The Involvement of miR-29b-3p in Arterial Calcification by ...downloads.hindawi.com/journals/bmri/2017/6713606.pdffication rat group, miR-29b-3p was significantly decreased (𝑃=0.0035)

Research ArticleThe Involvement of miR-29b-3p in Arterial Calcificationby Targeting Matrix Metalloproteinase-2

Wenhong Jiang1 Zhanman Zhang1 Han Yang1 Qiuning Lin1

Chuangye Han2 and Xiao Qin1

1Department of Vascular Surgery The First Affiliated Hospital of Guangxi Medical University Nanning Guangxi 530021 China2Department of Hepatobiliary Surgery The First Affiliated Hospital of Guangxi Medical University Nanning Guangxi 530021 China

Correspondence should be addressed to Xiao Qin dr qinxiaohotmailcom

Received 25 July 2016 Revised 5 November 2016 Accepted 15 November 2016 Published 9 January 2017

Academic Editor Diego Franco

Copyright copy 2017 Wenhong Jiang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Vascular calcification is a risk predictor and common pathological change in cardiovascular diseases that are associated with elastindegradation and phenotypic transformation of vascular smooth muscle cells via gelatinase matrix metalloproteinase-2 (MMP2)However the mechanisms involved in this process remain unclear In this study we investigated the relationships between miR-29b-3p and MMP2 to confirmmiR-29b-3p-mediated MMP2 expression at the posttranscriptional level in arterial calcification Inmale Sprague Dawley rats arterial calcification was induced by subcutaneous injection of a toxic dose of cholecalciferol In vivo thequantitative real-time polymerase chain reaction (qRT-PCR) showed that MMP2 expression was upregulated in calcified arterialtissues and miR-29b-3p expression was downregulated There was a negative correlation between MMP2 mRNA expression andmiR-29b-3p levels (119875 = 00014 1198772 = 0481) Western blotting showed that MMP2 expression was significantly increased in ratstreated with cholecalciferol In vitro overexpression of miR-29b-3p led to decreased MMP2 expression in rat vascular smoothmuscle cells while downregulation of miR-29b-3p expression led to increasedMMP2 expression Moreover the luciferase reporterassay confirmed that MMP2 is the direct target of miR-29b-3p Together our results demonstrated that a role of miR-29b-3p invascular calcification involves targeting MMP2

1 Introduction

Arterial calcification increases morbidity and mortality incardiovascular diseases This process is also called ectopic orpathological mineralization of soft tissue and is considered aresult of calciumphosphate crystal deposition on the vascularwalls This process can also be divided into two types basedon the site of mineral deposits including intimal calcificationand medial calcification The former attributed to inflam-mation oxidative stress and disorders of lipid metabolismwithin the vascular walls is common in atherosclerosis andcoronary artery disease and contributes directly to vascularstenosis myocardial ischemia and infarction The latternamed Monckebergrsquos medial sclerosis or elastocalcinosis isa common pathological characteristic of vessels associatedwith aging diabetes renal failure and vascular injuriesThe two processes are followed by elastic fiber degradationand mineral deposits as linear deposits along the elastic

lamellae leading to arterial stiffening hypertension andmyocardial hypertrophy [1ndash3] Using different calcificationmodels researchers have recently conducted numerous stud-ies involving the pathogenesis of vascular calcification Theyhave found that vascular calcification is a complex activecell-regulated process which shares numerous similarities tophysiological mineralization found in bones teeth [4] andcartilage tissue which have been detected by imaging [5]Accumulating evidence has suggested that vascular calcifi-cation is related to transdifferentiation to a chondrocyte orosteoblast-like phenotype of vascular smooth muscle cells(VSMCs) [6 7] andmatrix vesicle or apoptotic bodies whichprovides binding sites for calcium phosphate [8 9] Elastindegradation which has a high affinity for calcium [10] caninduce a osteogenic response in VSMCs [11 12] Previousstudies have provided abundant information about the patho-genesis of vessel calcification but the precise mechanismshave yet to be fully characterized

HindawiBioMed Research InternationalVolume 2017 Article ID 6713606 9 pageshttpsdoiorg10115520176713606

2 BioMed Research International

Matrix metalloproteinases-2 (MMP2) a gelatinase thathas an important role in matrix degradation and vascularremodeling is involved in many cardiovascular diseasesincluding aortic aneurysm formation atherosclerosis andmyocardial fibrosis [13] Upregulation of MMP2 expressionand activity have been reported in VSMCs and animal calcifi-cationmodels whichmediate elastin degradation producingsoluble elastin peptides and causing release and activationof the latent TGF-1205731 complex from the extracellular matrixof vessel walls In cultured rat VSMCs [14] a soluble elastinpeptide upregulated the expression of typical bone proteinswith synergism of TGF-1205731 via elastin laminin receptorsignaling [15ndash17] Furthermore TGF-1205731 and its mediatedsignaling pathway have important roles in physiological andvascular calcification [18] MMP2 upregulation contributesto bone morphogenetic protein 2 (BMP2) expression in a120573-glycerophosphate-induced VSMC calcification model [19]that could increaseRUNX andMSX2 expression and promoteVSMC calcification [20]The regulation ofMMP2 expressionis therefore associated with the prevention and treatment ofvascular calcification However few studies have reportedthat posttranscriptional regulation of MMP2 is involved invascular calcification

In recent years microRNAs a novel class of endogenoussmall single-stranded noncoding RNAs that mediate epi-genetic regulation have been shown to play a role in geneexpression in many diseases The microRNAs induce specificmRNA degradation or block translation by binding to the31015840-UTR of the targeting mRNA [21] In vascular biologymicroRNA dysregulation was found to contribute to variouspathological conditions (atherosclerosis and a proliferativethickening and restenosis after vessel injury) through themodification of vessel cell functions [22 23] There has beenevidence that specific miRNAs also play critical roles in theregulation of vascular calcification both in vitro and in vivoOsteoblast-like phenotypic VSMC and elastin degradationplay central roles in vessel calcification Gain- and loss-of-function miRNA studies and luciferase reporter assaysreported that miR-204 [24] miR-133a [25] and miR-30b-c[26] inhibited the osteogenic differentiation of mice VSMCsand human coronary artery smoothmuscle cells (SMCs) dur-ing 120573-glycerophosphate-induced calcification through thetargeting of RUNX2 However miR-mediated MMP expres-sion in arterial calcification needs to be further characterized

The miR-29 family (miR-29a miR-29b-3p and miR-29c) has been previously implicated in multiple pathologicalchanges in cardiovascular diseases It has been reported thatperipheral plasma levels of miR-29a in hypertrophic car-diomyopathy patients were significantly upregulated whencompared with a control group and these levels correlatedwith both hypertrophy and fibrosis [27] Furthermore allmembers of the miR-29 family were downregulated inmyocardial tissue adjacent to the infarct and during cardiacfibrosis after acutemyocardial infarction inmice and humans[28] In addition accumulating evidence suggests that miR-29 downregulation inhibits dilation of aneurysms and is help-ful in an early fibrotic response at the aortic wall by increasingtarget gene expression in murine models of experimentalaneurysms and human aneurysm tissues [29] In recent years

some studies have reported that miR-29 is a novel diagnosticbiomarker and therapeutic target for cancer because it playsan important role in angiogenesis invasion andmetastasis oftumors by regulating MMP2 expression [30]

