Research Article Deregulated Cardiac Specific...

Research ArticleDeregulated Cardiac Specific MicroRNAs inPostnatal Heart Growth

Pujiao Yu1 Hongbao Wang2 Yuan Xie1 Jinzhe Zhou1 Jianhua Yao2 and Lin Che1

1Department of Cardiology Tongji Hospital Tongji University School of Medicine Shanghai 200065 China2Department of Cardiology Yangpu Hospital Tongji University School of Medicine Shanghai 200090 China

Correspondence should be addressed to Jianhua Yao yaojianhuatongjieducn and Lin Che linchering163com

Received 28 September 2016 Accepted 13 November 2016

Academic Editor Diego Franco

Copyright copy 2016 Pujiao Yu et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The heart is recognized as an organ that is terminally differentiated by adulthood However during the process of humandevelopment the heart is the first organ with function in the embryo and grows rapidly during the postnatal period MicroRNAs(miRNAs miRs) as regulators of gene expression play important roles during the development of multiple systems However therole of miRNAs in postnatal heart growth is still unclear In this study by using qRT-PCR we compared the expression of sevencardiac- or muscle-specific miRNAs that may be related to heart development in heart tissue from mice at postnatal days 0 3 8and 14 Four miRNAsmdashmiR-1a-3p miR-133b-3p miR-208b-3p and miR-206-3pmdashwere significantly decreased while miR-208a-3pwas upregulated during the postnatal heart growth period Based on these results GeneSpring GX was used to predict potentialdownstream targets by performing a 3-way comparison of predictions from the miRWalk PITA and microRNAorg databasesGene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to identify potential functionalannotations and signaling pathways related to postnatal heart growth This study describes expression changes of cardiac- andmuscle-specific miRNAs during postnatal heart growth and may provide new therapeutic targets for cardiovascular diseases

1 Introduction

Although the heart is recognized as a nearly terminally differ-entiated organ its weight increases rapidly by approximately20-fold from birth to adulthood (ie the period of postnatalheart growth) The development of the heart is a precise andcomplex process that is subjected to the regulation of manymolecules

MicroRNAs (miRNAs miRs) are endogenous highlyconserved short noncoding RNAs that can posttranscrip-tionally regulate gene expression by binding to complemen-tary sequences on target mRNAs [1ndash5] miRNAs play impor-tant roles in many biological processes including develop-ment differentiation proliferation apoptosis metabolismand tissue remodeling [6] and the expression patterns ofmiRNAs are restricted spatially and temporally in differenttissues and during various developmental stages The highconservation of all types of miRNAs is closely related to their

important functions especially regarding the evolution oftheir target genes

Overwhelming studies have demonstrated that miRNAsexert functions during development and that regulating theexpression of their target genes is essential for the devel-opment of multiple systems including the gastrointestinalsystem and neural system [7ndash12] However the role ofmiRNAs in postnatal heart growth is seldom discussed Theintegration and interaction of the effects of miRNAs onthe heart provide a regulatory guarantee of cardiac geneexpression [13]These miRNAsmay also have a potential rolein regulating postnatal heart growth

In the present study we explored the temporal patternof cardiac- and muscle-specific miRNAs during postnatalheart growth as well as the potential target genes of thesemiRNAs as regulators of postnatal heart growthThe data willprovide novel insights for the development of postnatal heartgrowth

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 6241763 6 pageshttpdxdoiorg10115520166241763

2 BioMed Research International

miR-133b-3p

miR-206-3p

miR-208a-3p

miR-208b-3p

miR-499-5p

miR-1a-3p miR-133a-3p

00

05

10

15Re

lativ

e to

U6

00

05

10

15

20

Rela

tive t

o U

6

00

05

10

15

Rela

tive t

o U

6

00

05

10

15

20

25

Rela

tive t

o U

6

00

05

10

15

Rela

tive t

o U

6

0

1

2

3

Rela

tive t

o U

6

00

05

10

15

Rela

tive t

o U

6

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

lowast

lowast

lowast

lowast

lowast

lowast

lowast

Figure 1 Validation of time-varying miRNAs using qRT-PCR miRNAs levels in mice heart at days 0 3 8 and 14 are shown lowast indicatessignificant differences (119901 lt 005) between two groups

