Research Article Circulating C19MC MicroRNAs in...

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 186041 12 pageshttpdxdoiorg1011552013186041

Research ArticleCirculating C19MC MicroRNAs in Preeclampsia GestationalHypertension and Fetal Growth Restriction

Ilona Hromadnikova1 Katerina Kotlabova1 Marketa Ondrackova1 Andrea Kestlerova2

Veronika Novotna2 Lucie Hympanova12 Jindrich Doucha3 and Ladislav Krofta2

1 Department of Molecular Biology and Cell Pathology Third Faculty of Medicine Charles UniversityRuska 87 100 00 Prague Czech Republic

2 Institute for the Care of the Mother and Child Third Faculty of Medicine Charles University Podolske Nabrezi 15736147 00 Prague Czech Republic

3 Clinic of Obstetrics and Gynecology Second Faculty of Medicine Charles University V Uvalu 84 150 06 Prague Czech Republic

Correspondence should be addressed to Ilona Hromadnikova ilonahromadnikovalf3cunicz

Received 22 July 2013 Revised 24 September 2013 Accepted 30 September 2013

Academic Editor Jyoti J Watters

Copyright copy 2013 Ilona Hromadnikova et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The objective of the study was to identify the profile of circulating C19MC microRNAs (miR-516-5p miR-517lowast miR-518b miR-520alowast miR-520h miR-525 and miR-526a) in patients with established preeclampsia (119899 = 63) fetal growth restriction (119899 = 27)and gestational hypertension (119899 = 23) We examined the correlation between plasmatic concentrations and expression levels ofmicroRNAs and the severity of the disease with respect to clinical signs requirements for the delivery and Doppler ultrasoundparameters Using absolute and relative quantification approaches increased extracellular C19MC microRNA levels (miR-516-5p119875 = 0037 119875 = 0009 miR-517lowast 119875 = 0033 119875 = 0043 miR-520alowast 119875 = 0001 119875 = 0009 miR-525 119875 = 0026 119875 = 001 miR-526a119875 = 003 119875 = 0035) were detected in patients with preeclampsia The association analysis pointed to no relationship betweenC19MC microRNA plasmatic concentrations and expression profile and identified risk factors for a poorer perinatal outcomeHowever the dependence between the levels of plasmatic C19MC microRNAs and the pulsatility index in the middle cerebralartery and the values of cerebroplacental ratio was demonstrated The study brought the interesting finding that the upregulationof miR-516-5p miR-517lowast miR-520alowast miR-525 and miR-526a is a characteristic phenomenon of established preeclampsia

1 Introduction

Normal pregnancy is associated with a systemic inflamma-tory response Many of the physiologic changes of normalpregnancy are part of an acute-phase reaction which isgenerated by an inflammatory response The placenta is theproximal cause of these problems [1] Since the placentais being continuously remodelled during normal placentaldevelopment extracellular nucleic acids of both fetal and pla-cental origin packed into either trophoblast-derived apop-totic bodies or shedding syncytiotrophoblast microparticlesmay be detected in maternal circulation during the course ofnormal gestation [2ndash6]

Circulating syncytiotrophoblast debris has been hypoth-esized to contribute to maternal inflammation and someof the features of the maternal syndrome [7] Signs of

maternal inflammation which appear to be present in normalpregnancies at term are exaggerated in preeclampsia (PE)and fetal growth restriction (FGR) and can account for theirclinical features [1 8]

Preeclampsia and fetal growth restriction (FGR) aremajor complications affecting about 2ndash10 of pregnanciesresponsible for maternal and perinatal morbidity and mor-tality [9 10] Preeclampsia usually develops after 20 weeksof gestation and is characterized by chronic or gestationalhypertension combined with proteinuria which results fromdefective placentation eliciting inadequate uteroplacentalblood perfusion and ischemia [8 11 12] The causes ofpreeclampsia and FGR remain unknown Trophoblasticdebris and the microparticles shed during normal preg-nancy are proinflammatory and this process is amplified inpreeclampsia [13] A hypoxic environment induces excessive

2 Mediators of Inflammation

trophoblast cell death and increased shedding of placentadebris into the maternal circulation as a result placentalinsufficiency related pregnancy complications are associatedwith abnormal levels of extracellular fetal DNA and mRNAtranscripts [5 14]

Recent evidence suggests that preeclampsia can be furthersubdivided into early PE (before 34 weeks of gestation)intermediate PE (between 34 and 37 weeks of gestation) andlate PE (after 37 weeks of gestation) [15 16] The conceptof early and late PE is modern and it is widely acceptedthat these two entities have different etiologies and should beregarded as different forms of the disease where early onsetsof PE and IUGR are considered to be placenta-mediateddiseases [17ndash19]

There has been a trend over the last 10 years to developnoninvasive methods utilizing quantification of cell-freenucleic acids inclusive of microRNAs in maternal circulation[6 20ndash39] The diagnostic potential of particular molec-ular biomarkers and their implementation in the currentpredictive and diagnostic algorithms for pregnancy relatedcomplications is subject of interest [6]

MicroRNAs belong to a family of small noncoding RNAsthat regulate gene expression at the posttranscriptional levelby degrading or blocking translation of messenger RNA(mRNA) targets [40 41]

Recent studies have shown that preeclampsia is associatedwith alterations in extracellular microRNA expression Usingreal-time PCR analysis Gunel et al [42] demonstratedthe upregulation of miR-210 and downregulation of miR-152 in patients with preeclampsia The application of nextgeneration sequencing technology revealed a broader pro-file of dysregulated circulating microRNAs in preeclampsiaCompared to controls 15 microRNAs were found to beupregulated (miR-521 miR-520h miR-517c miR-519d miR-520g miR-517b miR-542-3p miR-136 let-7f-1lowast miR-518elet-7alowast miR-125bmiR-125 a-5pmiR-519a andmiR-29a) and7 microRNAs were found to be downregulated (let-7f miR-223 miR-1260 let-7d miR-320c miR-185 and miR-1272) infour examined preeclamptic serum samples [43]

Later using microarray analysis Wu et al [44] reportedthe upregulation of 13 microRNAs (miR-574-5p miR-26amiR-151-3p miR-130a miR-181a miR-130b miR-30d miR-145 miR-103 miR-425 miR-221 miR-342-3p and miR-24)and down-regulation of 2 microRNAs (miR-144 miR-16) inpatients with severe preeclampsia Seven of these 13 microR-NAs (miR-574-5p miR-26a miR-130b miR-181a miR-342-3p miR-103 and miR-24) were validated by real-time PCRanalysis to be elevated in plasma from severe preeclampticpregnancies

In a small-scale analysis Mouillet et al [45] did notobserve any differentiation between pregnancies with normaland fetal growth restricted fetuseswhen compared circulatingmicroRNA expression levels (miR-27a miR-30d miR-141miR-200c miR-205 miR-424 miR-451 miR-491 miR-517amiR-518b miR-518e and miR-524)

However most of investigators focused on the study ofthose microRNAs whose genes are located outside chromo-some 19 miRNA clusters (C19MC and miR-371-3 cluster) or

the chromosome 14 miRNA cluster (C14MC) that encodepregnancy-associated microRNAs [46ndash50]

We have previously identified C19MC microRNAs (miR-516-5p miR-517lowast miR-518b miR-520alowast miR-520h miR-525and miR-526a) present in maternal plasma differentiatingbetween normal pregnancies and nonpregnant individuals[51]We selected from the chromosome 19microRNA clusterwhich involves 46 microRNA genes altogether [48ndash50 52]preferentially those microRNAs that were previously demon-strated to be exclusively expressed in placental tissues (miR-520alowast miR-516-5p miR-517lowast miR-518b miR-519a miR-524-5p miR-525 miR-526a miR-526b and miR-520h) and thosemicroRNAs that were reported to be highly expressed inplacental tissues (miR-512-5p miR-515-5p miR-518flowast miR-519d and miR-519elowast) [51 53 54]

Later we demonstrated significant increases in extracel-lular C19MCmicroRNAs levels (miR-516-5p miR-517lowast miR-518b miR-520alowast miR-520h miR-525 and miR-526a) overtime in normally progressing pregnancies [51 54]

