-miR-107

hsa-miR-185-5pPTEN


hsa-miR-29b-1-5p


hsa-miR-7-5p

hsa-miR-9-3p

hsa-miR-7-1-3p

hsa-miR-29b-1-5p

hsa-let-7e-5p

hsa-miR-505-5pAPAF1


hsa-miR-221-5p

hsa-miR-376a-3p

hsa-miR-9-3pTP53BP1

hsa-miR-9-3p

hsa-miR-29b-1-5p

GADD45A


hsa-miR-9-3p

hsa-miR-378a-3p

hsa-miR-378a-3p

hsa-miR-155-5p

hsa-miR-1225-5p


hsa-miR-107GPNMB


PDCD4

hsa-miR-200a-3p

hsa-miR-135a-3p

hsa-miR-532-5p

hsa-miR-629-5p

hsa-miR-362-5p

minus51 0 75

hsa-miR-378a-5p


hsa-let-7i-5p



0

05

1

15

2

25

3

35

4

45

miR-9-5p miR-155-5p

MMG

Relat

ive e

xpre

ssio

n

lowastlowastlowast

lowastlowast

1g

0

02

04

06

08

1

12

miR-378a miR-150-3p

Relat

ive e

xpre

ssio

n


MMG1g

(a)

0

5

10

15

20

25

IFNG IL17F BCL6

80

120

140

Relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

MMG1g

0

02

04

06

08

1

12

TLR4 HLA-DRB1

Relat

ive e

xpre

ssio


MMG1g

(b)


119875 lt 005 t-test)


4 Discussion




25

20

15

10

5

0

lowast

24 48 72


Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)