BioMed Research International 3

PITA

microRNAorg miRWalk

PITA

microRNAorgmiRWalk

1028

52856

226

10142

18691374

Figure 2 A 3-way comparison of predictions from microRNAorgmiRWalk and PITA The red green and blue sets stand for targetgenes predicted by databases microRNAorg miRWalk and PITArespectively

2 Materials and Methods

21 Mice Male and female wild-type C57BL6 mice werepurchased from the Shanghai Laboratory Animal Center(SLAC Shanghai China) and housed in specific pathogen-free (SPF) conditions on a 12 h light12 h dark cycle in atemperature-controlled room (21ndash23∘C) All animal experi-ments were conducted under the guidelines of the humaneuse and care of laboratory animals for biomedical researchpublished by the National Institutes of Health (no 85-23revised 1996) This study was approved by the Local EthicCommittee of Animal Experiments at Tongji University

22 Quantitative Reverse Transcriptase-Polymerase ChainReaction (qRT-PCR) To collect heart tissues for qRT-PCRanalysis mice were sacrificed by cervical dislocation atpostnatal days 0 3 8 and 14 The tissues were iso-lated rapidly removed frozen fresh in liquid nitrogenand stored at minus80∘C until use Total RNA was extractedusing TRIzol reagent (Invitrogen) For miRNA analysiscDNA was generated and the amplification and detec-tion of specific products were performed on an ABI7900 qPCR System U6 was used as an internal con-trol to normalize miRNA expression Primers sequencesof miRNAs (forward 51015840-31015840) were designed as followsmiR-1a-3p ACGATGGAATGTAAAGAAGT miR-133a-3p ACGATTTGGTCCCCTTCAAC miR-133b-3p ACG-ATTTGGTCCCCTTCAAC miR-208a-3p ACGAATAAG-ACGAGCAAAAA miR-208b-3p ACGAATAAGACG-AACAAAAG miR-206-3p ACGATGGAATGTAAGGAA-GT miR-499-5p ACGATTAAGACTTGCAGTG common

reverse GTGCAGGGTCCGAGGT primers sequences ofU6 (forward and reverse 51015840-31015840) GCTTCGGCAGCACAT-ATACTAAAAT and CGCTTCACGAATTTGCGTGTC-AT Expression values were presented as fold change 2minusΔ(ΔCT)

where ΔCT = (CT gene of interest minusCT internal control)

23 Bioinformatic Analysis GeneSpring GX software wasused to predict the target genes of the miRNAs and thesepredictions were compared with 3 databases (microRNAorgPITA and miRWalk) and integrated into a Venn diagramto demonstrate interactions among the databases The GeneOntology (GO) database was used to describe 3 attributes ofthe identified gene products molecular function subcellularlocation and related biological processes Molecule and genenetworks were analyzed by the Kyoto Encyclopedia of Genesand Genomes (KEGG) pathway database

24 Statistical Analysis One-way ANOVA was conductedwith a Bonferronirsquos post hoc test Data analyses were per-formed using SPSS 200 software and all statistical testswere two-sided 119901 lt 005 was considered to be statisticallysignificant

3 Results

31 Distinct Temporal miRNA Expression Profiles in Heartsduring Postnatal Cardiac Development in Mice The periodof cardiac muscle postnatal growth until 14 days after birthis defined as the maturation and differentiation stage To testwhether the observed cardiac or muscle miRNA expressionprofiles changes are temporal we compared the miRNAexpression profiles of mice hearts at postnatal days 0 3 8and 14 by using qRT-PCR and foundmiR-1a-3pmiR-133b-3pmiR-208b-3p and miR-206-3p were significantly decreasedwhile miR-208a-3p was upregulated (Figure 1)