The results of our pilot study indicated no differentiationbetween normal and complicated pregnancies but couldnot come to definitive conclusions due to the low numberof studied subjects involved [51 54] The current study isa followup of our previous studies [51 54] and describescomprehensively for the first time the expression profileof circulating C19MC microRNAs (miR-516-5p miR-517lowastmiR-518b miR-520alowast miR-520h miR-525 and miR-526a)in the entirely new sample set of patients with clinicallyestablished preeclampsia andor fetal growth restriction Toour knowledge no study describing the profile of circulatingC19MC microRNAs in gestational hypertension has beencarried out

2 Materials and Methods

21 Patients The studied cohort consisted of Caucasianwomen involving 63 preeclampsia (PE) w or wo fetal growthrestriction (FGR) 27 FGR 23 gestational hypertension(GH) and 55 controls Twenty-four women had signs ofmild preeclampsia 39 women were diagnosed with severepreeclampsia 24 preeclamptic patients required the deliverybefore 34 weeks of gestation and 39 patients delivered after 34weeks of gestation In 18 cases preeclampsia superposed onprevious hypertension otherwise it occurred in normoten-sive patients (45 cases) Eight growth-retarded foetuses weredelivered before 34 weeks of gestation and 19 those after 34weeks of gestation Oligohydramnios or anhydramnios werepresent in 7 growth restricted foetuses

Doppler studies showed an abnormal pulsatility index(PI) in the umbilical artery (14 preeclampsia plusmn FGR and 14FGR) andor in themiddle cerebral artery (10 preeclampsia plusmnFGR and 11 FGR) Cerebroplacental ratio (CPR) expressedas a ratio between umbilical artery and middle cerebralartery pulsatility index was below the fifth percentile in21 cases (9 preeclampsia plusmn FGR and 12 FGR) Absent orreversed enddiastolic velocity waveforms in the umbilicalartery occurred in 8 cases (2 preeclampsia + FGR and 6 FGR)

Normal pregnancies were defined as those without com-plications who delivered full term singleton healthy infants

Mediators of Inflammation 3

Table 1 Correlation between Doppler ultrasonography parameters and C19MCmicroRNAs expression levels and plasmatic concentrations

Spearmanrsquos rank correlationAbsolute quantification Relative quantification

A umbilicalis PI Middle cerebralartery PI

Cerebroplacentalratio A umbilicalis PI Middle cerebral

artery PICerebroplacental

ratio120588 119875 120588 119875 120588 119875 120588 119875 120588 119875 120588 119875

miR-516-5p minus0023 0845 minus0393 0005 minus0219 0125 minus0040 0736 minus0307 0030 minus0190 0183miR-517lowast 0032 0791 minus0328 0020 minus0215 0131 0073 0542 minus0288 0041 minus0269 0060miR-518b minus0025 0834 minus0152 0269 minus0074 0606 minus0019 0872 minus0220 0120 minus0167 0242miR-520alowast 0092 0441 minus0314 0027 minus0261 0050 0085 0477 minus0339 0017 minus0339 0018miR-520h 0016 0893 minus0238 0093 minus0178 0214 0038 0748 minus0256 0071 minus0219 0126miR-525 0009 0941 minus0358 0011 minus0190 0183 minus0028 0812 minus0357 0012 minus0185 0194miR-526a 0156 0196 minus0304 0032 minus0340 0017 0131 0277 minus0286 0043 minus0329 0021The association between the plasmatic concentration (absolute quantification) and gene expression levels (relative quantification) of C19MC microRNAs andDoppler ultrasonography parameters such as arteria umbilicalis pulsatility index middle cerebral artery pulsatility index and cerebroplacental ratioPI pulsatility index 120588 Spearmanrsquos correlation coefficient P level of significance bold font statistically significant results

weighting gt2500 g after 37 completed weeks of gestationPreeclampsia was defined as blood pressure gt14090mmHgin two determinations 4 hours apart that was associated withproteinuria gt300mg24 h after 20 weeks of gestation Severepreeclampsiawas diagnosed by the presence of one ormore ofthe findings according to the guidelines of ACOGCommittee[11]

Fetal growth restriction was diagnosed when the esti-mated fetal weight (EFW) calculated using the Hadlockformula (Astraia Software GmbH) was below the tenthpercentile for the evaluated gestational age

Gestational hypertension was defined as high blood pres-sure that developed after the twentieth week of pregnancy

All patients provided written informed consent Thestudy was approved by the Ethics Committee of the ThirdFaculty of Medicine Charles University in Prague Thesamples for the study were chosen on the basis of equal timesin storage and gestation age Gestational age was assessedusing ultrasonography

22 Processing of Samples Nine millilitres of peripheralblood were collected into EDTA tubes and centrifuged twiceat 1200 g for 10min at room temperature Plasma sampleswere stored at minus80∘C until subsequent processing

Total RNA was extracted from 1mL of plasma and 25mgof normal placental tissue preserved in RNAlater (AmbionAustin USA) followed by an enrichment procedure for smallRNAs using a mirVana microRNA Isolation kit (AmbionAustin USA) Trizol LS reagent was used in plasma samplesfor total RNA extraction from biological fluids (InvitrogenCarlsbad USA) and preceded the small RNAs enrichmentprocedure To minimize DNA contamination we treated theeluted RNA with 5 120583L of DNase I (Fermentas InternationalON Canada) for 30min at 37∘C

23 Reverse Transcriptase Reaction Each microRNA wasreverse-transcribed into complementaryDNAusing TaqManMicroRNA Assay containing microRNA-specific stem-loop

RT primers (Table 1) and TaqMan MicroRNA Reverse Tran-scription Kit (Applied Biosystems Branchburg USA) in atotal reaction volume of 50120583L on a 7500 real-time PCRsystem (Applied Biosystems Branchburg USA) with thefollowing thermal cycling parameters 30 minutes at 16∘C 30minutes at 42∘C 5 minutes at 85∘C and then held at 4∘C

24 Quantification of MicroRNAs 15 120583L of cDNA corre-sponding to each microRNA was mixed with componentsof TaqMan MicroRNA Assay and the ingredients of theTaqMan Universal PCR Master Mix (Applied BiosystemsBranchburg USA) in a total reaction volume of 35120583LTaqMan PCR conditions were set as described in the TaqManguidelines The analysis was performed using a 7500 real-time PCR system All PCRs were performed in duplicatesA sample was considered positive if the amplification signaloccurred before the 40th threshold cycle Concentrations ofindividual microRNAs were expressed as pg of total RNAenriched for small RNAs per millilitre of plasma

The expression of particular microRNA in maternalplasma was determined using the comparative Ct method[55] relative to the expression of the same microRNA in thereference sample randomly selected placenta derived fromgestation with normal course

RNA fraction highly enriched for small RNA isolatedfrom the fetal part of the placenta (the part of the placentaderived from the chorionic sac that encloses the embryoconsisting of the chorionic plate and villi) was used to build-up the standard curves and as a reference sample for relativequantification throughout the study

Synthetic C elegans microRNA (cel-miR-39 QiagenHilden Germany) was used as an internal control for vari-ations during the preparation of RNA cDNA synthesis andreal-time PCR Due to a lack of generally accepted standardsall experimental real-time qRT-PCR data were normalizedto cel-miR-39 as it shows no sequence homology to anyhuman microRNA 1 120583l of 01 nM cel-miR-39 was spiked inafter incubation with Trizol LS reagent to the human plasmasamples


25 Statistical Analysis MicroRNA levels were comparedbetween groups by nonparametric tests (the Mann-WhitneyU test for the comparison between two groups and theKruskal-Wallis test for the comparison between three ormore groups) using Statistica software (StatSoft Inc USA)Correlation between variables including absolute andor rel-ativemicroRNAquantification andDoppler ultrasonographyparameters (the pulsatility index in the umbilical arterythe pulsatility index in the middle cerebral artery and thecerebroplacental ratio) was calculated using the Spearmanrsquosrank correlation coefficient (120588) If it varies from minus05 to 0there is a weak negative correlationThe significance level wasestablished at a 119875 value of 119875 lt 005

3 Results

31 Circulating C19MC MicroRNAs Differentiate betweenComplicated and Normal Pregnancies Overall increasedplasmatic levels of miR-516-5p (119875 = 0008) miR-517lowast (119875 =0003) miR-520alowast (119875 lt 0001) miR-525 (119875 = 0003)and miR-526a (119875 = 0004) were observed in women withpregnancy-related complications (gestational hypertensionpreeclampsia and fetal growth restriction) compared to nor-mal pregnancies

