Research Article Runx2/miR-3960/miR-2861 Positive Feedback Loop Is Responsible …downloads.hindawi.com/journals/bmri/2015/624037.pdf · 2019-07-31 · Research Article Runx2/miR-3960/miR-2861

Research ArticleRunx2miR-3960miR-2861 Positive FeedbackLoop Is Responsible for Osteogenic Transdifferentiation ofVascular Smooth Muscle Cells

Zhu-Ying Xia1 Yin Hu1 Ping-Li Xie2 Si-Yuan Tang3 Xiang-Hang Luo1

Er-Yuan Liao1 Fei Chen4 and Hui Xie1

1 Institute of Endocrinology and Metabolism Second Xiang-Ya Hospital of Central South University Changsha Hunan 410011 China2Department of Forensic Medicine School of Basic Medical Science of Central South University Changsha Hunan 410013 China3School of Nursing of Central South University Changsha Hunan 410013 China4Spine Surgery Department Second Xiang-Ya Hospital of Central South University Changsha Hunan 410011 China

Correspondence should be addressed to Fei Chen chenfei91138aliyuncom and Hui Xie hxie5 csu163com

Received 15 August 2014 Accepted 2 January 2015

Academic Editor Xiao-Bo Liao

Copyright copy 2015 Zhu-Ying Xia et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We previously reported that Runx2miR-3960miR-2861 regulatory feedback loop stimulates osteoblast differentiation Howeverthe effect of this feedback loop on the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) remains unclearOur recent study showed that miR-2861 and miR-3960 expression increases significantly during 120573-glycerophosphate-inducedosteogenic transdifferentiation of VSMCs Overexpression of miR-2861 or miR-3960 in VSMCs enhances 120573-glycerophosphate-induced osteoblastogenesis whereas inhibition of miR-2861 or miR-3960 expression attenuates it MiR-2861 or miR-3960 promotesosteogenic transdifferentiation of VSMCs by targeting histone deacetylase 5 or Homeobox A2 respectively resulting in increasedrunt-related transcription factor 2 (Runx2) protein production Furthermore overexpression of Runx2 induces miR-2861 andmiR-3960 transcription and knockdown of Runx2 attenuates 120573-glycerophosphate-induced miR-2861 and miR-3960 transcriptionin VSMCs Thus our data show that Runx2miR-3960miR-2861 positive feedback loop plays an important role in osteogenictransdifferentiation of VSMCs and contributes to vascular calcification

1 Introduction

Medial artery calcification is a common and serious problemwith ageing of the population It is a nonocclusive processwhich leads to decreased vessel elasticity increased bloodpressure and higher risk of cardiovascular mortality [1ndash3]Medial artery calcification is now recognized as a highlyregulated process which is similar in many ways to bonemineralization Osteoblast-like phenotypes transformationof vascular smoothmuscular cells (VSMCs) the predominantcells in the tunica media of arteries is currently consideredresponsible for the formation of medial artery calcification[4ndash6] However the specific mechanism governing this pro-cess is still elusive

MicroRNAs (miRNAs) are endogenous small (16sim25nucleotides) single-stranded noncoding RNAs [7] MicroR-NAsmediate translational repression or degradation of targettranscript by binding to sites of complementarity in the 31015840-UTRs of target mRNAs During the past decade they haveemerged as powerful posttranscriptional regulators of geneexpression [8] To date more than 3 of the genes in thehuman genome have been found to encode for miRNAs andover 30 of genes in the human genome are estimated tobe regulated by miRNAs [9] It has been documented thatmiRNAs participate in cellular proliferation differentiationmigration and apoptosis [10ndash12] However their roles inosteogenic transdifferentiation or calcification of VSMC havejust begun to be understood

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 624037 7 pageshttpdxdoiorg1011552015624037

2 BioMed Research International

Recently we reported that Runx2miR-3960miR-2861positive feedback loop is responsible for osteoblast differ-entiation [13] Osteoblast differentiation signals lead to theactivation of Runx2 transcription factor in stromal cellsIn addition to induction of genes essential for osteoblastdifferentiation Runx2 transactivates miR-3960miR-2861 Inturn miR-3960 and miR-2861 maintain the levels of Runx2mRNA and protein via repressing Homeobox A2 (Hoxa2)and histone deacetylase 5 (HDAC5) and stabilizing theosteoblast differentiation The objective of this study was toelucidate the effect of Runx2miR-3960miR-2861 feedbackloop on regulation of the osteogenic transdifferentiation ofVSMCs

2 Materials and Methods

21 Cell Culture and Transfection To isolate primary cellseight-week-old C57BL6 male wild type mice received a150mgkg intraperitoneal dose of pentobarbital sodium priorto euthanasia which was confirmed by the absence of aheartbeat Vascular smooth muscle cells (VSMCs) were iso-lated from aorta as previously described [14] VSMCs werecultured in DMEM supplemented with 10 FBS To induceosteogenic transdifferentiation VSMCs between passages 3and 7 were cultured in DMEM supplemented with 10mM120573-glycerophosphate For transient transfection of miR-2861miR-3960 anti-miR-2861 anti-miR-3960 and Runx2 siRNAamixture of each of them or their control with Lipofectamine2000 (Invitrogen) was added to cells in 24-well plates at adensity of 3 times 104 cells per well for 48 hours

