Research Article Role of MicroRNA-103a Targeting ADAM10 in Abdominal Aortic Aneurysm

15 Pages·2017·3.06 MB·English

Hindawi
BioMed Research International
Volume 2017, Article ID 9645874, 14 pages
https://doi.org/10.1155/2017/9645874
Research Article
Role of MicroRNA-103a Targeting ADAM10 in Abdominal
Aortic Aneurysm
TongJiao,1YeYao,1BoZhang,1Da-ChengHao,2Qing-FengSun,1Jing-BoLi,1ChaoYuan,1
BaoJing,1Yun-PengWang,1andHai-YangWang1
1DepartmentofVascularSurgery,TheFirstAffiliatedHospitalofHarbinMedicalUniversity,Harbin150001,China
2BiotechnologyInstitute,SchoolofEnvironmentandChemicalEngineering,DalianJiaotongUniversity,Dalian116028,China
CorrespondenceshouldbeaddressedtoHai-YangWang;[email protected]
Received 27 October 2016; Revised 4 February 2017; Accepted 9 February 2017; Published 5 March 2017
AcademicEditor:XuweiHou
Copyright©2017TongJiaoetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
MicroRNAs (miRNAs) are deregulated in various vascular ailments including abdominal aortic aneurysm (AAA). MiR-103 is
involvedinvascular,metabolic,andmalignantdiseases,butwhetheritparticipatesinthepathogenesisofAAAremainselusive.
ADAM10playsavitalroleintheformationofaneurysm,butwhethermiRsmodulateitsactivityduringAAAformationistotally
unknown.Inthisstudy,wedetectedthesignificantlyincreasedproteinexpressionofADAM10inangiotensinIIinducedmurine
AAAspecimens,whilethemRNAexpressionofADAM10wassimilarbetweenAAAandcontrol,suggestingtheposttranscriptional
regulation.TheADAM10specificinhibitorGI254023Xdramaticallyreducedthemacrophageinfiltrationofmurineabdominal
aorta.BioinformaticpredictionssuggestthatADAM10isthetargetofmiR-103a/107butthebindingsiteisexclusive.Atthecellular
level,miR-103a-1suppressedtheproteinexpressionofADAM10,whileantisensemiR-103a-1increaseditsexpression.Particularly,
withtheprogressionofmurineAAA,themRNAexpressionofmiR-103a/107substantiallydecreasedandtheproteinexpressionof
ADAM10greatlyincreased.Together,ourdataaffordthenewinsightthatmiR-103ainhibitedAAAgrowthviatargetingADAM10,
whichmightbeapromisingtherapeuticstrategytoalleviateAAA.
1.Introduction Anaorticaneurysmisaballoon-likebulgeintheaorta,
thelargearterythatcarriesbloodfromtheheartthroughthe
MicroRNAs(miRNAs)arehighlyconserved,22-nucleotide- chest and torso. Aortic aneurysms were the primary cause
long noncodingRNAs that negativelyregulate gene expres- of 9,863 deaths in 2014 and a contributing cause in more
sion at the posttranscriptional level [1]. MiRNA exerts than17,215deathsintheUnitedStatesin2009[8].Although
the regulatory activity by interacting with complementary imaging and surgical techniques have substantial progress
󸀠 󸀠
sequences (frequently in the 3 -untranslated region (3 - in the last decades, no effective drug therapy is available
UTR))ofmRNAtargets.Theseinteractionsmayleadtothe untilrecently[4],andAAAisstilllife-threatening,especially
protein translation inhibition or target mRNA degradation amongelders.Itisvitaltodevelopearlydetectionmethodand
depending on whether miRNAs and their targets are per- efficientdrug-basedtherapiesforAAApatients[9,10].
fectly complementary. MiRNAs are dysregulated in various MiR-103 is a member of the miR-15/107 family [11].
vasculardiseasesandtheyplayanessentialroleinaneurysm AccumulatingdataindicatethatthederegulationofmiR-103
pathogenesis[2,3].AnumberofmiRNAshavebeeninferred contributestovariousdiseases,suchasmyocardialinfarction
to participate in the aneurysm formation [4, 5]. In recent [3], cancer [12], and diabetes [13]. However, whether it is
years,miRNAshavebeenrecognizedascriticalregulatorsin involvedinthepathogenesisofAAAhasneverbeenreported.
developmentandprogressionofabdominalaorticaneurysm The principal TNF-𝛼 (tumor necrosis factor-𝛼) converting
(AAA)[6,7]. enzyme, a disintegrin and metalloproteinase 17 (ADAM17),
2 BioMedResearchInternational
Table1:Primersforplasmidconstruction,qRT-PCR,andoligonucleotide.