Similarly the difference in gene expression of circulatingmicroRNAs between pregnancy-related complications andthe control cohort (normal pregnancies) achieves statisticalsignificance for miR-516-5p (119875 lt 0001) miR-517lowast (119875 =0005) miR-520alowast (119875 = 0001) miR-525 (119875 = 0001) andmiR-526a (119875 = 0004)

32 Upregulation of Circulating C19MC MicroRNAs in Preg-nancies with Established Preeclampsia Consecutive detailedgroup analysis confirmed a difference in the levels of extra-cellular microRNAs in 55 C19MC microRNAs (miR-516-5p119875 = 0037 miR-517lowast 119875 = 0015 miR-520alowast 119875 = 0003 miR-525 119875 = 0026 and miR-526a 119875 = 0032)

While plasmatic levels ofmicroRNAs between the controlcohort and the cohorts of patients with FGR and GH did notdiffer increased levels were detected in the group of patientswith established preeclampsia (miR-516-5p 119875 = 0037 miR-517lowast 119875 = 0033 miR-520alowast 119875 = 0001 miR-525 119875 = 0026and miR-526a 119875 = 0030) (Figures 1(a)ndash1(e))

Parallel significant difference in microRNA gene expres-sion was found between groups of preeclampsia gestationalhypertension fetal growth restriction and controls (miR-516-5p 119875 = 0005 miR-517lowast 119875 = 0028 miR-520alowast 119875 = 0011miR-525 119875 = 001 miR-526a 119875 = 0034) Again whilethe expression of microRNAs between the control cohortgestational hypertension and fetal growth restriction did notdiffer the highest expression was detected in the group ofpatients with preeclampsia (miR-516-5p 119875 = 0009 miR-517lowast 119875 = 0043 miR-520alowast 119875 = 0009 miR-525 119875 = 001miR-526a 119875 = 0035) (Figures 1(f)ndash1(j))

33 The Association Study of Circulating C19MC MicroRNAsand the Severity of the Disease with respect to Clinical Signsand Requirements for the Delivery Plasmatic concentrations

andor expression profile of C19MC microRNAs were anal-ysed in relation to the severity of the disease with respect tothe degree of clinical signs (mild and severe preeclampsia)and requirements for the delivery (before and after 34 weeksof gestation) No effect of the severity of the disease eitheron plasmatic C19MC microRNA concentrations (miR-516-5p 119875 = 0396 miR-517lowast 119875 = 0226 miR-520alowast 119875 = 008miR-525 119875 = 0237 and miR-526a 119875 = 0201) or C19MCmicroRNA expression levels (miR-516-5p 119875 = 0476 miR-517lowast 119875 = 058 miR-520alowast 119875 = 0239 miR-525 119875 = 0397miR-526a 119875 = 0646) was observed

Further the association between C19MC microRNAplasmatic levels andor gene expression and the occurrenceof previous hypertension in the cohort of patients withpreeclampsia was determined No difference between thegroup of preeclampsia superposed on chronic hypertensionandor gestational hypertension and the group of patientswith unexpected onset of preeclampsia was revealed (abso-lute quantification miR-516-5p 119875 = 0885 miR-517lowast 119875 =0538 miR-520alowast 119875 = 0342 miR-525 119875 = 0909 miR-526a119875 = 0273 relative quantification miR-516-5p 119875 = 0721miR-517lowast 119875 = 0621 miR-520alowast 119875 = 0885 miR-525 119875 =0568 miR-526a 119875 = 0201)

34 The Association Study of Circulating C19MC Micrornasand the Severity of the Disease with respect to DopplerUltrasonography Monitoring The association between theplasmatic concentration and gene expression levels of C19MCmicroRNAs and Doppler ultrasonography parameters (thepulsatility index in the umbilical artery the pulsatility indexin the middle cerebral artery and the cerebroplacental ratio)was studied in the cohort of pregnancies complicated withpreeclampsia andor fetal growth restriction

No difference within the group of complicated preg-nancies with normal and abnormal values of flow rate inthe umbilical artery was found out with the exception ofmiR-526a which was upregulated in the group of patientswith abnormal blood flow velocity waveforms (absolutequantification 119875 = 0038 relative quantification 119875 = 005)

Further the statistical analysis showed no effect of thepulsatility index in the middle cerebral artery and the cere-broplacental ratio on the plasmatic concentrations (A cerebrimedia 119875 = 0479 119875 = 0826 119875 = 0528 119875 = 0625119875 = 0154 CPR 119875 = 0426 119875 = 0479 119875 = 0443 119875 = 0867and 119875 = 0181) and expression levels (A cerebri media 119875 =0826 119875 = 0931 119875 = 0427 119875 = 0639 and 119875 = 0297 CPR119875 = 0517 119875 = 0288 119875 = 0198 119875 = 0984 and 119875 = 0195)of all microRNAs (miR-516-5p miR-517lowast miR-520alowast miR-525 and miR-526a) that were identified to be upregulated inplasma samples derived from preeclampsia with or withoutfetal growth restriction

The correlation between variables including absoluteandor relative quantification of particular microRNA inmaternal plasma and the values of flow rate in the umbilicalartery and the fetal blood vessel was calculated usingSpearmanrsquos rank correlation coefficient The pulsatility indexin the umbilical artery did not show any correlation withmicroRNA plasmatic concentrations andor microRNA geneexpressionHowever aweak negative correlation between the


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Figure 1 Upregulation of circulating C19MCmicroRNAs in pregnancies with preeclampsia Absolute ((a) (b) (c) (d) and (e)) and relative((f) (g) (h) (i) and (j)) quantification data were expressed as box plots of individual microRNAs in cohorts of normal and complicatedpregnancies using Statistica softwareThe upper and lower limits of the boxes represent the 75th and 25th percentiles respectivelyThe upperand lower whiskers represent the maximum and minimum values that are no more than 15 times the span of the interquartile range (rangeof the values between the 25th and the 75th percentiles) The median is indicated by the line in each box Outliers are indicated by circles andextremes by asterisks

pulsatility index in the middle cerebral artery andmicroRNAplasmatic concentrations (miR-516-5p 120588 = minus0393119875 = 0005 miR-517lowast 120588 = minus0328 119875 = 0020 miR-520alowast120588 = minus0314119875 = 0026 miR-525 120588 = minus0358119875 = 0011 miR-526a 120588 = minus0304 119875 = 0031) or microRNA gene expression(miR-516-5p 120588 = minus0307 119875 = 0030 miR-517lowast 120588 = minus0288119875 = 0041 miR-520alowast 120588 = minus0339 119875 = 0017 miR-525120588 = minus0357 119875 = 0012 miR-526a 120588 = minus0286 119875 = 0043)was observed Furthermore a weak negative correlationbetween cerebroplacental ratio and microRNA plasmaticconcentrations (miR-520alowast 120588 = minus0261 119875 = 0050 miR-526a 120588 = minus0340 119875 = 0017 Figure 2(a)) or microRNA geneexpression (miR-520alowast 120588 = minus0339 119875 = 0018 miR-526a120588 = minus0329 119875 = 0021 Figure 2(b)) was found (Table 1)

35 Function and Functional Relationship Analysis of Tar-get Genes of Differentially Expressed Extracellular C19MCMicroRNAs in Preeclampsia The function and functionalrelationship analysis of predicted targets of the five elevatedextracellular C19MCmicroRNAs in patients with establishedpreeclampsia indicated that a large group of genes was con-nected to the regulation of the immune system and inflam-matory response (Table 2) The data were collected frommiRDB database (httpmirdborgmiRDB) All the targetswere predicted by a bioinformatics tool MirTarget2 whichwas developed by analyzing thousands of genes impacted bymiRNAs with an SVM learning machine

4 Discussion

The results of our previous pilot study strongly supportedthe need for a more detailed exploration of extracellularmicroRNAs in maternal circulation with the view towardtheir recognition as potential biomarkers for placental insuf-ficiency related complications [51 54]