22 Northern Blot Total RNA was extracted from cells byTrizol reagent (Invitrogen) Northern blottingwas performedas described previously [15] 20120583g of RNA was separatedon a 15 urea polyacrylamide gel with 05x Tris borate-EDTA and transferred to a Hybond-N+ nylon membrane(Amersham Biosciences) using a semidry transfer cell (Bio-Rad) Hybridization was performed according to a standardprotocol 32P-labeled oligonucleotide probes complementaryto the mature miR-2861 and miR-3960 were used in thehybridization The probes for miR-2861 miR-3960 and U6were 51015840-CCGCCCGCCGCCAGGCCCC-31015840 51015840-GCCCCC-GCCTCCGCCGCCGCC-31015840 and 51015840-ATATGGAACGCT-TCACGAATT-31015840 respectively

23 Western Blot To detect Runx2 HDAC5 and Hoxa2protein expression Western blot analysis was performedas previously described [16 17] Briefly equal amountsof protein (40 120583glane) were subjected to SDS gel elec-trophoresis and transferred to a polyvinylidene difluoridemembrane (Amersham Pharmacia Biotech GE HealthcareBiosciences Pittsburgh PA USA) The membranes wereincubated with primary antibodies against Runx2 (SantaCruz) HDAC5 (Santa Cruz) Hoxa2 (Santa Cruz) and 120573-actin (Abcam) overnight at 4∘C and then incubated withappropriate horseradish peroxidase-conjugated secondaryantibodies (SantaCruz) for 1 h at room temperatureTheblotsthen were visualized with the chemiluminescent detection

method using the SuperSignal West Pico Substrate (PierceBiotechnology)

24 ALP Activity and Osteocalcin Secretion Assay Cells weregrown to confluence in 24-well plates The cells were thenwashed with PBS and scraped into a solution containing20mM Tris-HCl pH 80 and 150mM NaCl and 1 TritonX-100 002 NaN

3 and 1 120583gmL aprotinin The lysates

were homogenized then alkaline phosphatase (ALP) activ-ity was assayed by spectrophotometric measurement of p-nitrophenol release at 37∘C Osteocalcin released into theculturemediawasmeasured using a specific radioimmunoas-say kit (DiaSorin) To normalize protein expression to totalcellular protein a fraction of the lysate solution was used in aBradford protein assay

25 qRT-PCR Analysis Total RNA was isolated using theTrizol reagent (Invitrogen) and reverse transcription wasperformed using 10mg of total RNA and the SuperScriptII Kit (Invitrogen) qRT-PCR was performed using a RocheMolecular Light Cycler (Roche Molecular BiochemicalsIndianapolis IN USA) Amplification reactions were setup in 25 120583L reaction volumes containing SYBR Green PCRMaster Mix (PE Applied Biosystems Waltham MA USA)template cDNA and amplification primers The followingprimers were used 51015840-TGTCACCGCCAGATGTTTTG-3101584051015840-TGAGCAGAGCCGAGACACAG-31015840 Hoxa2 forward 51015840-GTCACTCTTTGAGCAAGCCC-31015840 reverse 51015840-TAGGCC-AGCTCCACAGTTCT-31015840 GAPDH sense 51015840-CACCAT-GGAGAAGGCCGGGG-31015840 and antisense 51015840-GACGGA-CACATTGGGGGTAG-31015840 PCR amplifications were per-formed and the amplification data were analyzed using thesequence detector system software (PE Applied Biosystems)Relative quantification was calculated by normalizing the testcrossing thresholds (Ct) with the GAPDH amplified controlCt

26 Statistical Analyses Data were presented as mean plusmnsem Comparisons were made using a one-way analysis ofvariance All experiments were repeated at least three timesand representative experimentswere shownDifferenceswereconsidered significant at 119875 lt 005

3 Results

31 Increased Expression of miR-2861 and miR-3960 duringthe Osteogenic Transdifferentiation of VSMCs We performedNorthern blot analysis to examine the expression of miR-2861 and miR-3960 in VSMCs during 120573-glycerophosphate-induced osteogenesis BothmiR-2861 andmiR-3960 could bedetected after treatmentwith120573-glycerophosphate for 12 h andincreasedwith time throughout osteoblastic differentiation ofVSMCs (Figure 1)

32 miR-2861 or miR-3960 Promotes Osteoblast Differentia-tion of Vascular Smooth Muscle Cells We next determinedthe role of miR-2861 or miR-3960 during osteogenic trans-differentiation of VSMCs by changing the functional levels

BioMed Research International 3

120573-Glycerophosphate (h)0 12 24 48

miR-2861

miR-3960

U6

Figure 1 Increased expression of miR-2861 and miR-3960 dur-ing 120573-glycerophosphate-induced osteogenic transdifferentiation ofVSMCs Northern blot analysis of miR-2861 and miR-3960 expres-sion in 10mM 120573-glycerophosphate-treated VSMCs for the indicatedtimes U6 snRNA is used as a loading control

of miR-2861 or miR-3960 in VSMCs Northern blot dataconfirmed overexpression of miR-2861 and miR-3960 inVSMCs stably transfected with miR-2861 precursor andmiR-3960 precursor respectively (Figure 2(a)) The transfectedVSMCs were treated with 120573-glycerophosphate for 48 h forinducing osteogenesis Then ALP activity osteocalcin secre-tion and Runx2 protein expression as markers of osteoblastdifferentiation were evaluatedThe levels of ALP osteocalcinand Runx2 were all elevated in cells with overexpression ofmiR-2861 or miR-3960 (Figure 2(b)) These results indicatedthat overexpression of miR-2861 or miR-3960 promotedosteogenic transdifferentiation of VSMCs (Figures 2(b) and2(c)) In contrast knockdown of miR-2861 or miR-3960reduced ALP osteocalcin and Runx2 levels induced by 120573-glycerophosphate (Figures 3(a) and 3(b)) All of these resultssuggest that both miR-2861 and miR-3960 act to promoteosteogenic transdifferentiation of VSMCs