0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



rece

ptor

casc

ade



minus026minus132

DA

P12

signa

ling

Cos

timul

atio

n by

the C

D28

fam

ily

TCR

signa

ling

Dow

nstre

am T

CR si

gnal

ing

MH

C cla

ss II

antig

en p

rese

ntat

ion

Gen

erat

ion

of se

cond

mes

seng

er m

olec

ules

Tran

sloca

tion

of Z

AP-70

to im

mun

olog

ical

syna

pse

Phos

phor

ylat

ion

of C

D3

and

TCR

zeta

chai

ns

Inte

rfero

n ga

mm

a sig

nalin

g

PD-1

signa

ling
















hsa-let-7i-3p

hsa-miR-200a-3p

TP73

CXCL10

hsa-miR-34a-5p

IRAK2

hsa-miR-663aIL18RAP

CCL19

hsa-miR-150-3p

BMP6

IL6

CXCL11CD180

CCR7

hsa-miR-378a-5p

FOS

FCN2

hsa-miR-125a-5p

hsa-miR-132-3pPDPN


hsa-miR-155-5p

hsa-miR-9-3p PRDX2

ATRN

FN1

SPP1HMOX1

RELAhsa-miR-7-5p

hsa-let-7i-5p

LYZ

CLEC7AVSIG4

hsa-miR-10a-5p

hsa-miR-940

hsa-miR-362-5p

hsa-miR-532-5pCD55

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-103a-3p

CXCL1

CIITA

TLR4

TLR7

hsa-miR-629-5p

hsa-miR-107

MEFV

hsa-miR-376a-3p

hsa-miR-376c-3p

IL17F

IL1A


hsa-miR-185-5p

CCL7

hsa-miR-135a-3p

SIGLEC1 TLR5

LIPA AIF1

CFP


hsa-miR-1225-5p

C1QCF2R

C5

C1QB

hsa-miR-378a-3p


GIMAP5

DDAH2 HMOX1

SOX4PRDX1

hsa-miR-505-5p

hsa-miR-223-3p

SNCA hsa-miR-221-5p

hsa-miR-192-5p

UNC13B

PTPRFRNF7

TXNIPAPAF1

RUNX3

MGMTPTENNOTCH2IFNG

MPOETS1

NRG1

PRDX2

ANXA4

TNFSF13

NEFL GPX1NAIP


STK17ATLR4


hsa-miR-135a-3p

hsa-miR-99b-5p

IL12A

ITGA1CHD8

RAG1

UACASH3RF1

IL1A

HGF

KCNMA1

HTATIP2

SMOCAMK1D

IL6NOL3

AVEN

APOE

hsa-miR-663a

hsa-miR-200a-3p



TAF9B

LTB

RASGRF2

LGALS1

TRIO

MEF2CBCL6

NQO1

ERBB3

AIFM3

TRAF1

FURIN

NR4A2

SERPINB2

FGD2

PIM2

NR4A1

TNFSF14

TIMP3

CEBPG

FGD4CARD6

RAB27A

ARHGEF3NF1

PPT1

F2RRELA

VAV2

SMAD6

ESR1

BMF ITSN1

MUC2

FOXO3

hsa-miR-132-3p

hsa-miR-423-3p

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-378a-3p

hsa-miR-629-5p

SLC25A4

hsa-let-7e-5p

MAP3K5

hsa-miR-181a-3p

TP73hsa-let-7i-3p


hsa-miR-940

hsa-miR-7-1-3p

hsa-miR-625-5p


hsa-miR-342-5p

hsa-miR-7-5p

hsa-miR-155-5p

hsa-miR-29b-1-5p

hsa-miR-10a-5p


VSIG4

hsa-a-423-3p

SMO

hsa-let-7i-5p

hsa-miR-629-3p

hsa-let-7e-5p

VEGFB

NCK2

hsa-miR-505-5pHMOX1

IL12RB2 IL6CEBPA

CD86

CYP27B1