Initially some extracellular placental specific microRNAs(miR-516-5p miR-520lowast miR-518b and miR-526a) trended

just to a higher level in the small cohort of patients with pla-cental insufficiency related complications (16 preeclampsia5 preeclampsia with IUGR and 11 IUGR) however did notreach statistical significance when compared to gestational-age-matched controls Although we have previously demon-strated that normal pregnancies and the manifestation ofplacental-insufficiency-related complications did not influ-ence the levels of miR-16 and let-7d in maternal plasma[51 54] it is nowno doubt that ubiquitously expressedmiR-16and let-7d should not be further used to normalize expressionprofiles of various extracellular microRNAs as was donebefore in our pilot study Our latest research revealed thatthe expression levels of miR-16 were significantly decreasedin placental tissues derived from patients with preeclampsia(data submitted for publication) SimilarlyMaccani et al [56]also reported that reduced expression of miR-16 in placentaltissue may be relevant to the low birth weight in terminfants born small for gestational age In contrast miR-16 waspreviously observed to be overexpressed in placental tissuesaffectedwith severe preeclampsia respectively [38]The lateststudy of Wu et al [44] brought the evidence of circulatingmir-16 down-regulation in patients with severe preeclampsiaFurthermore Yang et al [43] showed decreased expressionof circulating let-7d in preeclamptic patients applying moresophisticated approach such as next generation sequencingtechnology

For that reason alternative endogenous control candi-dates to normalize extracellular microRNA gene expressiondata should be used

In the current study the cohort of pregnancy relatedcomplicationswas expanded to achieve adequate power of thestudy This time the normalization of circulating microRNAexpression was done against synthetic C elegans microRNA(cel-miR-39) Using both absolute and relative quantificationapproaches the ability of extracellular C19MC microRNAs(miR-516-5p miR-517lowast miR-520alowast miR-525 and miR-526a)to differentiate between normal and complicated pregnancies


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Figure 2 The association between the plasmatic concentration (a) and expression levels (b) of C19MC microRNAs and Dopplerultrasonography parameters 120588 Spearmanrsquos correlation coefficient P level of significance

during the onset of preeclampsia w or wo fetal growthrestriction was confirmed

Unfortunately limited data comparing extracellularC19MC microRNA levels between the groups of normaland complicated pregnancies are available Our dataare inconsistent with Yang et al [43] who observed theupregulation of extracellular miR-520h in four patients withpreeclampsia

Our findings may be supported by Mouillet et al [45]who have recently also observed no significant differencein relative placental specific microRNA levels (miR-518b) inplasma samples from those with normally progressing andfetal growth restriction pregnancies

On the basis of the results of our study we further studiedthe association between circulating C19MC microRNAs andthe severity of the disease with respect to the degree of clinicalsigns requirements for the delivery (before and after 34weeksof gestation) and Doppler ultrasound examination

The association analysis pointed to no relationshipbetween C19MCmicroRNA plasmatic concentrations andorgene expression and identified risk factors for a poorerperinatal outcome There was no difference in microRNA

plasmatic levels andor gene expression between pregnancieswith mild and severe preeclampsia pregnancy-related com-plication with the need for the delivery before 34 weeks ofgestation and those who delivered after this critical periodand pregnancies with abnormal and normal blood flowvelocity waveforms Nevertheless the levels ofmiR-526aweresignificantly increased in the group of patients with abnormalvalues of flow rate in the umbilical artery

On the other hand the dependence between the levelsof plasmatic C19MC microRNAs and the pulsatility index inthe middle cerebral artery and the values of cerebroplacentalratio was demonstrated The relation between the increasedlevels of plasmatic C19MC microRNAs (miR-516-5p miR-517lowast miR-520alowast miR-525 and miR-526a) and decreasedvalues of flow rate in the middle cerebral artery reached astatistical significance in complicated pregnancies

Similarly the relationship between increased levels ofplasmatic C19MC microRNAs (miR-520alowast and miR-526a)and decreased values of cerebroplacental ratio was revealed

In conclusion microRNAs play a fundamental role in avariety of physiological and pathological processes involving


Table 2 (a) Function and functional relationship analysis of target genes of differentially expressed extracellular C19MC microRNAs inpreeclampsia in relation to immune system response (b) Function of target genes of differentially expressed extracellular C19MCmicroRNAsin preeclampsia in immune system response

(a)

microRNA miR-516-5p miR-517lowast miR-526a miR-525 miR-520alowast

Number of predicted target genes 349 179 212 340 352Unique target genes Shared with miR-525

CCR2 FAS BCAP29 TOX ACVR2BCD109 IL6ST CD24 AHSA2CD1A IL9R CD302 ATRN

DNAJC25 IRAK3 CFLAR CD2FLT1 LILRA2 DNAJC21 CD300LBIL17RE MTDH HSP90AA1 CD46IRAK1 PAPPA IGFBP1 CD93LILRB5 TLR2 HSF5

PDCD6IP TNFRSF19 IGF1RSOCS2 TNFSF15 IL10RA

TRAF6 MMD2PPARATLR7VSIG4

All the targets were predicted by a bioinformatics tool MirTarget2 using miRDB online database

(b)

Gene official symbol Gene full name The role in immune system responseACVR2B Activin A receptor type IIB Activins belong to the TGF-120573 superfamily

AHSA2AHA1 activator of heat shock90 kDa protein ATPase homolog 2(yeast)

Hsp90 is an inducible molecular chaperone protectingstressed cells

ATRN AttractinInvolvement in initial immune cell clustering duringinflammatory responses that may regulate thechemotactic activity of chemokines

BCAP29 B-cell receptor-associated protein29 Involvement in CASP8-mediated apoptosis

CCR2 Chemokine (C-C motif) receptor2

Binds monocyte chemoattractant protein-1 involved inmonocyte infiltration during inflammation

CD109 CD109 molecule Encodes GPI-linked glycoprotein that negativelyregulates signaling of TGF-120573

CD1A CD1a moleculeEncodes glycoproteins structurally related to MHCproteins mediating the presentation of lipid andglycolipid antigens

CD2 CD2 molecule A surface antigen of thymocytes T and NK cells

CD24 CD24 molecule Encodes a sialoglycoprotein expressed on maturegranulocytes and B cells

CD300LB CD300 molecule-like familymember b

A nonclassical activating receptor of the Ig superfamilyexpressed on myeloid cells

CD302 CD302 molecule A C-type lectin receptor involved in cell adhesionmigration endocytosis and phagocytosis

CD46 CD46 molecule complementregulatory protein

Has cofactor activity for inactivation of complementcomponents C3b and C4b by serum factor I

CD93 CD93 molecule Involvement in intercellular adhesion and in theclearance of apoptotic cells

CFLAR CASP8 and FADD-like apoptosisregulator Regulator of apoptosis structurally similar to caspase-8


(b) Continued

Gene official symbol Gene full name The role in immune system response

DNAJC21 DnaJ (Hsp40) homolog subfamilyC member 21

A molecular chaperone protein protecting againstcellular stress



FAS FAS cell surface death receptor(FAS)

Plays a central role in regulation of programmed celldeath

FLT1 fms-related tyrosine kinase 1A member of vascular endothelial growth factorreceptor (VEGFR) playing an important role inangiogenesis and vasculogenesis

HSF5 Heat shock transcription factorfamily member 5 A transcriptional activator of heat shock genes

HSP90AA1 Heat shock protein 90 kDa alpha(cytosolic) class A member 1

An inducible molecular chaperone protecting stressedcells

IGF1R Insulin-like growth factor 1receptor Antiapoptotic agent enhancing cell survival

IGFBP1 Insulin-like growth factor bindingprotein 1

Prolongs the half-time of IGFs in plasma that regulatecell growth and development

IL10RA Interleukin 10 receptor alpha Involvement in inhibition of the synthesis ofproinflammatory cytokines

IL17RE Interleukin 17 receptor E Participation in MAPK pathway

IL6ST Interleukin 6 signal transducer(gp130 oncostatin M receptor)

A signal transducer shared by IL-6 LIF and oncostatinM

IL9R Interleukin 9 receptor Mediates IL-9 effects like stimulation of cellproliferation and prevention of apoptosis

IRAK1 Interleukin-1 receptor-associatedkinase 1

Responsible for IL-1 induced upregulation of thetranscription factor NF-kappa B


Functions as a negative regulator of Toll-like receptorsignaling

LILRA2Leukocyte immunoglobulin-likereceptor subfamily A (with TMdomain) member 2

An activatory cell-surface receptor expressed onmonocytes B cells dendritic and NK cells