32 Target Gene GO and Pathway Analysis We used themicroRNAorg PITA and miRWalk databases to predict thetarget genes of differentially expressed miRNAs in hearttissues at different time points during postnatal developmentby usingGeneSpring software Next we examined the overlapof the resulting gene lists among these 3 databases andconstructed a Venn diagram There were 226 overlappinggenes that were most likely to be targets of miRNAs duringheart postnatal growth listed in the analysis results (Figure 2)

The function of up- and downregulated genes was classi-fied by Gene Ontology (httpgeneontologyorg) based on3 attributes molecular function biological processes andsubcellular location In this study differentially expressedmRNAs were enriched in numerous biological processesincluding the regulation of blood vessel size cell cycle andmigration transcription DNA replication ephrin receptorsignaling heart valve development and positive regulationofmyoblast proliferation (Figure 3(a)) Similarly the nucleusnucleoplasm and cytoplasmwere the identified cellular com-ponents affected (Figure 3(b)) whereas the affected molec-ular functions include the following poly(A) RNA bindingcytoskeletal adaptor activity and protein kinase binding(Figure 3(c))


Biological process

1 2 3 4 50

minuslog10 (p value)

Spleen developmentNegative regulation of transcription from RNA polymerase II promoter

Lymph vessel developmentBlood vessel remodeling

Blood vessel endothelial cell migrationPhosphate ion transport

Cell migrationSequestering of actin monomers

Blood vessel morphogenesisPositive regulation of myoblast proliferation

Membrane raft assemblyRegulation of potassium ion transport

Mesenchymal cell developmentRegulation of cell cycle

Heart valve developmentmRNA splice site selection

Regulation of transcription DNA-templatedEphrin receptor signaling pathway

Transcription DNA-templatedRegulation of blood vessel size

(a)

Cellular component

1 2 3 4 5 6 7 80


Dendritic spineNucleolus Podosome

Secretory granule Node of Ranvier

Midbody Cell cortex

Basement membrane Nuclear matrix

Collagen trimer Transcription factor complex

Cortical cytoskeleton Cleavage furrow

Neuromuscular junction Stress fiber

Cytosol Sarcoplasm Cytoplasm

Nucleoplasm Nucleus

(b)

Molecular function

1 2 3 4 50


Transcription factor activity sequence-specific DNA bindingActin binding

AU-rich element binding mRNA binding Protein binding

DNA binding Nucleocytoplasmic transporter activity

Rab GTPase binding mRNA 3998400-UTR AU-rich region binding

RNA binding Histone demethylase activity

Sequence-specific DNA bindingInositol-145-trisphosphate 3-kinase activity

Ephrin receptor binding Inositol hexakisphosphate 3-kinase activity Inositol hexakisphosphate 1-kinase activity Inositol hexakisphosphate 5-kinase activity

Protein kinase binding Cytoskeletal adaptor activity

Poly(A) RNA binding

(c)

Figure 3 GO analysis for differentially expressed mRNAs (andashc) GO analysis according to biological process cellular component andmolecular function respectively ranked by enrichment score (minuslog10 (119901 value))


KEGG

Pathways in cancerPhosphatidylinositol signaling system

Calcium signaling pathway Purine metabolism

Protein digestion and absorption Glycosaminoglycan biosynthesis chondroitin sulfatedermatan sulfate

Dilated cardiomyopathyECM-receptor interaction

Small cell lung cancer Hypertrophic cardiomyopathy (HCM)

Cardiac muscle contraction Renal cell carcinoma

Regulation of actin cytoskeleton Epstein-Barr virus infection

Adherens junction Dorsoventral axis formation

p53 signaling pathway Cell cycle

Axon guidance RNA transport

1 20


Figure 4 Pathway analysis based on the KEGG database Ranked by enrichment score (minuslog10 (119901 value))