33 miR-2861 and miR-3960 Posttranscriptionally RepressHDAC5 andHoxa2 Expression Wepreviously identified thatHDAC5 and Hoxa2 are the direct targets of miR-2861 andmiR-3960 respectively in a stromal cell line ST2 [13] Herewe overexpressed miR-2861 or miR-3960 to directly identifytheir actions on HDAC5 or Hoxa2 in VSMCs As expectedoverexpression of miR-2861 or miR-3960 resulted in adecrease in the HDAC5 or Hoxa2 protein levels (Figures 4(a)and 4(b)) However the HDAC5 or Hoxa2mRNA levels werenot affected (Figures 4(a) and 4(b)) These results revealedthat miR-2861 and miR-3960 posttranscriptionally repressedHDAC5 and Hoxa2 protein expression by inhibiting mRNAtranslation in VSMCs

34 Runx2 Stimulates miR-2861 and miR-3960 Expression inVascular Smooth Muscle We previously demonstrated thatRunx2 directly induces the expression of the miR-3960miR-2861 cluster by binding to the putative binding site of itspromoter in ST2 cells [13] Here in order to identify thedirect impact of Runx2 on miR-2861 and miR-3960 gene

expression in VSMCs we changed the functional levels ofRunx2 First we used a pcDNA-driven expression vector tooverexpress Runx2 protein levels as confirmed by Westernblot (Figure 5(a)) The results showed that Runx2 overex-pression could induce miR-2861 and miR-3960 expressionin VSMCs (Figure 5(a)) Furthermore we transfected siRNAdesigned against Runx2 into 120573-glycerophosphate-inducedVSMCs to specifically silence Runx2 expressionWestern blotanalysis revealed that si-Runx2 blocked Runx2 expressioncompared with the control (Figure 5(b)) The expression ofmiR-3960 and miR-2861 was inhibited by the knockdownof Runx2 (Figure 5(b)) Thus these results suggest a directstimulatory action of Runx2 on the expression of miR-2861and miR-3960 in VSMCs

4 Discussion

In our previous study we cloned both miR-2861 and miR-3960 and identified that miR-2861 induces osteoblast dif-ferentiation by repressing HDAC5 an enhancer of Runx2degradation andmiR-3960 induces osteoblast differentiationby repressing Hoxa2 a repressor of Runx2 expression [13 15]miR-2861 binds to the coding domain sequence (CDS) ofHDAC5 mRNA with complementarity to the miR-2861 seedregion and posttranscriptionally represses HDAC5 proteinexpression by inhibiting mRNA translation and not bymRNA degradation [15] Similarly miR-3960 directly targetsHoxa2 by specifically binding with the target CDS of Hoxa2and represses Hoxa2 expression at the posttranscriptionallevel [13] Thus miR-3960 and miR-2861 can coregulate theRunx2 during osteoblast differentiation In turn Runx2 bindsto the miR-2861miR-3960 cluster promoter to transcription-ally induce the expression of miR-2861 and miR-3960 [13]Runx2miR-3960miR-2861 is a critical positive feedbackloop for osteoblast differentiation

Recently several miRNAs have been reported to beinvolved in osteogenic transdifferentiation and calcifica-tion of VSMCs [18ndash22] It has been reported that miR-221 and miR-222 increase runt-related transcription factor2 (Runx2) expression and calcium deposition in VSMCs[18] On the contrary miR-204 miR-133a and miR-125bsuppress osteoblastic differentiation of VSMCs by inhibitingRunx2 protein expression whereas miR-204 miR-133a andmiR-125b inhibition enhance osteoblastic differentiation ofVSMCs [19ndash22] In this study we show that Runx2miR-3960miR-2861 is also responsible for stimulating osteogenictransdifferentiation of VSMCs At first we demonstratedthat the expression of both miR-2861 and miR-3960 wassignificantly increased during 120573-glycerophosphate-inducedosteogenic transdifferentiation of VSMCs indicating thatmiR-2861 and miR-3960 might play a role in vascular cal-cification To prove whether miR-2861 and miR-3960 aredirectly associated with the osteogenic transdifferentiation ofVSMCs we evaluated the effect of miR-2861 and miR-3960in the process of 120573-glycerophosphate-induced osteogenictransdifferentiation of VSMCs Runx2 has been identified asa ldquomaster transcription factorrdquo simulating osteogenic trans-differentiation of precursor cells [4 23] It plays an essential


miR-2861 miR-3960

U6 U6

Pre-

miR

-2861

Pre-

miR

-3960

miR

-C

miR

-C

(a)

0

100

200

300

400

500

Oste

ocal

cin

(ng

mL

prot

ein)

Pre-miR-2861 Pre-miR-2861 Pre-miR-3960 Pre-miR-3960

0 48

(h)0 48

(h)

0

100

200

300

400

500

0 48

(h)0 48

(h)

0

5

10

15

20

25

Oste

ocal

cin

(ng

mL

prot

ein)

0

5

10

15

20

25

ALP

(nm

olm

inmiddotm

g pr

otei

n)

ALP

(nm

olm

inmiddotm

g pr

otei

n)

miR-C miR-C miR-C miR-C

lowastlowast

lowastlowast

(b)

Runx2

120573-actin

120573-GP (h) 0 48 0 48

miR

-C

miR

-C

miR

-C

miR

-C

Pre-

miR

-3960

Pre-

miR

-3960

Pre-

miR

-2861

Pre-

miR

-2861

(c)