LILRB5Leukocyte immunoglobulin-likereceptor subfamily B (with TMand ITIM domains) member 5

An inhibitory cell-surface receptor expressed onimmune cells

MMD2 Monocyte to macrophagedifferentiation-associated 2 Modulates Ras signaling

MTDH Metadherin Involvement in HIF-1 alpha mediated angiogenesis andRNA-induced silencing complex and miRNA functions

PAPPA Pregnancy-associated plasmaprotein A pappalysin-1

Involvement in local proliferative processes such aswound healing

PDCD6IP Programmed cell death 6interacting protein Protects against cell death

PPARA Peroxisome proliferator-activatedreceptor alpha

Affects the expression of genes involved in cellproliferation cell differentiation and in immune andinflammation responses

SOCS2 Suppressor of cytokine signaling 2 A negative regulator of JAKSTAT cytokine signalingpathway

TLR2 Toll-like receptor 2 Plays a fundamental role in activation of innateimmunity stimulates NF-kappa B

TLR7 Toll-like receptor 7 Plays a fundamental role in activation of innateimmunity

TNFRSF19 Tumor necrosis factor receptorsuperfamily member 19

Interacts with TRAF family members inducesapoptosis by a caspase-independent mechanism


(b) Continued


TNFSF15 Tumor necrosis factor (ligand)superfamily member 15

A cytokine induced by TNF and IL-1 alpha activatingNF-kappa B and MAP kinases inducing apoptosis inendothelial cells

TOX Thymocyte selection-associatedhigh mobility group box

Highly expressed in thymus the site of development ofT cells

TRAF6 TNF receptor-associated factor 6E3 ubiquitin protein ligase

Functions as a signal transducer in the NF-kappa Bpathway activates Ikappa B kinase in response toproinflammatory cytokines

VSIG4 V-set and immunoglobulindomain containing 4

A negative regulator of T-cell responses structurallyrelated to the B7 family of immune regulatory proteins

pregnancy-related complications Current study demon-strated for the first time that circulating C19MC microRNAsmight play a role in the pathogenesis of preeclampsia butnot in the pathogenesis of gestational hypertension and fetalgrowth restriction The study brought interesting findingthat the upregulation of circulating C19MC microRNAs(miR-516-5p miR-517lowast miR-520alowast miR-525 and miR-526a)is a characteristic phenomenon of established preeclampsia

5 Conclusion

The study brought the interesting finding that the upregu-lation of circulating C19MC microRNAs (miR-516-5p miR-517lowast miR-520alowast miR-525 and miR-526a) is a characteristicphenomenon of established preeclampsia

Conflict of Interests

The authors report no conflict of interests

Acknowledgments

The work was exclusively supported by the Grant Agency ofCzech Republic (no 304121352) and partially supported bythe Charles University Research Program PRVOUK P32

References

[1] C W G Redman and I L Sargent ldquoPreeclampsia and the sys-temic inflammatory responserdquo Seminars in Nephrology vol 24no 6 pp 565ndash570 2004

[2] D M Nelson ldquoApoptotic changes occur in syncytiotrophoblastof human placental villi where fibrin type fibrinoid is depositedat discontinuities in the villous trophoblastrdquoPlacenta vol 17 no7 pp 387ndash391 1996

[3] C B M Oudejans M L Tjoa B A Westerman M A MMulders I J Van Wijk and J M G Van Vugt ldquoCirculatingtrophoblast in maternal bloodrdquo Prenatal Diagnosis vol 23 no2 pp 111ndash116 2003

[4] B Huppertz and J C P Kingdom ldquoApoptosis in the tro-phoblastmdashrole of apoptosis in placental morphogenesisrdquo Jour-nal of the Society for Gynecologic Investigation vol 11 no 6 pp353ndash362 2004

[5] A F Orozco F Z Bischoff C Horne E Popek J L Simpsonand D E Lewis ldquoHypoxia-induced membrane-bound apop-totic DNA particles potential mechanism of fetal DNA inmaternal plasmardquo Annals of the New York Academy of Sciencesvol 1075 pp 57ndash62 2006

[6] I Hromadnikova ldquoExtracellular nucleic acids in maternalcirculation as potential biomarkers for placental insufficiencyrdquoDNA and Cell Biology vol 31 no 7 pp 1221ndash1232 2012

[7] CWRedman and I L Sargent ldquoLatest advances in understand-ing preeclampsiardquo Science vol 308 no 5728 pp 1592ndash15942005

[8] T YKhong FDeWolfW B Robertson and I Brosens ldquoInade-quatematernal vascular response to placentation in pregnanciescomplicated by pre-eclampsia and by small-for-gestational ageinfantsrdquo British Journal of Obstetrics and Gynaecology vol 93no 10 pp 1049ndash1059 1986

[9] WHO ldquoWorldHealthOrganization International CollaborativeStudy of Hypertensive Disorders in Pregnancy Geographicvariation in the incidence of hypertension in pregnancyrdquoAmerican Journal of Obstetrics and Gynecology vol 158 no 1pp 80ndash83 1988

[10] J E Bamfo and A O Odibo ldquoDiagnosis and management offetal growth restrictionrdquo Journal of Pregnancy vol 2011 ArticleID 640715 15 pages 2011

[11] ACOGCommittee on Practise Bulletins-Obstetrics ldquoDiagnosisand management of preeclampsia and eclampsiardquo Obstetricsand Gynecology vol 99 no 1 pp 159ndash167 2002

[12] K S Khan D Wojdyla L Say A M Gulmezoglu and P F VanLook ldquoWHO analysis of causes of maternal death a systematicreviewrdquoThe Lancet vol 367 no 9516 pp 1066ndash1074 2006

[13] S J Germain G P Sacks S R Soorana I L Sargent andC W Redman ldquoSystemic inflammatory priming in normalpregnancy and preeclampsia the role of circulating syncytiotro-phoblast microparticlesrdquo Journal of Immunology vol 178 no 9pp 5949ndash5956 2007

[14] A Reddy X Y Zhong C Rusterholz et al ldquoThe effect of labourand placental separation on the shedding of syncytiotropho-blast microparticles cell-free DNA and mRNA in normalpregnancy and pre-eclampsiardquo Placenta vol 29 no 11 pp 942ndash949 2008

[15] L C Y Poon R Akolekar R Lachmann J Beta and K HNicolaides ldquoHypertensive disorders in pregnancy screening bybiophysical and biochemical markers at 11ndash13 weeksrdquo Ultra-sound in Obstetrics and Gynecology vol 35 no 6 pp 662ndash6702010


[16] R Akolekar A Syngelaki R Sarquis M Zvanca and K HNicolaides ldquoPrediction of early intermediate and late pre-eclampsia from maternal factors biophysical and biochemicalmarkers at 11ndash13 weeksrdquo Prenatal Diagnosis vol 31 no 1 pp66ndash74 2011

[17] P von Dadelszen L A Magee and J M Roberts ldquoSubclassifi-cation of preeclampsiardquo Hypertension in Pregnancy vol 22 no2 pp 143ndash148 2003

[18] B Huppertz ldquoPlacental origins of preeclampsia challenging thecurrent hypothesisrdquo Hypertension vol 51 no 4 pp 970ndash9752008

[19] HValensise B Vasapollo G Gagliardi andG PNovelli ldquoEarlyand late preeclampsia two different maternal hemodynamicstates in the latent phase of the diseaserdquo Hypertension vol 52no 5 pp 873ndash880 2008

[20] YMDennis Lo N Corbetta P F Chamberlain et al ldquoPresenceof fetal DNA in maternal plasma and serumrdquo The Lancet vol350 no 9076 pp 485ndash487 1997

[21] M Smid A Vassallo F Lagona et al ldquoQuantitative analysisof fetal DNA in maternal plasma in pathological conditionsassociatedwith placental abnormalitiesrdquoAnnals of the NewYorkAcademy of Sciences vol 945 pp 132ndash137 2001

[22] E Caramelli N Rizzo M Concu et al ldquoCell-free fetal DNAconcentration in plasma of patients with abnormal uterineartery Doppler waveform and intrauterine growth restrictionmdasha pilot studyrdquo Prenatal Diagnosis vol 23 no 5 pp 367ndash3712003