In this study we elucidated the relationships betweenMMP2 and miR-29b-3p during arterial calcification andcharacterized the effects and mechanisms for regulation ofcalcification in vitro and in vivo Taken together these resultsprovide a candidate molecule and model for therapeuticintervention of vascular calcification

2 Materials and Methods

21 Ethics Statement All animal studies were approved by theEthics Committee of Guangxi Medical University (ApprovalNumber 201511018) The studies conformed with the guidefor the care and use of laboratory animals published by theChina National Institutes of Health

22 Rat Thoracic Aorta Calcification Model Male SpragueDawley rats which were from the experimental animalcenter of Guangxi Medical University with a mean bodyweight of 150ndash200 g were used for the studies The animalshad free access to water and food After an acclimatizationperiod of 2 days the animals were randomly allocated toexperimental groups The rat thoracic aorta calcificationmodel was established as previously described [31 32] Ratswere killed 7 days after the first vitamin D3 injection bycervical dislocation while they were undermetofane-inducedanesthesia The aortic tissues between the aortic arch and1 cm below the renal branch were immediately removed afterdeath The tissues of the thoracic aorta were fixed in 4paraformaldehyde for at least 24 h at room temperatureSectioning and histological staining (HE and Von Kossastaining) of the paraformaldehyde-fixed tissues were per-formed for histological analyses ofmineral accumulation andfor assessment of success in creating a rat thoracic aortacalcificationmodelTheother aortic tissueswere immediatelyfrozen in liquid nitrogen until they were analyzed for RNAand protein content

23 Von Kossa and HE Staining Paraffin blocks of aortictissue were cut into 8 um sections and subjected to Von Kossastaining by following the protocol of the Von Kossa stainingkit (Jianchengbio Nanjing China) In brief the slides weredeparaffinized hydrated with distilled water and incubatedin 5 silver nitrate solution for 1 h under a strong sunlightThe silver nitrate solution was then diluted with distilledwater and the sections were incubated with a 5 thiosulphatesolution for 10min Other sections were stained with HEImages were captured using a light microscope and calciumphosphate deposits were detected as black-stained areas

24 Cell Culturing and Transfection VSMC lines (A7R5ATCC CRL-1444) (Aolu Biotech Shanghai China) werecultured in a humidified atmosphere at 37∘C and 5 CO

2

Standard culture medium consisted of Dulbeccorsquos ModifiedEagle Medium (DMEM) with 50 120583gmL streptomycin and50 unitsmL penicillin and was supplemented with 10

BioMed Research International 3

fetal bovine serum (FBS) (Grand Island Biological GrandIsland NY USA) Cell layers were detached with 025trypsin002 EDTA solution (Solarbio Beijing China)Cells were maintained at 80ndash90 confluency by passagingas needed A7R5 VSMCs were used from passages 5ndash10

Mimics (51015840-UUGUGACUAAAGUUUACCACGAU-31015840)and inhibitors of rno-miR-29b-3p (51015840-AACACUGAU-UUCAAAUGGUGCUA-31015840) and the negative control(51015840-UCACAACCUCCUAGAAAGAGUAGA-31015840) weretransfected into A7R5 VSMCs The transfection used a CPtransfection kit (Ribobio Guangzhou China) according tothe manufacturerrsquos protocol The cells were plated in six-wellplates with DMEM supplemented with 10 FBS at a densityof 1 times 105 cells per well 24 h prior to the transfection Forexpression miR-29b-3p and mimics of miR-29b-3p weretransfected into cells at a final concentration of 50 nmAfter transfection for 48 h RNA was extracted usingTRIzol (Invitrogen Carlsbad CA USA) and successfultransfection was confirmed by the quantitative real-timepolymerase chain reaction (qRT-PCR) for miR-29b-3pRandom sequence miR was used as a negative control Toinhibit miR-29b-3p expression cells were transfected with150 nm inhibitors of miR-29b-3p using a similar method asdescribed for the transfection of mimics

25 qRT-PCR for MMP2 and rno-miR-29b-3p Total RNAcontaining the miRs was isolated from cells and frozen aortatissues using TRIzol according to themanufacturerrsquos instruc-tions (Invitrogen) For each sample the total RNA contentwas determined by measuring the absorbance at 260 nm andthe purity was determined using A260A280 ratios For qRT-PCR the first strand cDNAofmiRNAandmRNAwas reversetranscribed from total RNA using the Mir-XTM miRNAfirst strand synthesis kit and PrimeScript RT reagent kitwith gDNA eraser (Takara Biotechnology Dalian China)respectively and 2 120583L of the first strand cDNA was used fordetecting mRNA and miRNA expression using the real-timepolymerase chain reaction (RT-PCR) (ABI 7500 Fast AppliedBiosystems Darmstadt Germany) using the FastStart SYBRGreen Kit (Roche East Sussex UK) The specific PCRprimers used were rat MMP2 (forward primer 51015840-ACCTTG-ACCAGAACACCATCGAG-31015840 reverse primer 51015840-CAG-GGTCCAGGTCAGGTGTGTA-31015840) and rat 120573-actin (for-ward primer 51015840-GGAGATTACTGCCCTGGCTCCTA-31015840reverse primer 51015840-GACTCATCGTACTCCTGCTTGCTG-31015840) The rat U6 primer and miR-29b-3p were purchasedfrom Genecopoeia (Guangzhou China) Expression of MP2and miR-29b-3p were normalized with 120573-actin and U6respectively and the relative expression was determined bythe comparative CT (2minusΔΔCt) method

26 Western Blot Analysis To investigate rat aorta andcellular MMP2 protein expression western blotting wasperformed according to the Abcam western blot instruc-tions Western blots were performed with 50 120583g of totalprotein The antibodies used were anti-MMP2 (ab37150Abcam Cambridge MA USA) and anti-actin (KC-5A08Kang Cheng Bio-tech Shanghai China) Image-Pro Plus 60software (Media Cybernetics lnc Maryland USA) was used

for analyzing the results of densitometric scans of the bandsThe results were normalized to total protein levels andexpressed as a ratio of 120573-actin