hsa-miR-7-5p

RARRES1

hsa-miR-940

hsa-miR-629-5p

hsa-miR-146b-5p

KLF11

RAC2

APOEPDCD1LG2

IFI30

F2R

hsa-miR-342-5p

hsa-miR-625-5pIL1A

SYKKLF4

HHEX

BCL6

AIF1

PDGFRB

IL12A

IFNGCTTNBP2

GPNMB

EREG

HES1

ETS1

hsa-miR-378a-5p

hsa-miR-155-5p

hsa-miR-378a-3p

NKX3-1

PPARG

CD33

NOTCH4

hsa-miR-221-5p

hsa-miR-135a-3p

ERBB3

CXCL10

COMTTNFRS13B

hsa-miR-34a-5p

hsa-miR-376c-3p

TNFSF13

hsa-miR-1225-5p

GPX1

hsa-miR-132-3p


KLF5FIGF

SOX2

KIFAP3 DLG3

hsa-miR-532-5p

hsa-miR-192-5p

hsa-miR-193b-3pPLAU

PTGS1PTEN

PDGFBIRS1

hsa-miR-10a-5p

SOX4NDN

hsa-miR-223-3p

hsa-miR-29b-1-5p

NOTCH2

hsa-miR-7-1-3pTXNIP

hsa-miR-107

CD9

NF1

NRG1

SLAMF1

RUNX3

PTPRF

MUC2

hsa-miR-125a-5p

hsa-miR-103a-3p

CHRNA10

RELA

PDGFCIL13RA1

hsa-miR-9-3p


GRN

hsa-miR-9-5p

hsa-miR-362-5pTIMP2





0




Expression value

Regu

latio

n of

cell

prol

ifera

tion

Regu

latio

n of

pro

gram

med

cell

deat

hIm

mun

ein

flam

mat

ory

resp

onse

hsa-miR-155-5pIFNG


hsa-miR-150-3p

hsa-miR-150-3p

hsa-miR-9-3p


hsa-miR-135a-3p

hsa-miR-378a-5p

hsa-miR-135a-3p

IL17F

hsa-miR-155-5p

hsa-miR-9-3p

CXCL5

IL1A

hsa-miR-7-5pRELA





hsa-miR-629-5p

hsa-miR-34a-5p

hsa-miR-185-5p

hsa-miR-223-3p

hsa-miR-107

hsa-miR-185-5pPTEN


hsa-miR-29b-1-5p


hsa-miR-7-5p

hsa-miR-9-3p

hsa-miR-7-1-3p

hsa-miR-29b-1-5p

hsa-let-7e-5p

hsa-miR-505-5pAPAF1


hsa-miR-221-5p

hsa-miR-376a-3p

hsa-miR-9-3pTP53BP1

hsa-miR-9-3p

hsa-miR-29b-1-5p

GADD45A


hsa-miR-9-3p

hsa-miR-378a-3p

hsa-miR-378a-3p

hsa-miR-155-5p

hsa-miR-1225-5p


hsa-miR-107GPNMB


PDCD4

hsa-miR-200a-3p

hsa-miR-135a-3p

hsa-miR-532-5p

hsa-miR-629-5p

hsa-miR-362-5p

minus51 0 75

hsa-miR-378a-5p


hsa-let-7i-5p



0

05

1

15

2

25

3

35

4

45

miR-9-5p miR-155-5p

MMG

Relat

ive e

xpre

ssio

n

lowastlowastlowast

lowastlowast

1g

0

02

04

06

08

1

12

miR-378a miR-150-3p

Relat

ive e

xpre

ssio

n


MMG1g

(a)

0

5

10

15

20

25

IFNG IL17F BCL6

80

120

140

Relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

MMG1g

0

02

04

06

08

1

12

TLR4 HLA-DRB1

Relat

ive e

xpre

ssio


MMG1g

(b)


119875 lt 005 t-test)


4 Discussion




25

20

15

10

5

0

lowast

24 48 72


Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)