[23] J-M Costa A Benachi and E Gautier ldquoNew strategy forprenatal diagnosis of X-linked disordersrdquo New England Journalof Medicine vol 346 no 19 p 1502 2002

[24] R J Rijnders C E van der Schoot B Bossers M A deVroede and G C Christiaens ldquoFetal sex determination frommaternal plasma in pregnancies at risk for congenital adrenalhyperplasiardquoObstetetrics andGynecology vol 98 no 3 pp 374ndash378 2001

[25] B HW Faas E A Beuling G C M L Christiaens A E G KVon Dem Borne and C E Van Der Schoot ldquoDetection of fetalRHD-specific sequences in maternal plasmardquo The Lancet vol352 no 9135 p 1196 1998

[26] Y M D Lo N M Hjelm C Fidler et al ldquoPrenatal diagnosisof fetal RhD status by molecular analysis of maternal plasmardquoNewEngland Journal ofMedicine vol 339 no 24 pp 1734ndash17381998

[27] I Hromadnikova L Vechetova K Vesela B Benesova JDoucha and R Vlk ldquoNon-invasive fetal RHD and RHCEgenotyping using real-time PCR testing of maternal plasmain RhD-negative pregnanciesrdquo Journal of Histochemistry andCytochemistry vol 53 no 3 pp 301ndash305 2005

[28] K C A Chan C Ding A Gerovassili et al ldquoHypermethylatedRASSF1A in maternal plasma a universal fetal DNA markerthat improves the reliability of noninvasive prenatal diagnosisrdquoClinical Chemistry vol 52 no 12 pp 2211ndash2218 2006

[29] Y M D Lo T N Leung M S C Tein et al ldquoQuantitativeabnormalities of fetal DNA inmaternal serum in preeclampsiardquoClinical Chemistry vol 45 no 2 pp 184ndash188 1999

[30] T-W Lau T N Leung L Y S Chan et al ldquoFetal DNA clearancefrom maternal plasma is impaired in preeclampsiardquo ClinicalChemistry vol 48 no 12 pp 2141ndash2146 2002

[31] B M Byrne A Crowley F Taulo J Anthony J J OrsquoLearyand C OrsquoHerlihy ldquoFetal DNA quantitation in peripheral bloodis not useful as a marker of disease severity in women with

preeclampsiardquoHypertension in Pregnancy vol 22 no 2 pp 157ndash164 2003

[32] A SekizawaM JimboH Saito et al ldquoCell-free fetalDNA in theplasma of pregnant womenwith severe fetal growth restrictionrdquoAmerican Journal of Obstetrics and Gynecology vol 188 no 2pp 480ndash484 2003

[33] D W Y Tsui K C A Chan S S C Chim et al ldquoQuantitativeaberrations of hypermethylated RASSF1A gene sequences inmaternal plasma in pre-eclampsiardquo Prenatal Diagnosis vol 27no 13 pp 1212ndash1218 2007

[34] I Hromadnikova M Benesova L Zejskova et al ldquoThe effectof DYS-14 copy number variations on extracellular fetal DNAquantification in maternal circulationrdquo DNA and Cell Biologyvol 28 no 7 pp 351ndash358 2009

[35] I Hromadnikova L Zejskova K Kotlabova et al ldquoQuantifi-cation of extracellular DNA using hypermethylated RASSF1ASRY andGLO sequencesmdashevaluation of diagnostic possibilitiesfor predicting placental insufficiencyrdquo DNA and Cell Biologyvol 29 no 6 pp 295ndash301 2010

[36] B L Pineles R Romero D Montenegro et al ldquoDistinctsubsets of microRNAs are expressed differentially in the humanplacentas of patients with preeclampsiardquo American Journal ofObstetrics and Gynecology vol 196 no 3 pp 261e1ndash261e62007

[37] X-M Zhu T Han I L Sargent G-W Yin and Y-Q YaoldquoDifferential expression profile of microRNAs in human pla-centas from preeclamptic pregnancies vs normal pregnanciesrdquoAmerican Journal of Obstetrics and Gynecology vol 200 no 6pp 661e1ndash661e7 2009

[38] Y Hu P Li S Hao L Liu J Zhao and Y Hou ldquoDifferentialexpression of microRNAs in the placentae of Chinese patientswith severe pre-eclampsiardquo Clinical Chemistry and LaboratoryMedicine vol 47 no 8 pp 923ndash929 2009

[39] S S C Chim T K F Shing E CWHung et al ldquoDetection andcharacterization of placental microRNAs in maternal plasmardquoClinical Chemistry vol 54 no 3 pp 482ndash490 2008

[40] E C Lai ldquoMicro RNAs are complementary to 31015840 UTR sequencemotifs that mediate negative post-transcriptional regulationrdquoNature Genetics vol 30 no 4 pp 363ndash364 2002

[41] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[42] T Gunel Z G Zeybek P Akcakaya et al ldquoSerum microRNAexpression in pregnancies with preeclampsiardquo Genetics andMoecular Research vol 10 no 4 pp 4034ndash4040 2011

[43] Q Yang J Lu S Wang H Li Q Ge and Z Lu ldquoApplicationof next-generation sequencing technology to profile the circu-lating microRNAs in the serum of preeclampsia versus normalpregnant womenrdquo Clinica Chimica Acta vol 412 no 23-24 pp2167ndash2173 2011

[44] L Wu H Zhou H Lin et al ldquoCirculating microRNAs areelevated in plasma from severe preeclamptic pregnanciesrdquoReproduction vol 143 no 3 pp 389ndash397 2012

[45] J-F Mouillet T Chu C A Hubel D M Nelson W T Parksand Y Sadovsky ldquoThe levels of hypoxia-regulated microRNAsin plasma of pregnant women with fetal growth restrictionrdquoPlacenta vol 31 no 9 pp 781ndash784 2010

[46] H Seitz H Royo M-L Bortolin S-P Lin A C Ferguson-Smith and J Cavaille ldquoA large imprinted microRNA genecluster at the mouse Dlk1-Gtl2 domainrdquo Genome Research vol14 no 9 pp 1741ndash1748 2004


[47] Y Liang D Ridzon LWong andC Chen ldquoCharacterization ofmicroRNA expression profiles in normal human tissuesrdquo BMCGenomics vol 8 article 166 2007

[48] I Bentwich A Avniel Y Karov et al ldquoIdentification ofhundreds of conserved and nonconserved humanmicroRNAsrdquoNature Genetics vol 37 no 7 pp 766ndash770 2005

[49] S Lin W K C Cheung S Chen et al ldquoComputational identi-fication and characterization of primate-specific microRNAs inhuman genomerdquoComputational Biology and Chemistry vol 34no 4 pp 232ndash241 2010

[50] D M Morales-Prieto S Ospina-Prieto W ChaiwangyenM Schoenleben and U R Markert ldquoPregnancy-associatedmiRNA-clustersrdquo Journal of Reproductive Immunology vol 97no 1 pp 51ndash61 2013

[51] K Kotlabova J Doucha and I Hromadnikova ldquoPlacental-specific microRNA in maternal circulationmdashidentification ofappropriate pregnancy-associated microRNAs with diagnosticpotentialrdquo Journal of Reproductive Immunology vol 89 no 2pp 185ndash191 2011

[52] M-L Bortolin-Cavaille M Dance M Weber and J CavailleldquoC19MCmicroRNAs are processed from introns of large Pol-IInon-protein-coding transcriptsrdquoNucleic Acids Research vol 37no 10 pp 3464ndash3473 2009

[53] MiRNAMap 20 [Internet] Department of Biological Scienceand Technology Institute of Bioinformatics National ChiaoTung University Hsinchu Taiwan 2013 httpmirnamapmbcnctuedutw

[54] I Hromadnikova K Kotlabova J Doucha K Dlouha andL Krofta ldquoAbsolute and relative quantification of placenta-specific microRNAs in maternal circulation with placen-tal insufficiencymdashrelated complicationsrdquo Journal of MolecularDiagnostics vol 14 no 2 pp 160ndash167 2012

[55] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[56] M AMaccani J F Padbury and C J Marsit ldquomiR-16 andmiR-21 expression in the placenta is associated with fetal growthrdquoPLoS ONE vol 6 no 6 Article ID e21210 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


trophoblast cell death and increased shedding of placentadebris into the maternal circulation as a result placentalinsufficiency related pregnancy complications are associatedwith abnormal levels of extracellular fetal DNA and mRNAtranscripts [5 14]