27 Luciferase Reporter Assay The public databases (Tar-getScan httpwwwtargetScanorg and mirbase httpwwwmirbaseorg) were searched for predicting the miRNA forregulating rat MMP2 To confirm the regulation betweenmiR-29b-3p and MMP2 a luciferase reporter assay wasperformed in HEK293T cells The cells were grown to 60confluency in 24-well plates in complete culture medium[high glucoseDMEM (Gibco NewYork USA)with 10 FBS50 120583gmL streptomycin and 50 unitsmL penicillin] withluciferase reporter plasmids carrying wild-type MMP2 31015840-UTR mutant MMP2 31015840-UTR and a 31015840-UTR negative controland rno-miR-29b-3p or rno-miR negative control plasmidand renilla plasmid cotransfected into HEK 293T cells Forassessing the efficacy of the transfection system the TRAF631015840-UTR plasmid and the rno-mir negative control plasmidor has-miR-146b plasmid were also cotransfected into cellsCells were collected 48 h after transfection and analyzed usingthe Luciferase Assay System according to the manufacturerrsquosinstructions (Promega Corporation Madison WisconsinUSA)

28 Statistical Analyses SPSS statistical software for Win-dows version 200 (SPSS Chicago IL USA) was usedfor all analyses Data were expressed as the mean plusmn SDComparisons were made using a two-sample test in twogroups and one-way ANOVAwas used for multiple compar-isons The relationship between rno-miR-29b-3p and MMP2mRNA and protein was determined using linear least squaresregression analysesThe results were reported graphically anda correlation constant (1198772) and a probability value 119875 lt 005was considered statistically significant

3 Results

31 Establishment of a Thoracic Aortic Calcification ModelVon Kossa staining and hematoxylin and eosin (HE) stainingshowed extensive mineral deposition elastic fibers andextracellular matrix degradation in aorta media in the groupreceiving cholecalciferol These pathological changes werenot found in structural features of the vascular walls inthe control group (Figure 1) The results suggested that thevascular calcification model was successfully induced in ratsafter injection with cholecalciferol

32 MMP2 and miR-29b-3p Are Involved in Arterial Calcifi-cation MMP2 and miR-29b-3p are associated with vascularcalcification induced by cholecalciferol in rats Comparedwith the control group the expression of MMP2 mRNAand protein were upregulated in the cholecalciferol treatmentgroup (both 119875 lt 00001 Figure 2) However in the calci-fication rat group miR-29b-3p was significantly decreased(119875 = 00035) Moreover we investigated whether MMP2expression correlated with miR-29b-3p levels which may beinvolved in regulating target gene expression at the post-transcriptional level Correlation analyses showed that there


Con

trol

(a)

Con

trol

(b)

Vita

min

D3

(c)

Vita

min

D3

(d)

Figure 1 Von Kossa and hematoxylin and eosin (HE) staining for aortic sections from the rat (a) Von Kossa staining for aortic sections inthe control group (b) HE staining for aortic sections in the control group (c) Von Kossa staining for aortic sections in the vitamin D3 group(d) HE staining for aortic sections in the vitamin D3 group The black arrow designates excessive mineral deposits

was a negative correlation between MMP2 protein levels andmRNA and miR-29b-3p expression (1198772 = 0443 and 0481resp 119875 = 00026 and 00014 resp) as shown in Figure 2These data suggested thatmiR-29may be involved in vascularcalcification through mediation of MMP2 expression

33 The Effect of Inhibition and Overexpression of miR-29b-3pon MMP2 Expression in Rat VSMCs To further characterizethe relationship between miR-29b-3p and MMP2 we usedbioinformatics and a target prediction database of miRsto determine that MMP2 was a potential target of miR-29b-3p Furthermore exogenous transfection of miRs wasused to determine whether miR-29b-3p can regulate MMP2expression in rat VSMCs The mimic inhibitor and controlsequences of miR-29b-3p were transfected in VSMCs tochange the levels of cellular miR-29b-3p (Figure 3) Theresults of this transfection showed that an increase of miR-29b-3p inhibited MMP2 expression and loss of miR-29b-3p-induced MMP2 expression in rat VSMCs indicatingthat miR-29b-3p had an effect on MMP2 expression eitherdirectly or indirectly (Figure 4)

34 miR-29b-3p Directly Regulates MMP2 Expression Tofurther characterize the mechanisms involving miR-29b-3p-mediatedMMP2 expression a luciferase assaywas performedto determine whether miR-29 can directly target MMP2Figure 5 shows that seven bases of rno-miR-29b-3p in the

seed region are complementary with MMP2 31015840-UTR basesat positions 269ndash275 The relative luciferase activity wassignificantly inhibited by overexpression of miR-29b-3p inthe wild-type 31015840-UTR of the MMP2 group compared withthe MMP2 31015840-UTR-negative control group (119875 lt 0001) andMMP2 31015840-UTR-mutant group (119875 lt 0001) In additiongain of position miR (has-miR-146b) decreased the relativeluciferase activity in the 31015840-UTR (TRAF6) group confirmingthat the transfection system was functional These resultsindicated that MMP2 is a direct target gene of miR-29b-3p

4 Discussion

In the present study we showed that the expression of miR-29b-3p was downregulated in rat models of vascular calcifi-cation induced with cholecalciferol when compared with thecontrol group Furthermore there was a negative correlationbetween miR-29b-3p expression and MMP2 expression

Overexpression of miR-29b-3p or inhibition of miR-29b-3p in rat VSMCs resulted in MMP2 downregulation orupregulation at the protein level respectively Moreover aluciferase reporter assay was used to show that miR-29b-3p directly inhibited MMP2 expression Taken together theresults showed that miR-29b-3p was involved in vascularcalcification via targeting of MMP2 expression

It is well-known that MMP2 plays a pivotal role inthe pathogenesis of vascular calcification involved in elastin


β-Ac

tinM

MP2

C1 M1 C2 M2 C3 M3

(a)

0

2

4

6

8

10

rno-miR-29b MMP2 protein MMP2 mRNA

Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Relat

ive e

xpre

ssio

n

P = 00035

P lt 00001

P lt 00001

(b)

0 1 2 3 4 5 6 7 8

0123456789

10

Relative rno-miR-29b expression

Rela

tive M

MP2

mRN

A ex

pres

sion

P = 00014

R2= 0481

(c)

0 1 2 3 4 5 6 7 8

00

03

06

09

12

15

18


Rela

tive M

MP2

pro

tein

expr

essio

n

P = 00026

R2= 0443

(d)