Figure 2 miR-2861 or miR-3960 promoted 120573-glycerophosphate-induced osteoblast differentiation of vascular smooth muscle cells (a)Northern blot analysis of miR-2861 expression in VSMCs transfected with miR-C (control) or miR-2861 precursor (pre-miR-3861) miR-Cor pre-miR-3960 ((b) and (c)) ALP activity and osteocalcin secretion (b) andWestern blot analysis of Runx2 protein expression (c) in 10mM120573-glycerophosphate- (120573-GP-) treated VSMCs stably transfected with miR-C or pre-miR-2861 miR-C or pre-miR-3960 Data are shown asmean plusmn sem lowast119875 lt 005

role in the osteogenic transdifferentiation of VSMCs [24 25]Knockdown of Runx2 significantly inhibits the ALP expres-sion and the calcification in the senescent VSMCs [26] ALPis a prophase (proliferative phase)marker of osteoblast differ-entiation Osteocalcin is ametaphase (bonematrix formationperiod) marker of osteoblast differentiation Both miR-2861and miR-3960 promoted osteogenic transdifferentiation of

VSMCs as indicated by increased levels of ALP activityosteocalcin secretion and Runx2 protein expression

HDAC5 is an enhancer of Runx2 degradation It deacety-lates Runx2 allowing the protein to undergo Smurf-mediateddegradation and inhibition of HDAC5 increases Runx2acetylation [27 28] Hoxa2 a member of the Hox home-odomain family of transcription factors that regulate skeletal


0

100

200

300

400

0

100

200

300

400

0

5

10

15

0

5

10

15

Oste

ocal

cin

(ng

mL

prot

ein)

Oste

ocal

cin

(ng

mL

prot

ein)

Anti-miR-CAnti-miR-2861

Anti-miR-CAnti-miR-CAnti-miR-2861

0 48

(h)0 48

(h)0 48

(h)0 48

(h)

Anti-miR-3960Anti-miR-CAnti-miR-3960

ALP

(nm

olm

inmiddotm

g pr

otei

n)

ALP

(nm

olm

inmiddotm

g pr

otei

n)

lowastlowast lowast

lowast

(a)

Runx2

120573-actin

120573-GP (h)0 48 0 48

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-3960

Ant

i-miR

-3960

(b)

Figure 3 Inhibition of miR-2861 or miR-3960 inhibited 120573-glycerophosphate-induced osteoblast differentiation of vascular smooth musclecells ALP activity and osteocalcin secretion (a) and Western blot analysis of Runx2 protein expression (b) in 10mM 120573-glycerophosphate-(120573-GP-) treated VSMCs transfected with anti-miR-C (control) or anti-miR-2861 anti-miR-C or anti-miR-3960 Data are shown as mean plusmnsem lowast119875 lt 005

0

05

1

15

2

25

HD

AC5

relat

ive m

RNA

leve

l

120573-actin

HDAC5

miR

-C

Pre-

miR

-2861

miR

-C

Pre-

miR

-2861

(a)

0

1

2

3

Hox

a2re

lativ

e mRN

A le

vel

120573-actin

Hoxa2

miR

-C

Pre-

miR

-3960

miR

-C

Pre-

miR

-3960

(b)

Figure 4 miR-2861 and miR-3960 posttranscriptionally repressed HDAC5 and Hoxa2 expression respectively ((a) and (b)) QuantitativeRT-PCR analysis of mRNA levels of HDAC5 and Hoxa2 (left) and Western blot analysis of protein levels of HDAC5 and Hoxa2 (right) inVSMCs transfected with miR-C (control) or pre-miR-3960 (a) miR-C or miR-3960 (b) For quantitative RT-PCR analysis mRNA levelsnormalized to GAPDH Data are shown as means plusmn sem For Western blot analysis 120573-actin acts as a loading control


Runx2

120573-actin

miR-2861

miR-3960

U6

pcD

NA31

pcD

NA31

-Run

x2

pcD

NA31

pcD

NA31

-Run

x2(a)

Runx2

120573-actin

miR-2861

miR-3960

U6

Si-C

Si-R

unx2

Si-C

Si-R

unx2

(b)

Figure 5 Runx2 stimulates miR-2861 and miR-3960 expression in vascular smooth muscle cells (a) Western blot analysis of Runx2 proteinlevels (left) andNorthern blot analysis of miR-2861 andmiR-3960 levels (right) in VSMCs transfected with pcDNA31 vector control or Runx2pcDNA31 vector (pcDNA31-Runx2) for 48 h (b) Western blot analysis of Runx2 protein levels (left) and Northern blot analysis of miR-2861andmiR-3960 levels (right) in VSMCs transfected with siRNA control (si-C) or si-Runx2 and then cultured with 120573-glycerophosphate for 48 hFor Western blot analysis 120573-actin acts as a loading control For Northern blot analysis U6 snRNA acts as a loading control

patterning is found to control Runx2 expression and isrequired in skeletalmorphogenesis [29]Hoxa2minusminusmice showan upregulation of the Runx2 level and Hoxa2 inhibitsbone formation by repressing Runx2 expression [29] In ourprevious study ST2 cells transfected with a Hoxa2 expressionvector showed reduced Runx2 expression and ALP activityconfirming that Hoxa2 can suppress Runx2 expression andfurther inhibits osteoblast differentiation [13] In this studywe detected HDAC5 expression in VSMCs inconsistent withseveral previous papers [29ndash32] Moreover we identifiedHoxa2 expression in VSMCs Thus miR-2861 and miR-3960directly target HDAC5 and Hoxa2 respectively to increaseRunx2 levels in VSMCs