Recent evidence suggests that preeclampsia can be furthersubdivided into early PE (before 34 weeks of gestation)intermediate PE (between 34 and 37 weeks of gestation) andlate PE (after 37 weeks of gestation) [15 16] The conceptof early and late PE is modern and it is widely acceptedthat these two entities have different etiologies and should beregarded as different forms of the disease where early onsetsof PE and IUGR are considered to be placenta-mediateddiseases [17ndash19]

There has been a trend over the last 10 years to developnoninvasive methods utilizing quantification of cell-freenucleic acids inclusive of microRNAs in maternal circulation[6 20ndash39] The diagnostic potential of particular molec-ular biomarkers and their implementation in the currentpredictive and diagnostic algorithms for pregnancy relatedcomplications is subject of interest [6]

MicroRNAs belong to a family of small noncoding RNAsthat regulate gene expression at the posttranscriptional levelby degrading or blocking translation of messenger RNA(mRNA) targets [40 41]

Recent studies have shown that preeclampsia is associatedwith alterations in extracellular microRNA expression Usingreal-time PCR analysis Gunel et al [42] demonstratedthe upregulation of miR-210 and downregulation of miR-152 in patients with preeclampsia The application of nextgeneration sequencing technology revealed a broader pro-file of dysregulated circulating microRNAs in preeclampsiaCompared to controls 15 microRNAs were found to beupregulated (miR-521 miR-520h miR-517c miR-519d miR-520g miR-517b miR-542-3p miR-136 let-7f-1lowast miR-518elet-7alowast miR-125bmiR-125 a-5pmiR-519a andmiR-29a) and7 microRNAs were found to be downregulated (let-7f miR-223 miR-1260 let-7d miR-320c miR-185 and miR-1272) infour examined preeclamptic serum samples [43]

Later using microarray analysis Wu et al [44] reportedthe upregulation of 13 microRNAs (miR-574-5p miR-26amiR-151-3p miR-130a miR-181a miR-130b miR-30d miR-145 miR-103 miR-425 miR-221 miR-342-3p and miR-24)and down-regulation of 2 microRNAs (miR-144 miR-16) inpatients with severe preeclampsia Seven of these 13 microR-NAs (miR-574-5p miR-26a miR-130b miR-181a miR-342-3p miR-103 and miR-24) were validated by real-time PCRanalysis to be elevated in plasma from severe preeclampticpregnancies

In a small-scale analysis Mouillet et al [45] did notobserve any differentiation between pregnancies with normaland fetal growth restricted fetuseswhen compared circulatingmicroRNA expression levels (miR-27a miR-30d miR-141miR-200c miR-205 miR-424 miR-451 miR-491 miR-517amiR-518b miR-518e and miR-524)

However most of investigators focused on the study ofthose microRNAs whose genes are located outside chromo-some 19 miRNA clusters (C19MC and miR-371-3 cluster) or

the chromosome 14 miRNA cluster (C14MC) that encodepregnancy-associated microRNAs [46ndash50]

We have previously identified C19MC microRNAs (miR-516-5p miR-517lowast miR-518b miR-520alowast miR-520h miR-525and miR-526a) present in maternal plasma differentiatingbetween normal pregnancies and nonpregnant individuals[51]We selected from the chromosome 19microRNA clusterwhich involves 46 microRNA genes altogether [48ndash50 52]preferentially those microRNAs that were previously demon-strated to be exclusively expressed in placental tissues (miR-520alowast miR-516-5p miR-517lowast miR-518b miR-519a miR-524-5p miR-525 miR-526a miR-526b and miR-520h) and thosemicroRNAs that were reported to be highly expressed inplacental tissues (miR-512-5p miR-515-5p miR-518flowast miR-519d and miR-519elowast) [51 53 54]

Later we demonstrated significant increases in extracel-lular C19MCmicroRNAs levels (miR-516-5p miR-517lowast miR-518b miR-520alowast miR-520h miR-525 and miR-526a) overtime in normally progressing pregnancies [51 54]

The results of our pilot study indicated no differentiationbetween normal and complicated pregnancies but couldnot come to definitive conclusions due to the low numberof studied subjects involved [51 54] The current study isa followup of our previous studies [51 54] and describescomprehensively for the first time the expression profileof circulating C19MC microRNAs (miR-516-5p miR-517lowastmiR-518b miR-520alowast miR-520h miR-525 and miR-526a)in the entirely new sample set of patients with clinicallyestablished preeclampsia andor fetal growth restriction Toour knowledge no study describing the profile of circulatingC19MC microRNAs in gestational hypertension has beencarried out

2 Materials and Methods

21 Patients The studied cohort consisted of Caucasianwomen involving 63 preeclampsia (PE) w or wo fetal growthrestriction (FGR) 27 FGR 23 gestational hypertension(GH) and 55 controls Twenty-four women had signs ofmild preeclampsia 39 women were diagnosed with severepreeclampsia 24 preeclamptic patients required the deliverybefore 34 weeks of gestation and 39 patients delivered after 34weeks of gestation In 18 cases preeclampsia superposed onprevious hypertension otherwise it occurred in normoten-sive patients (45 cases) Eight growth-retarded foetuses weredelivered before 34 weeks of gestation and 19 those after 34weeks of gestation Oligohydramnios or anhydramnios werepresent in 7 growth restricted foetuses

Doppler studies showed an abnormal pulsatility index(PI) in the umbilical artery (14 preeclampsia plusmn FGR and 14FGR) andor in themiddle cerebral artery (10 preeclampsia plusmnFGR and 11 FGR) Cerebroplacental ratio (CPR) expressedas a ratio between umbilical artery and middle cerebralartery pulsatility index was below the fifth percentile in21 cases (9 preeclampsia plusmn FGR and 12 FGR) Absent orreversed enddiastolic velocity waveforms in the umbilicalartery occurred in 8 cases (2 preeclampsia + FGR and 6 FGR)

Normal pregnancies were defined as those without com-plications who delivered full term singleton healthy infants







ratio120588 119875 120588 119875 120588 119875 120588 119875 120588 119875 120588 119875
















3 Results














7000

6000

4000

3000

2000

1000

0

Preeclampsia GH FGR

miR-516-5p

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0037

Normalpregnancy

(a)

1400

400

200

0

Normalpregnancy

Preeclampsia GH FGR

miR-517lowast

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0033

(b)

2750

750

500

250

0

Normalpregnancy

Preeclampsia GH FGR

miR-520alowast

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0001

(c)

3000

1000

500

0

Normalpregnancy

Preeclampsia GH FGR

miR-525

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0026

(d)

1500

1000

500

0

Normalpregnancy

Preeclampsia GH FGR

miR-526a

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0036500

3000

2000

(e)

000018

000016

000008000006

000004

000002

000000

Normalpregnancy

Preeclampsia GH FGR

miR-516-5p

P = 0009

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(f)

000055

000050

000035

000025

000020

000015

000010

000005

000000

Normalpregnancy

Preeclampsia GH FGR

miR-517lowast

P = 0043

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(g)

000060

000030

000020

000015

000010

000005

000000

Normalpregnancy

Preeclampsia GH FGR

miR-520alowast

P = 0009

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(h)

Figure 1 Continued


00011

00008

00006

00005

00004

00003

00002

00001

00000Normal


miR-525P = 001

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(i)

00026

00012

00008

00006

00004

00002

00000

Normalpregnancy

Preeclampsia GH FGR

miR-526aP = 0035

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(j)




4 Discussion







miR-520alowastC

ereb

ropl

acen

tal r

atio


302826242220181614121008060402

0 100 200 300 400 500 600 700

miR-526a

Cer

ebro

plac

enta

l rat

io


302826242220181614121008060402

0 200 400 600 800 1000 1200 1400 1600 1800

120588 = minus0261 P = 0050 120588 = minus0340 P = 0017

(a)

miR-520alowast

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00001 00002 00003

miR-526a

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00002 00004 00006 00008


120588 = minus0339 P = 0018 120588 = minus0329 P = 0021

(b)













(a)








(b)




