Figure 2 Analyses ofMMP2 andmiR-29b-3p expression in the vitaminD3 group and the control group (a)Western blot analyses forMMP2protein in rat aortas from the control group (C1 C2 and C3) and the vitamin D3-treated group (M1 M2 and M3) (b) Scatter diagram forexpression of MMP2 and miR-29b-3p MMP2 expression was significantly increased and miR-29b-3p was significantly downregulated inthe vitamin D3 group (c d) Correlation analyses to determine the relationships between miR-29b-3p and MMP2 mRNA levels and MMP2protein in the control and vitamin D3 groups (MMP2mRNA versus miR-29b-3p 1198772 = 0481 119875 = 00014 MMP2 protein versus miR-29b-3p1198772 = 0443 119875 = 00026)

degradation and VSMC transdifferentiation to an osteoblast-like phenotype [19 33ndash35] Inhibition or knock-down ofMMP2 in vitro and in vivo decreased arterial and VSMCscalcification in multiple calcification models [11 12 36ndash40] Similarly in our study miR-29b-3p was downregulatedduring rat calcification compared with the control groupimplying that miR-29b-3p inhibited vascular calcification bysome unknownmechanism Notably upregulation of MMP2negatively correlated with miR-29b-3p in calcified arterialtissues We therefore speculate that miR-29b-3p plays a rolein MMP2 expression and is involved in vascular calcifica-tion Subsequent studies reported that miR-29b-3p directly

regulated MMP2 expression as shown by overexpressionand inhibition of miR-29b-3p with mimics inhibitors anda luciferase reporter assay The relationship between miR-29b-3p andMMP2 was consistent with our finding that miR-29b-3p downregulation resulted in MMP2 upregulation andpromoted vascular calcification in rats

It has been generally accepted that miRs are involved inthe pathology of many cardiovascular diseases through theirexpression regulation of different target genes [41] The ratmiR-29 family includes three members miR-29a miR-29b-3p and miR-29c whose dysregulation has been reported inmany studies Some studies indirectly supported our results


NC miR-NC Inhibitor Mimics00

05

10

15

2030405060

Relat

ive m

iR-2

9b ex

pres

sion

P = 00004

P = 00005

Figure 3 Transfection of miR-29b-3p mimics into vascular smoothmuscle cells increases the miR-29b-3p expression and the inhibitordownregulates miR-29b-3p expression as determined by the quan-titative real-time polymerase chain reaction Data are expressed asthe mean plusmn SD and each experiment was repeated three times

β-Ac

tinM

MP2

NC miR-NC Inhibitor mimics

0

1

2

3

4

Relat

ive M

MP2

pro

tein

expr

essio

n

NC miR-NC Inhibitor Mimics

P = 00067

P = 00119

Figure 4 The effect of inhibition and overexpression of miR-29b-3p on MMP2 expression in rat vascular smooth muscle cells ThemiR-29b-3p mimics and inhibitors regulate MMP2 expression asdetermined by western blot analyses

although there were some differences in some of the studiesOur finding that miR-29b-3p was downregulated in vascularcalcification was consistent with the results of Du et al[42] who reported that miR-29b-3p inhibits vascular smoothmuscle cell calcification by decreasing the target genes dis-integrin and metalloproteinase with thrombospondin motif-7 (ADAMTS-7) expression in an inorganic phosphorus andchronic kidney disease-induced rat calcification model Thedifference of miR-29b-3p target genes in the two studiescan be explained by the observation that specific miRNAs

may target multiple mRNAs and that a specific mRNA maycontain binding sites for different miRNAs In additionthe miR-29 family is involved in aneurysm developmentby mediating extracellular matrix protein synthesis includ-ing Col1a1 Col3a1 Col5a1 and Eln Maegdefessel et al[29] reported that miR-29b-3p downregulation significantlyinhibited abdominal aortic aneurysm (AAA) expansion andprogression and promoted fibrosis within AAA walls todecrease the aneurysm rupture rate in twomice AAAmodelsinduced with porcine pancreatic elastase and angiotensin II(angII) Similar results were observed in human thoracicaneurysm tissue It has been reported that MMPs synthesisand activity are increased in rat and human aneurysm tissueespecially MMP2 and MMP9 which can degrade elastinresulting in less flexibility and loss of the integrity of thevessel walls Elastic fiber degradation is the earliest step inmedial calcification in chronic kidney disease and diabeticpatients Surprisingly there was also a negative correlationbetween miR-29a and total MMP2 expression in humanthoracic aortic aneurysm tissue [43] Furthermore miR-29b-3p largely decreased resulting in myocardial fibrosis ofangiotensin II involved in the TGF-1205731Smad signal pathwayin the angII-induced hypertensive heart diseasemousemodel[44] However many studies have reported that increasingangII can induce MMP2 upregulation and activity whichactivates TGF-1205731Smad signaling and is associated with acentral arterial wall stiffness such as fibrosis and vascularcalcification [45] We therefore speculate that there is anopposite trend among expressions of miR-29b-3p MMP2TGF-beta1 andmiR-29b-3p downregulation contributing tovascular calcification by upregulation of MMP2 and TGF-beta1 in the angII-induced calcification model Recently mclan antiapoptotic Bcl-2 familymember has been also reportedto be a target of miR-29b-3p in HeLa and KMCH cells [46]and miR-29b-3p downregulation inhibited apoptosis andpromoted cancer cell proliferation Apoptotic bodies fromcell apoptosis provide binding sites for minerals involvedin the first step of vascular calcification [47] The resultsalso suggested that miR-29b-3p can negatively regulate vas-cular calcification Moreover in the chronic kidney diseasemice miR-29ab was significantly suppressed in muscle [48]However medial elastin-specific calcification is a prevalentcomplication in patients and animal models with chronickidney disease

Although our results are consistent with other studiesthere is still controversy It has been reported that increasedmiR-29b-3p expression promoted VSMC calcification bydecreasing elastin expression in the inorganic phosphorus-induced VSMC calcification model [49] These differenceswith our study results can be explained in two ways Firstcalcification models can be induced in different ways thatinvolve different mechanisms Many possible mechanismshave not been identified in vitamin D3 intoxication-inducedarterial calcification Nonetheless this process can stimulatebone resorption and elevate serum calcium which playimportant roles in VSMC apoptosis and vesicle releasewhile increased inorganic phosphorus has a major rolein osteogenicchondrogenic differentiation of VSMCs [50]Second we induced the aortic calcification model in rats in


00

05

10

15

Rela

tive l

ucife

rase

activ

ity

P lt 001

P lt 001

P lt 001

Posit

ive3

998400-U

TR+m

iRN

A

3998400-U

TR+m

iRN

A

3998400-U

TR-M

U+m

iRN

A

3998400 -U

TR-N

C+m

iRN

A

Posit

ive 3

998400-U

TR+m

iRN

A-N

C

UCCUUUGGGCUGCCCUGGUGCUC

UUGUGACUAAAGUUUACCACGAU rno-miR-29b3998400

5998400

3998400

5998400

3998400 UTR

position 269ndash275 of MMP2

Figure 5 Target verification between miR-29b-3p and MMP2 in HEK293T cells The luciferase reporter plasmids (31015840-UTR wild-type 31015840-UTR mutant of MMP2 and the 31015840-UTR negative control) and the renilla plasmid were cotransfected into HEK293T cells The TRAF6 andhas-miR-146b genes were used for the positive control group Data was expressed as the mean plusmn SD and each experiment was repeated threetimes

vivo which was regulated by different factors compared withVSMC calcification in vitro These findings are indicative ofthe complex mechanism of gene regulation