Moreover KEGG pathway analysis identified the fol-lowing significantly (119901 lt 005) affected pathways cAMPsignaling vascular smooth muscle contraction regulation ofactin cytoskeleton neurotrophin signaling and cGMP-PKGsignaling (Figure 4)

4 Discussion

The heart is the first functional organ during embryonicdevelopment and plays an important role in the growthand maintenance of higher organisms In contrast to mostorgans the heart is more sensitive to small changes in thegene expression during development Even subtle biologicalperturbations in cardiac structure may result in catastrophicconsequences [14] Elucidating the effects of those subtleperturbations is key to fully understanding the molecularmechanisms of heart development

MiRNAs which are involved in a variety of mechanismsthat regulate gene expression play vital roles in heart forma-tion and cardiovascular disease [15ndash18] Increasing evidencehas indicated the diagnostic value and clinical implicationsof several miRNAs in heart diseases such as miR-433 miR-21 miR-378 and miR-940 [19ndash21] An increasing numberof miRNAs with different functions in heart developmenthave also been identified including miR-1 miR-208 miR-133miR-206miR-126miR-143miR-145 andmiR-499 fromthis group we analyzed the 7 miRNAs most relevant topostnatal heart growth Here we found that the time afterbirth is responsible for the changes of the observed miRNAexpression profiles These results indicated that the miRNAexpression levels present significant differences as a restrictedtemporal expression pattern during different developmentalstages

Previous studies showed that miR-1 and miR-133 arehighly correlated with heart development and miR-1 was

the first miRNA to be implicated in heart development [22]Several studies showed that these two gene clusters wererelated to processes involved in cardiac muscle develop-ment including mediated embryonic development embry-onic stem cell differentiation proliferation and apoptosissarcomere disarray cardiac fibrosis cardiac rhythm controland remodeling [23ndash26] However more subtle regulation ofthese gene clusters results in antagonistic effects in contrastto their more established roles [27 28] The functions of theidentified up- and downregulated geneswere classified byGOanalysis The results showed a series of potential regulatoryfunctions in cell development cytoskeleton formation andangiogenesis which suggest that these miRNAs may beinvolved in postnatal heart development When excludingthe two miRNAs with the strongest influence on geneexpression in the heart there are many other miRNAs thathave been functionally analyzed in the cardiovascular systemand commonly promote more balanced development of theheart Finally only fully understanding and appreciating themicroRNA regulatory networks in cardiovascular develop-ment can provide a new perspective on heart disorders as wellas new therapeutic targets for congenital heart disease

In conclusion the present study explores the temporalpattern of several cardiac- and muscle-specific miRNAs inpostnatal heart development By improving our understand-ing of cardiac growth these results provide new insights inthe diagnosis and treatment of heart diseases

Competing Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contributions

Pujiao Yu HongbaoWang and Yuan Xie contributed equallyto this work


Acknowledgments

This study was supported by the grants from Natural ScienceFoundation of Shanghai (14ZR1438300 to Hongbao Wang14ZR1437900 to Lin Che) and National Natural ScienceFoundation of China (81472158 to Lin Che 81541007 toHongbao Wang)

References

[1] J Li J Xu Y Cheng F Wang Y Song and J Xiao ldquoCirculatingmicroRNAs as mirrors of acute coronary syndromes MiRacleor quagMirerdquo Journal of Cellular and Molecular Medicine vol17 no 11 pp 1363ndash1370 2013

[2] J Xiao D Liang H Zhang et al ldquoMicroRNA-204 is requiredfor differentiation of human-derived cardiomyocyte progenitorcellsrdquo Journal of Molecular and Cellular Cardiology vol 53 no6 pp 751ndash759 2012

[3] J Xu J Zhao G Evan C Xiao Y Cheng and J XiaoldquoCirculating microRNAs novel biomarkers for cardiovasculardiseasesrdquo Journal of Molecular Medicine vol 90 no 8 pp 865ndash875 2012