Together our study shows that Runx2miR-3960miR-2861 positive feedback loop plays an important role inosteogenic transdifferentiation of VSMCs and contributes tovascular calcification

Conflict of Interests

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

Acknowledgments

This study was supported by the China National NaturalScientific Foundation (Grants nos 81000122 81370974 and81400858) the Science and Technology Project of ChangshaCity (Grant no K1207043-31) the Specialized Research Fundfor the Doctoral Program of Higher Education (SRFDPGrant no 20120162110067) the National Basic Research

Program of China (Grant no 2014CB942903) the ChinaNational Funds forDistinguishedYoung Scientists (Grant no81125006)

References

[1] M Wu C Rementer and C M Giachelli ldquoVascular calcifica-tion an update on mechanisms and challenges in treatmentrdquoCalcified Tissue International vol 93 no 4 pp 365ndash373 2013

[2] D A Chistiakov I A Sobenin A N Orekhov and Y VBobryshev ldquoMechanisms of medial arterial calcification indiabetesrdquo Current Pharmaceutical Design vol 20 no 37 pp5870ndash5883 2014

[3] M R Brodeur C Bouvet M Barrette and PMoreau ldquoPalmiticacid increases medial calcification by inducing oxidative stressrdquoJournal of Vascular Research vol 50 no 5 pp 430ndash441 2013

[4] R C Johnson J A Leopold and J Loscalzo ldquoVascular calcifi-cation pathobiological mechanisms and clinical implicationsrdquoCirculation Research vol 99 no 10 pp 1044ndash1059 2006

[5] R J Guzman ldquoClinical cellular and molecular aspects ofarterial calcificationrdquo Journal of Vascular Surgery vol 45 no6 pp A57ndashA63 2007

[6] L L Demer and Y Tintut ldquoVascular calcification pathobiologyof a multifaceted diseaserdquo Circulation vol 117 no 22 pp 2938ndash2948 2008

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

[8] R W Carthew and E J Sontheimer ldquoOrigins and mechanismsof miRNAs and siRNAsrdquo Cell vol 136 no 4 pp 642ndash655 2009

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


[10] V Ambros and X Chen ldquoThe regulation of genes and genomesby small RNAsrdquo Development vol 134 no 9 pp 1635ndash16412007

[11] J W Hagen and E C Lai ldquoMicroRNA control of cell-cellsignaling during development and diseaserdquoCell Cycle vol 7 no15 pp 2327ndash2332 2008

[12] P Trang J BWeidhaas and F J Slack ldquoMicroRNAs as potentialcancer therapeuticsrdquoOncogene vol 27 no 2 pp S52ndashS57 2008

[13] R Hu W Liu H Li et al ldquoA Runx2miR-3960miR-2861 reg-ulatory feedback loop during mouse osteoblast differentiationrdquoThe Journal of Biological Chemistry vol 286 no 14 pp 12328ndash12339 2011

[14] X B Liao Y Q Peng X M Zhou et al ldquoTaurine restoresAxlGas6 expression in vascular smooth muscle cell calcifica-tion modelrdquo Amino Acids vol 39 no 2 pp 375ndash383 2010

[15] H Li H Xie W Liu et al ldquoA novel microRNA targetingHDAC5 regulates osteoblast differentiation in mice and con-tributes to primary osteoporosis in humansrdquo The Journal ofClinical Investigation vol 119 pp 3666ndash3677 2009

[16] H Xie M Sun X-B Liao et al ldquoEstrogen receptor 12057236 medi-ates a bone-sparing effect of 17120573-estrodiol in postmenopausalwomenrdquo Journal of Bone and Mineral Research vol 26 no 1pp 156ndash168 2011

[17] H Xie P-L Xie X-P Wu et al ldquoOmentin-1 attenuates arterialcalcification and bone loss in osteoprotegerin-deficient mice byinhibition of RANKL expressionrdquo Cardiovascular Research vol92 no 2 pp 296ndash306 2011

[18] N C W Mackenzie K A Staines D Zhu P Genever andV E MacRae ldquomiRNA-221 and miRNA-222 synergisticallyfunction to promote vascular calcificationrdquo Cell Biochemistryand Function vol 32 no 2 pp 209ndash216 2014

[19] P Wen H Cao L Fang et al ldquomiR-125bEts1 axis regulatestransdifferentiation and calcification of vascular smoothmusclecells in a high-phosphate environmentrdquo Experimental CellResearch vol 322 no 2 pp 302ndash312 2014

[20] 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

[21] 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

[22] C Goettsch M Rauner N Pacyna U Hempel S R Bornsteinand L C Hofbauer ldquoMiR-125b regulates calcification of vascu-lar smoothmuscle cellsrdquoAmerican Journal of Pathology vol 179no 4 pp 1594ndash1600 2011

[23] T Komori ldquoRegulation of osteoblast differentiation by tran-scription factorsrdquo Journal of Cellular Biochemistry vol 99 no5 pp 1233ndash1239 2006

[24] H B Chang A Javed Q Dai et al ldquoOxidative stress inducesvascular calcification through modulation of the osteogenictranscription factor Runx2 by AKT signalingrdquo The Journal ofBiological Chemistry vol 283 no 22 pp 15319ndash15327 2008

[25] N X Chen D Duan K D OrsquoNeill et al ldquoThe mechanisms ofuremic serum-induced expression of bone matrix proteins inbovine vascular smooth muscle cellsrdquoKidney International vol70 no 6 pp 1046ndash1053 2006

[26] R Nakano-Kurimoto K Ikeda M Uraoka et al ldquoReplicativesenescence of vascular smooth muscle cells enhances the calci-fication through initiating the osteoblastic transitionrdquoAmericanJournal of PhysiologymdashHeart and Circulatory Physiology vol297 no 5 pp H1673ndashH1684 2009