Our study had some limitations First the levels of pre-mRNA for MMP2 were not measured in our studies Thetranscriptional activity of a MMP2 decrease may have a rolefor MMP2 downregulation at mRNA and proteins levels inthe cholecalciferol-induced rat calcificationmodel but it doesnot affect the miR-29b-3p inhibition of MMP2 expressionat posttranscriptional levels as shown by transfecting withmiR-29b-3pmimics and inhibitors and the luciferase reporterassay Furthermore whether miR-29b-3p is a treatment forvascular calcification in vivo and in vitro needs furtherstudies In future studies we plan to overexpress and knock-out miR-29b-3p with lentivirus in VSMCs and then testwhether miR-29b-3p plays a role in potential mechanisms ofvascular calcification

5 Conclusion

Our results indicated that miR-29b-3p is involved in theprogression of pathological vascular calcification by targetingMMP2 This process may be a treatment option for vascularcalcification However whether miR-29b-3p can be used as atreatment for vascular calcification both in vivo and in vitroneeds to be confirmed with further studies

Abbreviations

MMP2 Matrix metalloproteinase-2qRT-PCR Quantitative real-time polymerase chain

reactionVSMCs Vascular smooth muscle cellsAAA Abdominal aortic aneurysm

Competing Interests

The authors declare no conflict of interests

Authorsrsquo Contributions

Wenhong Jiang andZhanmanZhang contributed to thisworkequally

Acknowledgments

This work was supported in part by the National NatureScience Foundation of China (no 81160047)

References

[1] A Avogaro and G P Fadini ldquoMechanisms of ectopic calcifi-cation implications for diabetic vasculopathyrdquo CardiovascularDiagnosis and Therapy vol 5 pp 343ndash352 2015


[2] P Lanzer M Boehm V Sorribas et al ldquoMedial vascularcalcification revisited review and perspectivesrdquo EuropeanHeartJournal vol 35 no 23 pp 1515ndash1525 2014

[3] D Proudfoot and C M Shanahan ldquoBiology of calcification invascular cells intima versusmediardquoHerz vol 26 no 4 pp 245ndash251 2001

[4] TMDoherty H Uzui L A Fitzpatrick et al ldquoRationale for therole of osteoclast-like cells in arterial calcificationrdquo The FASEBJournal vol 16 no 6 pp 577ndash582 2002

[5] E Neven S Dauwe M E De Broe P C DrsquoHaese and VPersy ldquoEndochondral bone formation is involved in mediacalcification in rats and in menrdquo Kidney International vol 72no 5 pp 574ndash581 2007

[6] M Y Speer H-Y Yang T Brabb et al ldquoSmooth muscle cellsgive rise to osteochondrogenic precursors and chondrocytes incalcifying arteriesrdquoCirculation Research vol 104 no 6 pp 733ndash741 2009

[7] S A Steitz M Y Speer G Curinga et al ldquoSmooth muscle cellphenotypic transition associated with calcification upregula-tion of Cbfa1 and downregulation of smooth muscle lineagemarkersrdquo Circulation Research vol 89 no 12 pp 1147ndash11542001

[8] L Cui D A Houston C Farquharson and V E MacRaeldquoCharacterisation of matrix vesicles in skeletal and soft tissuemineralisationrdquo Bone vol 87 pp 147ndash158 2016

[9] D Proudfoot J N Skepper L Hegyi A Farzaneh-Far C MShanahan and P L Weissberg ldquoThe role of apoptosis in theinitiation of vascular calcificationrdquo Zeitschrift fur Kardiologievol 90 supplement 3 pp 43ndash46 2001

[10] DW Urry ldquoNeutral sites for calcium ion binding to elastin andcollagen a charge neutralization theory for calcification andits relationship to atherosclerosisrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 68 no4 pp 810ndash814 1971

[11] N Vyavahare P L Jones S Tallapragada and R J Levy ldquoInhi-bition of matrix metalloproteinase activity attenuates tenascin-C production and calcification of implanted purified elastin inratsrdquoAmerican Journal of Pathology vol 157 no 3 pp 885ndash8932000

[12] N Vyavahare M Ogle F J Schoen and R J Levy ldquoElastincalcification and its prevention with aluminum chloride pre-treatmentrdquo The American Journal of Pathology vol 155 no 3pp 973ndash982 1999

[13] A Azevedo A F Prado R C Antonio J P Issa and R F Ger-lach ldquoMatrix metalloproteinases are involved in cardiovasculardiseasesrdquo Basic and Clinical Pharmacology and Toxicology vol115 no 4 pp 301ndash314 2014

[14] M Wang D Zhao G Spinetti et al ldquoMatrix metalloproteinase2 activation of Transforming Growth Factor-1205731 (TGF-1205731) andTGF-1205731-type II receptor signaling within the aged arterial wallrdquoArteriosclerosis Thrombosis and Vascular Biology vol 26 no 7pp 1503ndash1509 2006

[15] A Simionescu K Philips and N Vyavahare ldquoElastin-derivedpeptides and TGF-1205731 induce osteogenic responses in smoothmuscle cellsrdquo Biochemical and Biophysical Research Communi-cations vol 334 no 2 pp 524ndash532 2005

[16] N Hosaka M Mizobuchi H Ogata et al ldquoElastin degrada-tion accelerates phosphate-induced mineralization of vascularsmooth muscle cellsrdquo Calcified Tissue International vol 85 no6 pp 523ndash529 2009

[17] D M Basalyga D T Simionescu W Xiong B T Baxter BC Starcher and N R Vyavahare ldquoElastin degradation and

calcification in an abdominal aorta injury model role of matrixmetalloproteinasesrdquo Circulation vol 110 no 22 pp 3480ndash34872004

[18] N Wang X Wang C Xing et al ldquoRole of TGF-1205731 in bonematrix production in vascular smooth muscle cells inducedby a high-phosphate environmentrdquo NephronmdashExperimentalNephrology vol 115 no 3 pp e60ndashe68 2010

[19] Y-G Zhao F-X Meng B-W Li et al ldquoGelatinases promotecalcification of vascular smooth muscle cells by up-regulatingbone morphogenetic protein-2rdquo Biochemical and BiophysicalResearch Communications vol 470 no 2 pp 287ndash293 2016

[20] S Rong X Zhao X Jin et al ldquoVascular calcification in chronickidney disease is induced by bone morphogenetic protein-2 viaa mechanism involving the Wnt120573-catenin pathwayrdquo CellularPhysiology and Biochemistry vol 34 no 6 pp 2049ndash2060 2014

[21] E Huntzinger and E Izaurralde ldquoGene silencing by microR-NAs contributions of translational repression and mRNAdecayrdquo Nature Reviews Genetics vol 12 no 2 pp 99ndash110 2011

[22] G Condorelli M V G Latronico and E Cavarretta ldquoMicroR-NAs in cardiovascular diseases current knowledge and the roadaheadrdquo Journal of theAmericanCollege of Cardiology vol 63 no21 pp 2177ndash2187 2014