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

[5] A Kozomara and S Griffiths-Jones ldquoMiRBase annotating highconfidence microRNAs using deep sequencing datardquo NucleicAcids Research vol 42 no 1 pp D68ndashD73 2014

[6] S Matkovich ldquoMicroRNAs in the stressed heart sorting thesignal from the noiserdquo Cells vol 3 no 3 pp 778ndash801 2014

[7] T Thum D Catalucci and J Bauersachs ldquoMicroRNAs novelregulators in cardiac development and diseaserdquo CardiovascularResearch vol 79 no 4 pp 562ndash570 2008

[8] S C Morgan H-Y Lee F Relaix L L Sandell J M Levorseand M R Loeken ldquoCardiac outflow tract septation failure inPax3-deficient embryos is due to p53-dependent regulation ofmigrating cardiac neural crestrdquo Mechanisms of Developmentvol 125 no 9-10 pp 757ndash767 2008

[9] P A M Roest D G M Molin C G Schalkwijk et alldquoSpecific local cardiovascular changes of N120576-(carboxymethyl)lysine vascular endothelial growth factor and Smad2 in thedeveloping embryos coincide with maternal diabetes-inducedcongenital heart defectsrdquoDiabetes vol 58 no 5 pp 1222ndash12282009

[10] F Wang S A Fisher J Zhong Y Wu and P Yang ldquoSuperoxidedismutase 1 in vivo ameliorates maternal diabetes mellitus-induced apoptosis and heart defects through restoration ofimpaired Wnt signalingrdquo Circulation Cardiovascular Geneticsvol 8 no 5 pp 665ndash676 2015

[11] F Wang E A Reece and P Yang ldquoOxidative stress is responsi-ble for maternal diabetes-impaired transforming growth factorbeta signaling in the developingmouse heartrdquoAmerican Journalof Obstetrics and Gynecology vol 212 no 5 pp 650e1ndash650e112015

[12] F Wang Y Wu M J Quon X Li and P Yang ldquoASK1mediates the teratogenicity of diabetes in the developing heartby inducing ER stress and inhibiting critical factors essentialfor cardiac developmentrdquo American Journal of PhysiologymdashEndocrinology and Metabolism vol 309 no 5 pp E487ndashE4992015

[13] N Liu and E N Olson ldquoMicroRNA regulatory networks incardiovascular developmentrdquo Developmental Cell vol 18 no 4pp 510ndash525 2010

[14] E N Olson ldquoGene regulatory networks in the evolution anddevelopment of the heartrdquo Science vol 313 no 5795 pp 1922ndash1927 2006

[15] S Ikeda S W Kong J Lu et al ldquoAltered microRNA expressionin human heart diseaserdquo Physiological Genomics vol 31 no 3pp 367ndash373 2007

[16] KOnoYKuwabara and JHan ldquoMicroRNAs and cardiovascu-lar diseasesrdquo FEBS Journal vol 278 no 10 pp 1619ndash1633 2011

[17] M Basson ldquoMicroRNAs loom large in the heartrdquo NatureMedicine vol 13 no 5 p 541 2007

[18] S P R Romaine M Tomaszewski G Condorelli and N JSamani ldquoMicroRNAs in cardiovascular disease an introduc-tion for cliniciansrdquo Heart vol 101 no 12 pp 921ndash928 2015

[19] T Xu Q Zhou L Che et al ldquoCirculating miR-21 miR-378 andmiR-940 increase in response to an acute exhaustive exercisein chronic heart failure patientsrdquo Oncotarget vol 7 no 11 pp12414ndash12425 2016

[20] L Tao Y Bei P Chen et al ldquoCrucial role of miR-433 inregulating cardiac fibrosisrdquoTheranostics vol 6 no 12 pp 2068ndash2083 2016

[21] H Wang Y Bei S Shen et al ldquomiR-21-3p controls sepsis-associated cardiac dysfunction via regulating SORBS2rdquo Journalof Molecular and Cellular Cardiology vol 94 pp 43ndash53 2016