[27] E-J Jeon K-Y Lee N-S Choi et al ldquoBone morphogeneticprotein-2 stimulates Runx2 acetylationrdquo Journal of BiologicalChemistry vol 281 no 24 pp 16502ndash16511 2006

[28] J S Kang T Alliston R Delston and R Derynck ldquoRepressionof Runx2 function by TGF-120573 through recruitment of class IIhistone deacetylases by Smad3rdquoThe EMBO Journal vol 24 no14 pp 2543ndash2555 2005

[29] B Kanzler S J Kuschert Y-H Liu and M Mallo ldquoHoxa-2restricts the chondrogenic domain and inhibits bone formationduring development of the branchial areardquo Development vol125 no 14 pp 2587ndash2597 1998

[30] R Ginnan L Y Sun J J Schwarz and H A Singer ldquoMEF2 isregulated by CaMKII120575

2and a HDAC4-HDAC5 heterodimer in

vascular smoothmuscle cellsrdquoBiochemical Journal vol 444 no1 pp 105ndash114 2012

[31] J Pang C Yan K Natarajan et al ldquoGIT1 mediates HDAC5activation by angiotensin II in vascular smooth muscle cellsrdquoArteriosclerosisThrombosis and Vascular Biology vol 28 no 5pp 892ndash898 2008

[32] X Xu C-H Ha C Wong et al ldquoAngiotensin II stimulatesprotein kinase D-dependent histone deacetylase 5 phosphory-lation and nuclear export leading to vascular smooth musclecell hypertrophyrdquo Arteriosclerosis Thrombosis and VascularBiology vol 27 no 11 pp 2355ndash2362 2007

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Recently we reported that Runx2miR-3960miR-2861positive feedback loop is responsible for osteoblast differ-entiation [13] Osteoblast differentiation signals lead to theactivation of Runx2 transcription factor in stromal cellsIn addition to induction of genes essential for osteoblastdifferentiation Runx2 transactivates miR-3960miR-2861 Inturn miR-3960 and miR-2861 maintain the levels of Runx2mRNA and protein via repressing Homeobox A2 (Hoxa2)and histone deacetylase 5 (HDAC5) and stabilizing theosteoblast differentiation The objective of this study was toelucidate the effect of Runx2miR-3960miR-2861 feedbackloop on regulation of the osteogenic transdifferentiation ofVSMCs

2 Materials and Methods

21 Cell Culture and Transfection To isolate primary cellseight-week-old C57BL6 male wild type mice received a150mgkg intraperitoneal dose of pentobarbital sodium priorto euthanasia which was confirmed by the absence of aheartbeat Vascular smooth muscle cells (VSMCs) were iso-lated from aorta as previously described [14] VSMCs werecultured in DMEM supplemented with 10 FBS To induceosteogenic transdifferentiation VSMCs between passages 3and 7 were cultured in DMEM supplemented with 10mM120573-glycerophosphate For transient transfection of miR-2861miR-3960 anti-miR-2861 anti-miR-3960 and Runx2 siRNAamixture of each of them or their control with Lipofectamine2000 (Invitrogen) was added to cells in 24-well plates at adensity of 3 times 104 cells per well for 48 hours

22 Northern Blot Total RNA was extracted from cells byTrizol reagent (Invitrogen) Northern blottingwas performedas described previously [15] 20120583g of RNA was separatedon a 15 urea polyacrylamide gel with 05x Tris borate-EDTA and transferred to a Hybond-N+ nylon membrane(Amersham Biosciences) using a semidry transfer cell (Bio-Rad) Hybridization was performed according to a standardprotocol 32P-labeled oligonucleotide probes complementaryto the mature miR-2861 and miR-3960 were used in thehybridization The probes for miR-2861 miR-3960 and U6were 51015840-CCGCCCGCCGCCAGGCCCC-31015840 51015840-GCCCCC-GCCTCCGCCGCCGCC-31015840 and 51015840-ATATGGAACGCT-TCACGAATT-31015840 respectively

23 Western Blot To detect Runx2 HDAC5 and Hoxa2protein expression Western blot analysis was performedas previously described [16 17] Briefly equal amountsof protein (40 120583glane) were subjected to SDS gel elec-trophoresis and transferred to a polyvinylidene difluoridemembrane (Amersham Pharmacia Biotech GE HealthcareBiosciences Pittsburgh PA USA) The membranes wereincubated with primary antibodies against Runx2 (SantaCruz) HDAC5 (Santa Cruz) Hoxa2 (Santa Cruz) and 120573-actin (Abcam) overnight at 4∘C and then incubated withappropriate horseradish peroxidase-conjugated secondaryantibodies (SantaCruz) for 1 h at room temperatureTheblotsthen were visualized with the chemiluminescent detection

method using the SuperSignal West Pico Substrate (PierceBiotechnology)

24 ALP Activity and Osteocalcin Secretion Assay Cells weregrown to confluence in 24-well plates The cells were thenwashed with PBS and scraped into a solution containing20mM Tris-HCl pH 80 and 150mM NaCl and 1 TritonX-100 002 NaN

3 and 1 120583gmL aprotinin The lysates

were homogenized then alkaline phosphatase (ALP) activ-ity was assayed by spectrophotometric measurement of p-nitrophenol release at 37∘C Osteocalcin released into theculturemediawasmeasured using a specific radioimmunoas-say kit (DiaSorin) To normalize protein expression to totalcellular protein a fraction of the lysate solution was used in aBradford protein assay