[23] E M Small and E N Olson ldquoPervasive roles of microRNAs incardiovascular biologyrdquoNature vol 469 no 7330 pp 336ndash3422011

[24] R-R Cui S-J Li L-J Liu et al ldquoMicroRNA-204 regulatesvascular smooth muscle cell calcification in vitro and in vivordquoCardiovascular Research vol 96 no 2 pp 320ndash329 2012

[25] X-B Liao Z-Y Zhang K Yuan et al ldquoMiR-133a modulatesosteogenic differentiation of vascular smooth muscle cellsrdquoEndocrinology vol 154 no 9 pp 3344ndash3352 2013

[26] J A F Balderman H-Y Lee C E Mahoney et al ldquoBonemorphogenetic protein-2 decreases microRNA-30b andmicroRNA-30c to promote vascular smooth muscle cellcalcificationrdquo Journal of the American Heart Association vol 1no 6 Article ID e003905 2012

[27] R Roncarati C V Anselmi M A Losi et al ldquoCirculatingmiR-29a among other up-regulated microRNAs is the onlybiomarker for both hypertrophy and fibrosis in patients withhypertrophic cardiomyopathyrdquo Journal of the American Collegeof Cardiology vol 63 no 9 pp 920ndash927 2014

[28] E van Rooij L B Sutherland J E Thatcher et al ldquoDysregula-tion of microRNAs after myocardial infarction reveals a role ofmiR-29 in cardiac fibrosisrdquo Proceedings of the National Academyof Sciences of the United States of America vol 105 no 35 pp13027ndash13032 2008

[29] L Maegdefessel J Azuma R Toh et al ldquoInhibition ofmicroRNA-29b reduces murine abdominal aortic aneurysmdevelopmentrdquo Journal of Clinical Investigation vol 122 no 2pp 497ndash506 2012

[30] J-H Fang H-C Zhou C Zeng et al ldquoMicroRNA-29bsuppresses tumor angiogenesis invasion and metastasis byregulating matrix metalloproteinase 2 expressionrdquo Hepatologyvol 54 no 5 pp 1729ndash1740 2011

[31] P A Price H H June J R Buckley and M K WilliamsonldquoOsteoprotegerin inhibits artery calcification induced by war-farin and by vitamin Drdquo Arteriosclerosis Thrombosis andVascular Biology vol 21 no 10 pp 1610ndash1616 2001

[32] P A Price J R Buckley and M K Williamson ldquoThe aminobisphosphonate ibandronate prevents vitamin D toxicity andinhibits vitamin D-induced calcification of arteries cartilage


lungs and kidneys in ratsrdquo Journal of Nutrition vol 131 no 11pp 2910ndash2915 2001

[33] T Liu J Lin T Ju L Chu and L Zhang ldquoVascular smoothmuscle cell differentiation to an osteogenic phenotype involvesmatrix metalloproteinase-2 modulation by homocysteinerdquoMolecular and Cellular Biochemistry vol 406 no 1-2 pp 139ndash149 2015

[34] N X Chen K D OrsquoNeill X Chen K Kiattisunthorn VH Gattone and S M Moe ldquoActivation of arterial matrixmetalloproteinases leads to vascular calcification in chronickidney diseaserdquo American Journal of Nephrology vol 34 no 3pp 211ndash219 2011

[35] A Sinha and N R Vyavahare ldquoHigh-glucose levels andelastin degradation products accelerate osteogenesis in vascularsmooth muscle cellsrdquo Diabetes and Vascular Disease Researchvol 10 no 5 pp 410ndash419 2013

[36] X Qin M A Corriere L M Matrisian and R J GuzmanldquoMatrixmetalloproteinase inhibition attenuates aortic calcifica-tionrdquoArteriosclerosisThrombosis and Vascular Biology vol 26no 7 pp 1510ndash1516 2006

[37] E Hecht C Freise K V Websky et al ldquoThe matrix metal-loproteinases 2 and 9 initiate uraemic vascular calcificationsrdquoNephrology Dialysis Transplantation vol 31 no 5 pp 789ndash7972016

[38] C Freise K Y Kim and U Querfeld ldquoA Lindera obtusilobaextract blocks calcium-phosphate-induced transdifferentia-tion and calcification of vascular smoothmuscle cells and inter-feres with matrix metalloproteinase-2 and metalloproteinase-9 and NF-120581Brdquo Evidence-Based Complementary and AlternativeMedicine vol 2015 Article ID 679238 8 pages 2015

[39] T Sasaki K Nakamura K Sasada et al ldquoMatrixmetalloproteinase-2 deficiency impairs aortic atheroscleroticcalcification in ApoE-deficient micerdquo Atherosclerosis vol 227no 1 pp 43ndash50 2013

[40] L Jiang J Zhang R E Monticone et al ldquoCalpain-1 regulationof matrix metalloproteinase 2 activity in vascular smoothmuscle cells facilitates age-associated aortic wall calcificationand fibrosisrdquo Hypertension vol 60 no 5 pp 1192ndash1199 2012

[41] N Papageorgiou D Tousoulis E Androulakis et al ldquoThe roleof microRNAs in cardiovascular diseaserdquo Current MedicinalChemistry vol 19 no 16 pp 2605ndash2610 2012

[42] Y Du C Gao Z Liu et al ldquoUpregulation of a disintegrinand metalloproteinase with thrombospondin motifs-7 by miR-29 repression mediates vascular smooth muscle calcificationrdquoArteriosclerosis Thrombosis and Vascular Biology vol 32 no11 pp 2580ndash2588 2012

[43] J A Jones R E Stroud E C OrsquoQuinn et al ldquoSelectivemicroRNA suppression in human thoracic aneurysms rela-tionship of miR-29a to aortic size and proteolytic inductionrdquoCirculation Cardiovascular Genetics vol 4 no 6 pp 605ndash6132011

[44] L-H Wei X-R Huang Y Zhang et al ldquoSmad7 inhibitsangiotensin II-induced hypertensive cardiac remodellingrdquoCar-diovascular Research vol 99 no 4 pp 665ndash673 2013

[45] M Wang J Zhang G Spinetti et al ldquoAngiotensin II activatesmatrix metalloproteinase type II and mimics age-associatedcarotid arterial remodeling in young ratsrdquo American Journal ofPathology vol 167 no 5 pp 1429ndash1442 2005

[46] J L Mott S Kobayashi S F Bronk and G J Gores ldquomir-29regulates Mcl-1 protein expression and apoptosisrdquo Oncogenevol 26 no 42 pp 6133ndash6140 2007

[47] D Proudfoot J N Skepper L Hegyi M R Bennett C MShanahan and P L Weissberg ldquoApoptosis regulates humanvascular calcification in vitro evidence for initiation of vascularcalcification by apoptotic bodiesrdquo Circulation Research vol 87no 11 pp 1055ndash1062 2000