[22] Y Zhao E Samal and D Srivastava ldquoSerum response factorregulates a muscle-specific microRNA that targets Hand2 dur-ing cardiogenesisrdquoNature vol 436 no 7048 pp 214ndash220 2005

[23] K N Ivey A Muth J Arnold et al ldquoMicroRNA regulation ofcell lineages in mouse and human embryonic stem cellsrdquo CellStem Cell vol 2 no 3 pp 219ndash229 2008

[24] Y Zhao J F Ransom A Li et al ldquoDysregulation of cardiogen-esis cardiac conduction and cell cycle in mice lacking miRNA-1-2rdquo Cell vol 129 no 2 pp 303ndash317 2007

[25] N Liu S Bezprozvannaya A H Williams et al ldquoMicroRNA-133a regulates cardiomyocyte proliferation and suppressessmooth muscle gene expression in the heartrdquo Genes amp Devel-opment vol 22 no 23 pp 3242ndash3254 2008

[26] N Xu T Papagiannakopoulos G Pan J AThomson and K SKosik ldquoMicroRNA-145 regulates OCT4 SOX2 and KLF4 andrepresses pluripotency in human embryonic stem cellsrdquo Cellvol 137 no 4 pp 647ndash658 2009

[27] C Xu Y Lu Z Pan et al ldquoThe muscle-specific microRNAsmiR-1 and miR-133 produce opposing effects on apoptosis bytargeting HSP60 HSP70 and caspase-9 in cardiomyocytesrdquoJournal of Cell Science vol 120 no 17 pp 3045ndash3052 2007

[28] J-F Chen E M Mandel J M Thomson et al ldquoThe role ofmicroRNA-1 andmicroRNA-133 in skeletalmuscle proliferationand differentiationrdquoNature Genetics vol 38 no 2 pp 228ndash2332006

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


miR-133b-3p

miR-206-3p

miR-208a-3p

miR-208b-3p

miR-499-5p

miR-1a-3p miR-133a-3p

00

05

10

15Re

lativ

e to

U6

00

05

10

15

20

Rela

tive t

o U

6

00

05

10

15

Rela

tive t

o U

6

00

05

10

15

20

25

Rela

tive t

o U

6

00

05

10

15

Rela

tive t

o U

6

0

1

2

3

Rela

tive t

o U

6

00

05

10

15

Rela

tive t

o U

6

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

3 d 8 d 2w0 d

lowast

lowast

lowast

lowast

lowast

lowast

lowast

Figure 1 Validation of time-varying miRNAs using qRT-PCR miRNAs levels in mice heart at days 0 3 8 and 14 are shown lowast indicatessignificant differences (119901 lt 005) between two groups


PITA

microRNAorg miRWalk

PITA

microRNAorgmiRWalk

1028

52856

226

10142

18691374









3 Results





Biological process

1 2 3 4 50












(a)

Cellular component

1 2 3 4 5 6 7 80




Midbody Cell cortex






Nucleoplasm Nucleus

(b)

Molecular function

1 2 3 4 50










Poly(A) RNA binding

(c)



KEGG











1 20




4 Discussion






Competing Interests





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PITA

microRNAorg miRWalk

PITA

microRNAorgmiRWalk

1028

52856

226

10142

18691374









3 Results





Biological process

1 2 3 4 50












(a)

Cellular component

1 2 3 4 5 6 7 80




Midbody Cell cortex






Nucleoplasm Nucleus

(b)

Molecular function

1 2 3 4 50










Poly(A) RNA binding

(c)



KEGG











1 20




4 Discussion






Competing Interests





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Biological process

1 2 3 4 50












(a)

Cellular component

1 2 3 4 5 6 7 80




Midbody Cell cortex






Nucleoplasm Nucleus

(b)

Molecular function

1 2 3 4 50










Poly(A) RNA binding

(c)



KEGG











1 20




4 Discussion






Competing Interests





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















KEGG











1 20




4 Discussion






Competing Interests





Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Deregulated Cardiac Specific...

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