0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









hsa-let-7i-3p

hsa-miR-200a-3p

TP73

CXCL10

hsa-miR-34a-5p

IRAK2

hsa-miR-663aIL18RAP

CCL19

hsa-miR-150-3p

BMP6

IL6

CXCL11CD180

CCR7

hsa-miR-378a-5p

FOS

FCN2

hsa-miR-125a-5p

hsa-miR-132-3pPDPN


hsa-miR-155-5p

hsa-miR-9-3p PRDX2

ATRN

FN1

SPP1HMOX1

RELAhsa-miR-7-5p

hsa-let-7i-5p

LYZ

CLEC7AVSIG4

hsa-miR-10a-5p

hsa-miR-940

hsa-miR-362-5p

hsa-miR-532-5pCD55

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-103a-3p

CXCL1

CIITA

TLR4

TLR7

hsa-miR-629-5p

hsa-miR-107

MEFV

hsa-miR-376a-3p

hsa-miR-376c-3p

IL17F

IL1A


hsa-miR-185-5p

CCL7

hsa-miR-135a-3p

SIGLEC1 TLR5

LIPA AIF1

CFP


hsa-miR-1225-5p

C1QCF2R

C5

C1QB

hsa-miR-378a-3p


GIMAP5

DDAH2 HMOX1

SOX4PRDX1

hsa-miR-505-5p

hsa-miR-223-3p

SNCA hsa-miR-221-5p

hsa-miR-192-5p

UNC13B

PTPRFRNF7

TXNIPAPAF1

RUNX3

MGMTPTENNOTCH2IFNG

MPOETS1

NRG1

PRDX2

ANXA4

TNFSF13

NEFL GPX1NAIP


STK17ATLR4


hsa-miR-135a-3p

hsa-miR-99b-5p

IL12A

ITGA1CHD8

RAG1

UACASH3RF1

IL1A

HGF

KCNMA1

HTATIP2

SMOCAMK1D

IL6NOL3

AVEN

APOE

hsa-miR-663a

hsa-miR-200a-3p



TAF9B

LTB

RASGRF2

LGALS1

TRIO

MEF2CBCL6

NQO1

ERBB3

AIFM3

TRAF1

FURIN

NR4A2

SERPINB2

FGD2

PIM2

NR4A1

TNFSF14

TIMP3

CEBPG

FGD4CARD6

RAB27A

ARHGEF3NF1

PPT1

F2RRELA

VAV2

SMAD6

ESR1

BMF ITSN1

MUC2

FOXO3

hsa-miR-132-3p

hsa-miR-423-3p

hsa-miR-9-5p

hsa-miR-146b-5p


hsa-miR-378a-3p

hsa-miR-629-5p

SLC25A4

hsa-let-7e-5p

MAP3K5

hsa-miR-181a-3p

TP73hsa-let-7i-3p


hsa-miR-940

hsa-miR-7-1-3p

hsa-miR-625-5p


hsa-miR-342-5p

hsa-miR-7-5p

hsa-miR-155-5p

hsa-miR-29b-1-5p

hsa-miR-10a-5p


VSIG4

hsa-a-423-3p

SMO

hsa-let-7i-5p

hsa-miR-629-3p

hsa-let-7e-5p

VEGFB

NCK2

hsa-miR-505-5pHMOX1

IL12RB2 IL6CEBPA

CD86

CYP27B1

hsa-miR-7-5p

RARRES1

hsa-miR-940

hsa-miR-629-5p

hsa-miR-146b-5p

KLF11

RAC2

APOEPDCD1LG2

IFI30

F2R

hsa-miR-342-5p

hsa-miR-625-5pIL1A

SYKKLF4

HHEX

BCL6

AIF1

PDGFRB

IL12A

IFNGCTTNBP2

GPNMB

EREG

HES1

ETS1

hsa-miR-378a-5p

hsa-miR-155-5p

hsa-miR-378a-3p

NKX3-1

PPARG

CD33

NOTCH4

hsa-miR-221-5p

hsa-miR-135a-3p

ERBB3

CXCL10

COMTTNFRS13B

hsa-miR-34a-5p

hsa-miR-376c-3p

TNFSF13

hsa-miR-1225-5p

GPX1

hsa-miR-132-3p


KLF5FIGF

SOX2

KIFAP3 DLG3

hsa-miR-532-5p

hsa-miR-192-5p

hsa-miR-193b-3pPLAU

PTGS1PTEN

PDGFBIRS1

hsa-miR-10a-5p

SOX4NDN

hsa-miR-223-3p

hsa-miR-29b-1-5p

NOTCH2

hsa-miR-7-1-3pTXNIP

hsa-miR-107

CD9

NF1

NRG1

SLAMF1

RUNX3

PTPRF

MUC2

hsa-miR-125a-5p

hsa-miR-103a-3p

CHRNA10

RELA

PDGFCIL13RA1

hsa-miR-9-3p


GRN

hsa-miR-9-5p

hsa-miR-362-5pTIMP2





0




Expression value

Regu

latio

n of

cell

prol

ifera

tion

Regu

latio

n of

pro

gram

med

cell

deat

hIm

mun

ein

flam

mat

ory

resp

onse

hsa-miR-155-5pIFNG


hsa-miR-150-3p

hsa-miR-150-3p

hsa-miR-9-3p


hsa-miR-135a-3p

hsa-miR-378a-5p

hsa-miR-135a-3p

IL17F

hsa-miR-155-5p

hsa-miR-9-3p

CXCL5

IL1A

hsa-miR-7-5pRELA





hsa-miR-629-5p

hsa-miR-34a-5p

hsa-miR-185-5p

hsa-miR-223-3p

hsa-miR-107

hsa-miR-185-5pPTEN


hsa-miR-29b-1-5p


hsa-miR-7-5p

hsa-miR-9-3p

hsa-miR-7-1-3p

hsa-miR-29b-1-5p

hsa-let-7e-5p

hsa-miR-505-5pAPAF1


hsa-miR-221-5p

hsa-miR-376a-3p

hsa-miR-9-3pTP53BP1

hsa-miR-9-3p

hsa-miR-29b-1-5p

GADD45A


hsa-miR-9-3p

hsa-miR-378a-3p

hsa-miR-378a-3p

hsa-miR-155-5p

hsa-miR-1225-5p


hsa-miR-107GPNMB


PDCD4

hsa-miR-200a-3p

hsa-miR-135a-3p

hsa-miR-532-5p

hsa-miR-629-5p

hsa-miR-362-5p

minus51 0 75

hsa-miR-378a-5p


hsa-let-7i-5p



0

05

1

15

2

25

3

35

4

45

miR-9-5p miR-155-5p

MMG

Relat

ive e

xpre

ssio

n

lowastlowastlowast

lowastlowast

1g

0

02

04

06

08

1

12

miR-378a miR-150-3p

Relat

ive e

xpre

ssio

n


MMG1g

(a)