(b) Continued









































(b) Continued











5 Conclusion




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















ratio120588 119875 120588 119875 120588 119875 120588 119875 120588 119875 120588 119875
















3 Results














7000

6000

4000

3000

2000

1000

0

Preeclampsia GH FGR

miR-516-5p

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0037

Normalpregnancy

(a)

1400

400

200

0

Normalpregnancy

Preeclampsia GH FGR

miR-517lowast

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0033

(b)

2750

750

500

250

0

Normalpregnancy

Preeclampsia GH FGR

miR-520alowast

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0001

(c)

3000

1000

500

0

Normalpregnancy

Preeclampsia GH FGR

miR-525

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0026

(d)

1500

1000

500

0

Normalpregnancy

Preeclampsia GH FGR

miR-526a

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0036500

3000

2000

(e)

000018

000016

000008000006

000004

000002

000000

Normalpregnancy

Preeclampsia GH FGR

miR-516-5p

P = 0009

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(f)

000055

000050

000035

000025

000020

000015

000010

000005

000000

Normalpregnancy

Preeclampsia GH FGR

miR-517lowast

P = 0043

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(g)

000060

000030

000020

000015

000010

000005

000000

Normalpregnancy

Preeclampsia GH FGR

miR-520alowast

P = 0009

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(h)

Figure 1 Continued


00011

00008

00006

00005

00004

00003

00002

00001

00000Normal


miR-525P = 001

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(i)

00026

00012

00008

00006

00004

00002

00000

Normalpregnancy

Preeclampsia GH FGR

miR-526aP = 0035

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(j)




4 Discussion







miR-520alowastC

ereb

ropl

acen

tal r

atio


302826242220181614121008060402

0 100 200 300 400 500 600 700

miR-526a

Cer

ebro

plac

enta

l rat

io


302826242220181614121008060402

0 200 400 600 800 1000 1200 1400 1600 1800

120588 = minus0261 P = 0050 120588 = minus0340 P = 0017

(a)

miR-520alowast

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00001 00002 00003

miR-526a

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00002 00004 00006 00008


120588 = minus0339 P = 0018 120588 = minus0329 P = 0021

(b)













(a)








(b)




















(b) Continued









































(b) Continued











5 Conclusion




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Results














7000

6000

4000

3000

2000

1000

0

Preeclampsia GH FGR

miR-516-5p

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0037

Normalpregnancy

(a)

1400

400

200

0

Normalpregnancy

Preeclampsia GH FGR

miR-517lowast

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0033

(b)

2750

750

500

250

0

Normalpregnancy

Preeclampsia GH FGR

miR-520alowast

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0001

(c)

3000

1000

500

0

Normalpregnancy

Preeclampsia GH FGR

miR-525

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0026

(d)

1500

1000

500

0

Normalpregnancy

Preeclampsia GH FGR

miR-526a

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0036500

3000

2000

(e)

000018

000016

000008000006

000004

000002

000000

Normalpregnancy

Preeclampsia GH FGR

miR-516-5p

P = 0009

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(f)

000055

000050

000035

000025

000020

000015

000010

000005

000000

Normalpregnancy

Preeclampsia GH FGR

miR-517lowast

P = 0043

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(g)

000060

000030

000020

000015

000010

000005

000000

Normalpregnancy

Preeclampsia GH FGR

miR-520alowast

P = 0009

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(h)

Figure 1 Continued


00011

00008

00006

00005

00004

00003

00002

00001

00000Normal


miR-525P = 001

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(i)

00026

00012

00008

00006

00004

00002

00000

Normalpregnancy

Preeclampsia GH FGR

miR-526aP = 0035

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(j)




4 Discussion







miR-520alowastC

ereb

ropl

acen

tal r

atio


302826242220181614121008060402

0 100 200 300 400 500 600 700

miR-526a

Cer

ebro

plac

enta

l rat

io


302826242220181614121008060402

0 200 400 600 800 1000 1200 1400 1600 1800

120588 = minus0261 P = 0050 120588 = minus0340 P = 0017

(a)

miR-520alowast

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00001 00002 00003

miR-526a

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00002 00004 00006 00008


120588 = minus0339 P = 0018 120588 = minus0329 P = 0021

(b)













(a)








(b)




















(b) Continued









































(b) Continued











5 Conclusion




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















7000

6000

4000

3000

2000

1000

0

Preeclampsia GH FGR

miR-516-5p

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0037

Normalpregnancy

(a)

1400

400

200

0

Normalpregnancy

Preeclampsia GH FGR

miR-517lowast

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0033

(b)

2750

750

500

250

0

Normalpregnancy

Preeclampsia GH FGR

miR-520alowast

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0001

(c)

3000

1000

500

0

Normalpregnancy

Preeclampsia GH FGR

miR-525

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0026

(d)

1500

1000

500

0

Normalpregnancy

Preeclampsia GH FGR

miR-526a

pg o

f tot

al R

NA

enric

hed

for s

mal

l RN

As

per1

mL

of p

lasm

a

P = 0036500

3000

2000

(e)

000018

000016

000008000006

000004

000002

000000

Normalpregnancy

Preeclampsia GH FGR

miR-516-5p

P = 0009

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(f)

000055

000050

000035

000025

000020

000015

000010

000005

000000

Normalpregnancy

Preeclampsia GH FGR

miR-517lowast

P = 0043

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(g)

000060

000030

000020

000015

000010

000005

000000

Normalpregnancy

Preeclampsia GH FGR

miR-520alowast

P = 0009

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(h)

Figure 1 Continued


00011

00008

00006

00005

00004

00003

00002

00001

00000Normal


miR-525P = 001

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(i)

00026

00012

00008

00006

00004

00002

00000

Normalpregnancy

Preeclampsia GH FGR

miR-526aP = 0035

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(j)




4 Discussion







miR-520alowastC

ereb

ropl

acen

tal r

atio


302826242220181614121008060402

0 100 200 300 400 500 600 700

miR-526a

Cer

ebro

plac

enta

l rat

io


302826242220181614121008060402

0 200 400 600 800 1000 1200 1400 1600 1800

120588 = minus0261 P = 0050 120588 = minus0340 P = 0017

(a)

miR-520alowast

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00001 00002 00003

miR-526a

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00002 00004 00006 00008


120588 = minus0339 P = 0018 120588 = minus0329 P = 0021

(b)













(a)








(b)




















(b) Continued









































(b) Continued











5 Conclusion




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00011

00008

00006

00005

00004

00003

00002

00001

00000Normal


miR-525P = 001

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(i)

00026

00012

00008

00006

00004

00002

00000

Normalpregnancy

Preeclampsia GH FGR

miR-526aP = 0035

Rela

tive q

uant

ifica

tion

(Ct c

ompa

rativ

e met

hod)

(j)




4 Discussion







miR-520alowastC

ereb

ropl

acen

tal r

atio


302826242220181614121008060402

0 100 200 300 400 500 600 700

miR-526a

Cer

ebro

plac

enta

l rat

io


302826242220181614121008060402

0 200 400 600 800 1000 1200 1400 1600 1800

120588 = minus0261 P = 0050 120588 = minus0340 P = 0017

(a)

miR-520alowast

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00001 00002 00003

miR-526a

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00002 00004 00006 00008


120588 = minus0339 P = 0018 120588 = minus0329 P = 0021

(b)













(a)








(b)




















(b) Continued









































(b) Continued











5 Conclusion




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















miR-520alowastC

ereb

ropl

acen

tal r

atio


302826242220181614121008060402

0 100 200 300 400 500 600 700

miR-526a

Cer

ebro

plac

enta

l rat

io


302826242220181614121008060402

0 200 400 600 800 1000 1200 1400 1600 1800

120588 = minus0261 P = 0050 120588 = minus0340 P = 0017

(a)

miR-520alowast

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00001 00002 00003

miR-526a

Cer

ebro

plac

enta

l rat

io

30

28

26

24

22

20

18

16

14

12

10

08

06

04

0200000 00002 00004 00006 00008


120588 = minus0339 P = 0018 120588 = minus0329 P = 0021

(b)













(a)








(b)




















(b) Continued









































(b) Continued











5 Conclusion




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)








(b)




















(b) Continued









































(b) Continued











5 Conclusion




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(b) Continued









































(b) Continued











5 Conclusion




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(b) Continued











5 Conclusion




Acknowledgments


References

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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