25 qRT-PCR Analysis Total RNA was isolated using theTrizol reagent (Invitrogen) and reverse transcription wasperformed using 10mg of total RNA and the SuperScriptII Kit (Invitrogen) qRT-PCR was performed using a RocheMolecular Light Cycler (Roche Molecular BiochemicalsIndianapolis IN USA) Amplification reactions were setup in 25 120583L reaction volumes containing SYBR Green PCRMaster Mix (PE Applied Biosystems Waltham MA USA)template cDNA and amplification primers The followingprimers were used 51015840-TGTCACCGCCAGATGTTTTG-3101584051015840-TGAGCAGAGCCGAGACACAG-31015840 Hoxa2 forward 51015840-GTCACTCTTTGAGCAAGCCC-31015840 reverse 51015840-TAGGCC-AGCTCCACAGTTCT-31015840 GAPDH sense 51015840-CACCAT-GGAGAAGGCCGGGG-31015840 and antisense 51015840-GACGGA-CACATTGGGGGTAG-31015840 PCR amplifications were per-formed and the amplification data were analyzed using thesequence detector system software (PE Applied Biosystems)Relative quantification was calculated by normalizing the testcrossing thresholds (Ct) with the GAPDH amplified controlCt

26 Statistical Analyses Data were presented as mean plusmnsem Comparisons were made using a one-way analysis ofvariance All experiments were repeated at least three timesand representative experimentswere shownDifferenceswereconsidered significant at 119875 lt 005

3 Results

31 Increased Expression of miR-2861 and miR-3960 duringthe Osteogenic Transdifferentiation of VSMCs We performedNorthern blot analysis to examine the expression of miR-2861 and miR-3960 in VSMCs during 120573-glycerophosphate-induced osteogenesis BothmiR-2861 andmiR-3960 could bedetected after treatmentwith120573-glycerophosphate for 12 h andincreasedwith time throughout osteoblastic differentiation ofVSMCs (Figure 1)

32 miR-2861 or miR-3960 Promotes Osteoblast Differentia-tion of Vascular Smooth Muscle Cells We next determinedthe role of miR-2861 or miR-3960 during osteogenic trans-differentiation of VSMCs by changing the functional levels



miR-2861

miR-3960

U6






4 Discussion




miR-2861 miR-3960

U6 U6

Pre-

miR

-2861

Pre-

miR

-3960

miR

-C

miR

-C

(a)

0

100

200

300

400

500

Oste

ocal

cin

(ng

mL

prot

ein)


0 48

(h)0 48

(h)

0

100

200

300

400

500

0 48

(h)0 48

(h)

0

5

10

15

20

25

Oste

ocal

cin

(ng

mL

prot

ein)

0

5

10

15

20

25

ALP

(nm

olm

inmiddotm

g pr

otei

n)

ALP

(nm

olm

inmiddotm

g pr

otei

n)


lowastlowast

lowastlowast

(b)

Runx2

120573-actin

120573-GP (h) 0 48 0 48

miR

-C

miR

-C

miR

-C

miR

-C

Pre-

miR

-3960

Pre-

miR

-3960

Pre-

miR

-2861

Pre-

miR

-2861

(c)






0

100

200

300

400

0

100

200

300

400

0

5

10

15

0

5

10

15

Oste

ocal

cin

(ng

mL

prot

ein)

Oste

ocal

cin

(ng

mL

prot

ein)



0 48

(h)0 48

(h)0 48

(h)0 48

(h)


ALP

(nm

olm

inmiddotm

g pr

otei

n)

ALP

(nm

olm

inmiddotm

g pr

otei

n)

lowastlowast lowast

lowast

(a)

Runx2

120573-actin

120573-GP (h)0 48 0 48

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-3960

Ant

i-miR

-3960

(b)


0

05

1

15

2

25

HD

AC5

relat

ive m

RNA

leve

l

120573-actin

HDAC5

miR

-C

Pre-

miR

-2861

miR

-C

Pre-

miR

-2861

(a)

0

1

2

3

Hox

a2re

lativ

e mRN

A le

vel

120573-actin

Hoxa2

miR

-C

Pre-

miR

-3960

miR

-C

Pre-

miR

-3960

(b)



Runx2

120573-actin

miR-2861

miR-3960

U6

pcD

NA31

pcD

NA31

-Run

x2

pcD

NA31

pcD

NA31

-Run

x2(a)

Runx2

120573-actin

miR-2861

miR-3960

U6

Si-C

Si-R

unx2

Si-C

Si-R

unx2

(b)






Acknowledgments



References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















miR-2861

miR-3960

U6






4 Discussion




miR-2861 miR-3960

U6 U6

Pre-

miR

-2861

Pre-

miR

-3960

miR

-C

miR

-C

(a)

0

100

200

300

400

500

Oste

ocal

cin

(ng

mL

prot

ein)


0 48

(h)0 48

(h)

0

100

200

300

400

500

0 48

(h)0 48

(h)

0

5

10

15

20

25

Oste

ocal

cin

(ng

mL

prot

ein)

0

5

10

15

20

25

ALP

(nm

olm

inmiddotm

g pr

otei

n)

ALP

(nm

olm

inmiddotm

g pr

otei

n)


lowastlowast

lowastlowast

(b)

Runx2

120573-actin

120573-GP (h) 0 48 0 48

miR

-C

miR

-C

miR

-C

miR

-C

Pre-

miR

-3960

Pre-

miR

-3960

Pre-

miR

-2861

Pre-

miR

-2861

(c)






0

100

200

300

400

0

100

200

300

400

0

5

10

15

0

5

10

15

Oste

ocal

cin

(ng

mL

prot

ein)