[48] I Martinez D Cazalla L L Almstead J A Steitz and DDiMaio ldquomiR-29 and miR-30 regulate B-Myb expression dur-ing cellular senescencerdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 2 pp 522ndash527 2011

[49] R Sudo F Sato T Azechi and H Wachi ldquoMiR-29-mediatedelastin down-regulation contributes to inorganic phosphorus-induced osteoblastic differentiation in vascular smooth musclecellsrdquo Genes to Cells vol 20 no 12 pp 1077ndash1087 2015

[50] R C Shroff R McNair J N Skepper et al ldquoChronic mineraldysregulation promotes vascular smoothmuscle cell adaptationand extracellular matrix calcificationrdquo Journal of the AmericanSociety of Nephrology vol 21 no 1 pp 103ndash112 2010

Submit your manuscripts athttpswwwhindawicom

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


Matrix metalloproteinases-2 (MMP2) a gelatinase thathas an important role in matrix degradation and vascularremodeling is involved in many cardiovascular diseasesincluding aortic aneurysm formation atherosclerosis andmyocardial fibrosis [13] Upregulation of MMP2 expressionand activity have been reported in VSMCs and animal calcifi-cationmodels whichmediate elastin degradation producingsoluble elastin peptides and causing release and activationof the latent TGF-1205731 complex from the extracellular matrixof vessel walls In cultured rat VSMCs [14] a soluble elastinpeptide upregulated the expression of typical bone proteinswith synergism of TGF-1205731 via elastin laminin receptorsignaling [15ndash17] Furthermore TGF-1205731 and its mediatedsignaling pathway have important roles in physiological andvascular calcification [18] MMP2 upregulation contributesto bone morphogenetic protein 2 (BMP2) expression in a120573-glycerophosphate-induced VSMC calcification model [19]that could increaseRUNX andMSX2 expression and promoteVSMC calcification [20]The regulation ofMMP2 expressionis therefore associated with the prevention and treatment ofvascular calcification However few studies have reportedthat posttranscriptional regulation of MMP2 is involved invascular calcification

In recent years microRNAs a novel class of endogenoussmall single-stranded noncoding RNAs that mediate epi-genetic regulation have been shown to play a role in geneexpression in many diseases The microRNAs induce specificmRNA degradation or block translation by binding to the31015840-UTR of the targeting mRNA [21] In vascular biologymicroRNA dysregulation was found to contribute to variouspathological conditions (atherosclerosis and a proliferativethickening and restenosis after vessel injury) through themodification of vessel cell functions [22 23] There has beenevidence that specific miRNAs also play critical roles in theregulation of vascular calcification both in vitro and in vivoOsteoblast-like phenotypic VSMC and elastin degradationplay central roles in vessel calcification Gain- and loss-of-function miRNA studies and luciferase reporter assaysreported that miR-204 [24] miR-133a [25] and miR-30b-c[26] inhibited the osteogenic differentiation of mice VSMCsand human coronary artery smoothmuscle cells (SMCs) dur-ing 120573-glycerophosphate-induced calcification through thetargeting of RUNX2 However miR-mediated MMP expres-sion in arterial calcification needs to be further characterized

The miR-29 family (miR-29a miR-29b-3p and miR-29c) has been previously implicated in multiple pathologicalchanges in cardiovascular diseases It has been reported thatperipheral plasma levels of miR-29a in hypertrophic car-diomyopathy patients were significantly upregulated whencompared with a control group and these levels correlatedwith both hypertrophy and fibrosis [27] Furthermore allmembers of the miR-29 family were downregulated inmyocardial tissue adjacent to the infarct and during cardiacfibrosis after acutemyocardial infarction inmice and humans[28] In addition accumulating evidence suggests that miR-29 downregulation inhibits dilation of aneurysms and is help-ful in an early fibrotic response at the aortic wall by increasingtarget gene expression in murine models of experimentalaneurysms and human aneurysm tissues [29] In recent years

some studies have reported that miR-29 is a novel diagnosticbiomarker and therapeutic target for cancer because it playsan important role in angiogenesis invasion andmetastasis oftumors by regulating MMP2 expression [30]

In this study we elucidated the relationships betweenMMP2 and miR-29b-3p during arterial calcification andcharacterized the effects and mechanisms for regulation ofcalcification in vitro and in vivo Taken together these resultsprovide a candidate molecule and model for therapeuticintervention of vascular calcification

2 Materials and Methods

21 Ethics Statement All animal studies were approved by theEthics Committee of Guangxi Medical University (ApprovalNumber 201511018) The studies conformed with the guidefor the care and use of laboratory animals published by theChina National Institutes of Health

22 Rat Thoracic Aorta Calcification Model Male SpragueDawley rats which were from the experimental animalcenter of Guangxi Medical University with a mean bodyweight of 150ndash200 g were used for the studies The animalshad free access to water and food After an acclimatizationperiod of 2 days the animals were randomly allocated toexperimental groups The rat thoracic aorta calcificationmodel was established as previously described [31 32] Ratswere killed 7 days after the first vitamin D3 injection bycervical dislocation while they were undermetofane-inducedanesthesia The aortic tissues between the aortic arch and1 cm below the renal branch were immediately removed afterdeath The tissues of the thoracic aorta were fixed in 4paraformaldehyde for at least 24 h at room temperatureSectioning and histological staining (HE and Von Kossastaining) of the paraformaldehyde-fixed tissues were per-formed for histological analyses ofmineral accumulation andfor assessment of success in creating a rat thoracic aortacalcificationmodelTheother aortic tissueswere immediatelyfrozen in liquid nitrogen until they were analyzed for RNAand protein content

23 Von Kossa and HE Staining Paraffin blocks of aortictissue were cut into 8 um sections and subjected to Von Kossastaining by following the protocol of the Von Kossa stainingkit (Jianchengbio Nanjing China) In brief the slides weredeparaffinized hydrated with distilled water and incubatedin 5 silver nitrate solution for 1 h under a strong sunlightThe silver nitrate solution was then diluted with distilledwater and the sections were incubated with a 5 thiosulphatesolution for 10min Other sections were stained with HEImages were captured using a light microscope and calciumphosphate deposits were detected as black-stained areas

24 Cell Culturing and Transfection VSMC lines (A7R5ATCC CRL-1444) (Aolu Biotech Shanghai China) werecultured in a humidified atmosphere at 37∘C and 5 CO

2

Standard culture medium consisted of Dulbeccorsquos ModifiedEagle Medium (DMEM) with 50 120583gmL streptomycin and50 unitsmL penicillin and was supplemented with 10









3 Results




Con

trol

(a)

Con

trol

(b)

Vita

min

D3

(c)

Vita

min

D3

(d)






4 Discussion





β-Ac

tinM

MP2

C1 M1 C2 M2 C3 M3

(a)

0

2

4

6

8

10


Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Relat

ive e

xpre

ssio

n

P = 00035

P lt 00001

P lt 00001

(b)