0

5

10

15

20

25

IFNG IL17F BCL6

80

120

140

Relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

MMG1g

0

02

04

06

08

1

12

TLR4 HLA-DRB1

Relat

ive e

xpre

ssio


MMG1g

(b)


119875 lt 005 t-test)


4 Discussion




25

20

15

10

5

0

lowast

24 48 72


Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)











0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Expression value

Regu

latio

n of

cell

prol

ifera

tion

Regu

latio

n of

pro

gram

med

cell

deat

hIm

mun

ein

flam

mat

ory

resp

onse

hsa-miR-155-5pIFNG


hsa-miR-150-3p

hsa-miR-150-3p

hsa-miR-9-3p


hsa-miR-135a-3p

hsa-miR-378a-5p

hsa-miR-135a-3p

IL17F

hsa-miR-155-5p

hsa-miR-9-3p

CXCL5

IL1A

hsa-miR-7-5pRELA





hsa-miR-629-5p

hsa-miR-34a-5p

hsa-miR-185-5p

hsa-miR-223-3p

hsa-miR-107

hsa-miR-185-5pPTEN


hsa-miR-29b-1-5p


hsa-miR-7-5p

hsa-miR-9-3p

hsa-miR-7-1-3p

hsa-miR-29b-1-5p

hsa-let-7e-5p

hsa-miR-505-5pAPAF1


hsa-miR-221-5p

hsa-miR-376a-3p

hsa-miR-9-3pTP53BP1

hsa-miR-9-3p

hsa-miR-29b-1-5p

GADD45A


hsa-miR-9-3p

hsa-miR-378a-3p

hsa-miR-378a-3p

hsa-miR-155-5p

hsa-miR-1225-5p


hsa-miR-107GPNMB


PDCD4

hsa-miR-200a-3p

hsa-miR-135a-3p

hsa-miR-532-5p

hsa-miR-629-5p

hsa-miR-362-5p

minus51 0 75

hsa-miR-378a-5p


hsa-let-7i-5p



0

05

1

15

2

25

3

35

4

45

miR-9-5p miR-155-5p

MMG

Relat

ive e

xpre

ssio

n

lowastlowastlowast

lowastlowast

1g

0

02

04

06

08

1

12

miR-378a miR-150-3p

Relat

ive e

xpre

ssio

n


MMG1g

(a)

0

5

10

15

20

25

IFNG IL17F BCL6

80

120

140

Relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

MMG1g

0

02

04

06

08

1

12

TLR4 HLA-DRB1

Relat

ive e

xpre

ssio


MMG1g

(b)


119875 lt 005 t-test)


4 Discussion




25

20

15

10

5

0

lowast

24 48 72


Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)











0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

05

1

15

2

25

3

35

4

45

miR-9-5p miR-155-5p

MMG

Relat

ive e

xpre

ssio

n

lowastlowastlowast

lowastlowast

1g

0

02

04

06

08

1

12

miR-378a miR-150-3p

Relat

ive e

xpre

ssio

n


MMG1g

(a)

0

5

10

15

20

25

IFNG IL17F BCL6

80

120

140

Relat

ive e

xpre

ssio

n

lowast

lowastlowastlowast

MMG1g

0

02

04

06

08

1

12

TLR4 HLA-DRB1

Relat

ive e

xpre

ssio


MMG1g

(b)


119875 lt 005 t-test)


4 Discussion




25

20

15

10

5

0

lowast

24 48 72


Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)











0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


25

20

15

10

5

0

lowast

24 48 72


Clon

ing e

fficie

ncy (

)

1gMMG

(a)

)

5

4

3

2

1

024 48


Apop

totic

inde

x (

1gMMG

lowast lowast

(b)

60

50

40

30

20

10

0

24 48

70


Casp

ase-3

activ

ation

(au

)

lowast

1gMMG

(c)











0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







0




5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusions






Acknowledgments


References













































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Integration Analysis of MicroRNA and mRNA...

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Transcript of Research Article Integration Analysis of MicroRNA and mRNA...