Oste

ocal

cin

(ng

mL

prot

ein)



0 48

(h)0 48

(h)0 48

(h)0 48

(h)


ALP

(nm

olm

inmiddotm

g pr

otei

n)

ALP

(nm

olm

inmiddotm

g pr

otei

n)

lowastlowast lowast

lowast

(a)

Runx2

120573-actin

120573-GP (h)0 48 0 48

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-3960

Ant

i-miR

-3960

(b)


0

05

1

15

2

25

HD

AC5

relat

ive m

RNA

leve

l

120573-actin

HDAC5

miR

-C

Pre-

miR

-2861

miR

-C

Pre-

miR

-2861

(a)

0

1

2

3

Hox

a2re

lativ

e mRN

A le

vel

120573-actin

Hoxa2

miR

-C

Pre-

miR

-3960

miR

-C

Pre-

miR

-3960

(b)



Runx2

120573-actin

miR-2861

miR-3960

U6

pcD

NA31

pcD

NA31

-Run

x2

pcD

NA31

pcD

NA31

-Run

x2(a)

Runx2

120573-actin

miR-2861

miR-3960

U6

Si-C

Si-R

unx2

Si-C

Si-R

unx2

(b)






Acknowledgments



References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















miR-2861 miR-3960

U6 U6

Pre-

miR

-2861

Pre-

miR

-3960

miR

-C

miR

-C

(a)

0

100

200

300

400

500

Oste

ocal

cin

(ng

mL

prot

ein)


0 48

(h)0 48

(h)

0

100

200

300

400

500

0 48

(h)0 48

(h)

0

5

10

15

20

25

Oste

ocal

cin

(ng

mL

prot

ein)

0

5

10

15

20

25

ALP

(nm

olm

inmiddotm

g pr

otei

n)

ALP

(nm

olm

inmiddotm

g pr

otei

n)


lowastlowast

lowastlowast

(b)

Runx2

120573-actin

120573-GP (h) 0 48 0 48

miR

-C

miR

-C

miR

-C

miR

-C

Pre-

miR

-3960

Pre-

miR

-3960

Pre-

miR

-2861

Pre-

miR

-2861

(c)






0

100

200

300

400

0

100

200

300

400

0

5

10

15

0

5

10

15

Oste

ocal

cin

(ng

mL

prot

ein)

Oste

ocal

cin

(ng

mL

prot

ein)



0 48

(h)0 48

(h)0 48

(h)0 48

(h)


ALP

(nm

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inmiddotm

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otei

n)

ALP

(nm

olm

inmiddotm

g pr

otei

n)

lowastlowast lowast

lowast

(a)

Runx2

120573-actin

120573-GP (h)0 48 0 48

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-3960

Ant

i-miR

-3960

(b)


0

05

1

15

2

25

HD

AC5

relat

ive m

RNA

leve

l

120573-actin

HDAC5

miR

-C

Pre-

miR

-2861

miR

-C

Pre-

miR

-2861

(a)

0

1

2

3

Hox

a2re

lativ

e mRN

A le

vel

120573-actin

Hoxa2

miR

-C

Pre-

miR

-3960

miR

-C

Pre-

miR

-3960

(b)



Runx2

120573-actin

miR-2861

miR-3960

U6

pcD

NA31

pcD

NA31

-Run

x2

pcD

NA31

pcD

NA31

-Run

x2(a)

Runx2

120573-actin

miR-2861

miR-3960

U6

Si-C

Si-R

unx2

Si-C

Si-R

unx2

(b)






Acknowledgments



References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

400

0

100

200

300

400

0

5

10

15

0

5

10

15

Oste

ocal

cin

(ng

mL

prot

ein)

Oste

ocal

cin

(ng

mL

prot

ein)



0 48

(h)0 48

(h)0 48

(h)0 48

(h)


ALP

(nm

olm

inmiddotm

g pr

otei

n)

ALP

(nm

olm

inmiddotm

g pr

otei

n)

lowastlowast lowast

lowast

(a)

Runx2

120573-actin

120573-GP (h)0 48 0 48

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-C

Ant

i-miR

-2861

Ant

i-miR

-3960

Ant

i-miR

-3960

(b)


0

05

1

15

2

25

HD

AC5

relat

ive m

RNA

leve

l

120573-actin

HDAC5

miR

-C

Pre-

miR

-2861

miR

-C

Pre-

miR

-2861

(a)

0

1

2

3

Hox

a2re

lativ

e mRN

A le

vel

120573-actin

Hoxa2

miR

-C

Pre-

miR

-3960

miR

-C

Pre-

miR

-3960

(b)



Runx2

120573-actin

miR-2861

miR-3960

U6

pcD

NA31

pcD

NA31

-Run

x2

pcD

NA31

pcD

NA31

-Run

x2(a)

Runx2

120573-actin

miR-2861

miR-3960

U6

Si-C

Si-R

unx2

Si-C

Si-R

unx2

(b)






Acknowledgments



References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Runx2

120573-actin

miR-2861

miR-3960

U6

pcD

NA31

pcD

NA31

-Run

x2

pcD

NA31

pcD

NA31

-Run

x2(a)

Runx2

120573-actin

miR-2861

miR-3960

U6

Si-C

Si-R

unx2

Si-C

Si-R

unx2

(b)






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
















Documents

Research Article Runx2/miR-3960/miR-2861 Positive Feedback Loop Is Responsible …downloads.hindawi.com/journals/bmri/2015/624037.pdf · 2019-07-31 · Research Article Runx2/miR-3960/miR-2861