0 1 2 3 4 5 6 7 8

0123456789

10


Rela

tive M

MP2

mRN

A ex

pres

sion

P = 00014

R2= 0481

(c)

0 1 2 3 4 5 6 7 8

00

03

06

09

12

15

18


Rela

tive M

MP2

pro

tein

expr

essio

n

P = 00026

R2= 0443

(d)







05

10

15

2030405060

Relat

ive m

iR-2

9b ex

pres

sion

P = 00004

P = 00005


β-Ac

tinM

MP2


0

1

2

3

4

Relat

ive M

MP2

pro

tein

expr

essio

n


P = 00067

P = 00119






00

05

10

15

Rela

tive l

ucife

rase

activ

ity

P lt 001

P lt 001

P lt 001

Posit

ive3

998400-U

TR+m

iRN

A

3998400-U

TR+m

iRN

A

3998400-U

TR-M

U+m

iRN

A

3998400 -U

TR-N

C+m

iRN

A

Posit

ive 3

998400-U

TR+m

iRN

A-N

C



5998400

3998400

5998400

3998400 UTR





5 Conclusion


Abbreviations



Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results




Con

trol

(a)

Con

trol

(b)

Vita

min

D3

(c)

Vita

min

D3

(d)






4 Discussion





β-Ac

tinM

MP2

C1 M1 C2 M2 C3 M3

(a)

0

2

4

6

8

10


Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Relat

ive e

xpre

ssio

n

P = 00035

P lt 00001

P lt 00001

(b)

0 1 2 3 4 5 6 7 8

0123456789

10


Rela

tive M

MP2

mRN

A ex

pres

sion

P = 00014

R2= 0481

(c)

0 1 2 3 4 5 6 7 8

00

03

06

09

12

15

18


Rela

tive M

MP2

pro

tein

expr

essio

n

P = 00026

R2= 0443

(d)







05

10

15

2030405060

Relat

ive m

iR-2

9b ex

pres

sion

P = 00004

P = 00005


β-Ac

tinM

MP2


0

1

2

3

4

Relat

ive M

MP2

pro

tein

expr

essio

n


P = 00067

P = 00119






00

05

10

15

Rela

tive l

ucife

rase

activ

ity

P lt 001

P lt 001

P lt 001

Posit

ive3

998400-U

TR+m

iRN

A

3998400-U

TR+m

iRN

A

3998400-U

TR-M

U+m

iRN

A

3998400 -U

TR-N

C+m

iRN

A

Posit

ive 3

998400-U

TR+m

iRN

A-N

C



5998400

3998400

5998400

3998400 UTR





5 Conclusion


Abbreviations



Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

(a)

Con

trol

(b)

Vita

min

D3

(c)

Vita

min

D3

(d)






4 Discussion





β-Ac

tinM

MP2

C1 M1 C2 M2 C3 M3

(a)

0

2

4

6

8

10


Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Relat

ive e

xpre

ssio

n

P = 00035

P lt 00001

P lt 00001

(b)

0 1 2 3 4 5 6 7 8

0123456789

10


Rela

tive M

MP2

mRN

A ex

pres

sion

P = 00014

R2= 0481

(c)

0 1 2 3 4 5 6 7 8

00

03

06

09

12

15

18


Rela

tive M

MP2

pro

tein

expr

essio

n

P = 00026

R2= 0443

(d)







05

10

15

2030405060

Relat

ive m

iR-2

9b ex

pres

sion

P = 00004

P = 00005


β-Ac

tinM

MP2


0

1

2

3

4

Relat

ive M

MP2

pro

tein

expr

essio

n


P = 00067

P = 00119






00

05

10

15

Rela

tive l

ucife

rase

activ

ity

P lt 001

P lt 001

P lt 001

Posit

ive3

998400-U

TR+m

iRN

A

3998400-U

TR+m

iRN

A

3998400-U

TR-M

U+m

iRN

A

3998400 -U

TR-N

C+m

iRN

A

Posit

ive 3

998400-U

TR+m

iRN

A-N

C



5998400

3998400

5998400

3998400 UTR





5 Conclusion


Abbreviations



Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















β-Ac

tinM

MP2

C1 M1 C2 M2 C3 M3

(a)

0

2

4

6

8

10


Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Con

trol

Vita

min

D3

Relat

ive e

xpre

ssio

n

P = 00035

P lt 00001

P lt 00001

(b)

0 1 2 3 4 5 6 7 8

0123456789

10


Rela

tive M

MP2

mRN

A ex

pres

sion

P = 00014

R2= 0481

(c)

0 1 2 3 4 5 6 7 8

00

03

06

09

12

15

18


Rela

tive M

MP2

pro

tein

expr

essio

n

P = 00026

R2= 0443

(d)







05

10

15

2030405060

Relat

ive m

iR-2

9b ex

pres

sion

P = 00004

P = 00005


β-Ac

tinM

MP2


0

1

2

3

4

Relat

ive M

MP2

pro

tein

expr

essio

n


P = 00067

P = 00119






00

05

10

15

Rela

tive l

ucife

rase

activ

ity

P lt 001

P lt 001

P lt 001

Posit

ive3

998400-U

TR+m

iRN

A

3998400-U

TR+m

iRN

A

3998400-U

TR-M

U+m

iRN

A

3998400 -U

TR-N

C+m

iRN

A

Posit

ive 3

998400-U

TR+m

iRN

A-N

C



5998400

3998400

5998400

3998400 UTR





5 Conclusion


Abbreviations



Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















05

10

15

2030405060

Relat

ive m

iR-2

9b ex

pres

sion

P = 00004

P = 00005


β-Ac

tinM

MP2


0

1

2

3

4

Relat

ive M

MP2

pro

tein

expr

essio

n


P = 00067

P = 00119






00

05

10

15

Rela

tive l

ucife

rase

activ

ity

P lt 001

P lt 001

P lt 001

Posit

ive3

998400-U

TR+m

iRN

A

3998400-U

TR+m

iRN

A

3998400-U

TR-M

U+m

iRN

A

3998400 -U

TR-N

C+m

iRN

A

Posit

ive 3

998400-U

TR+m

iRN

A-N

C



5998400

3998400

5998400

3998400 UTR





5 Conclusion


Abbreviations



Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

05

10

15

Rela

tive l

ucife

rase

activ

ity

P lt 001

P lt 001

P lt 001

Posit

ive3

998400-U

TR+m

iRN

A

3998400-U

TR+m

iRN

A

3998400-U

TR-M

U+m

iRN

A

3998400 -U

TR-N

C+m

iRN

A

Posit

ive 3

998400-U

TR+m

iRN

A-N

C



5998400

3998400

5998400

3998400 UTR





5 Conclusion


Abbreviations



Competing Interests




Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

The Involvement of miR-29b-3p in Arterial Calcification by ...downloads.hindawi.com/journals/bmri/2017/6713606.pdffication rat group, miR-29b-3p was significantly decreased (𝑃=0.0035)