Primername Nucleotidesequence
Plasmidconstruction
󸀠 󸀠
ADAM10XhoI F 5CCGCTCGAGCTGCAGCTTTTGCCTTGGTTCTTC3
󸀠 󸀠
ADAM10NotI R 5ATAAGAATGCGGCCGCTTTTTGGGTGTTTTAAAAAGGTTTATTG3
󸀠 󸀠
mutADAM10 F 5CGTGTTCCCTGTTCTTCGTCAACAAATTTTCTTCACTTGCAGGCA3
󸀠 󸀠
mutADAM10 R 5TGCCTGCAAGTGAAGAAAATTTGTTGACGAAGAACAGGGAACACG3
qPCR
󸀠 󸀠
ADAM10 F 5CGTGTTCCCTGTTCTTCGTT3
󸀠 󸀠
ADAM10 R 5TAGCCTTGATTGGCAGTTGA3
󸀠 󸀠
miR103a F 5AGCAGCAUUGUACAGGGCUAUGA3
󸀠 󸀠
miR107 F 5AGCAGCAUUGUACAGGGCUAUCA3
󸀠 󸀠
snRNAU6 F 5GCTTCGGCAGCACATATACTAAAAT3
󸀠 󸀠
snRNAU6 R 5CGCTTCACGAATTTGCGTGTCAT3
󸀠 󸀠
GAPDH F 5TGACTTCAACAGCGACACCCA3
󸀠 󸀠
GAPDH R 5CACCCTGTTGCTGTAGCCAAA3
󸀠 󸀠
pri-miR-103a-1 F 5TGCTCACCACTATACCACCAG3
󸀠 󸀠
pri-miR-103a-1 R 5CAAAGAATCCCAACTATATGGTC3
󸀠 󸀠
pre-miR-103a-1 F 5CCCTCGGCTTCTTTACAGTG3
󸀠 󸀠
pre-miR-103a-1 R 5CAATGCCTTCATAGCCCTG3
isinvolvedinthedevelopmentofhumanAAA[9].Nostudy radiotherapy or chemotherapy before surgery. The study
suggeststheinvolvementofsomeothermembersofADAM was approved by the Ethics Committee of the 1st Affiliated
family,forexample,ADAM9and12,intheformationofAAA. HospitalofHarbinMedicalUniversity.
In recent years, ADAM10 has been found to participate in
thedevelopmentofAAA[14]andthoracicaorticaneurysm 2.2. Cell Culture. Smooth muscle cells (SMCs) were grown
[15], but its regulatory factor is elusive. Could ADAM10 in RPMI1640 medium (GIBCO Laboratories, NY, US)
be regulated by miRs? Does miR-103 suppress the AAA and HEK293T cells were grown in DMEM medium. All
formation by directly interacting with its targets of aortic media were supplemented with 10% fetal bovine serum
tissues?Todate,thereisnostudyreportingtheimplication (Sigma),100U/mLpenicillinG,and100𝜇g/mLstreptomycin
ofmiR-103intheprogressionofAAA. (GIBCO). All cells were cultured at 37∘C in a humidified
Inthepresentstudy,wedeterminedthepotentialeffectof incubatorcontaining5%CO2.
miR-103 on murine AAA progression. Our data confirmed
the direct interaction between miR-103a-3p and ADAM10 2.3. Vectors Construction, Oligonucleotide Synthesis, and
and showed that miR-103a-3p has lower expression level Transfection. Table1listedallrelatedDNAsequences.Cells
in AAA samples, accompanied by the increased expres- wereplatedat60%to70%confluencyonthedaybeforetrans-
sion of ADAM10 proteins. We found that miR-103a, whose fection. Human microRNA mimics and the corresponding
expressioncouldbeincreasedbyitsSNP,inhibitedADAM10 antisenseRNAsweresynthesizedbyTakara(Dalian,China).
proteinexpressioninvitro.GiventhattheADAM10inhibitor The microRNAs and antisense RNAs were transfected at a
reducedtheinflammationofmurineaorticwallandthefor- final concentration of 20nmol/L, using lipofectamine 2000
mationofAAAandenhancedexpressionofmiR-103aand/or (Invitrogen) according to the manufacturer’s recommenda-
interfering with ADAM10 function might be a promising tions.
AAAtherapeuticstrategy.
2.4. Quantitative Real-Time PCR (qRT-PCR). Quantitative
2.MaterialsandMethods real-time RT-PCR was conducted similar to that described
previously [9, 16, 17]. Total RNA, including ≥18nt miRs,
2.1.Angiotensin(Ang)IIInfusionModelandMurineSamples. was isolated using miRNeasy Mini Kit (Qiagen, Germany)
Osmotic pumps (Alzet model 2004) containing Ang II according to the manufacturer’s instructions. Afterwards,
(1𝜇g/kg per minute; Sigma-Aldrich) were implanted in 10- RNAwasreversetranscribed,usingReverTraAceqPCRRT
week-oldapoE−/−malemice(day0;C57BL/6Jbackground). Master Mix with gDNA Remover (Toyobo, Shanghai; for
Theaortadiameterwasdeterminedbyultrasonicimaging[7]. ADAM10)andmiScriptIIRTKit(Qiagen;formiRs),respec-
AAA samples and their adjacent normal tissues were tively. Subsequent qRT-PCR was performed using SYBR
collected from AAA mouse in the First Affiliated Hospital Green Real-Time PCR MasterMix-plus (Toyobo), miScript
of Harbin Medical University and immediately stored in SYBRGreenPCRKit,andmiScriptPrimerAssay(Qiagen),
liquid nitrogen until use. No mice had been treated with onaCFX96Real-TimePCRSystem(Bio-Rad,US)asfollows:
BioMedResearchInternational 3
∘ ∘ ∘
95 C for 30s and then 40 cycles of 95 C for 5s and 60 C US) was used, followed by the procedures as described
∘
for 30s and one step of 95 C for 15s. Each amplification above.
reactionwasperformedintriplicateinafinalvolumeof20𝜇L
containing 2𝜇L of the cDNA. Fluorescence was detected
2.6.LuciferaseReporterAssay. ToconstructthepsiCHECK2-
at the end of each cycle. The expression levels of miRNAs 󸀠
ADAM10-3 UTRplasmidthatcontainedthepotentialbind-
werenormalizedtoU6andwerecalculatedusingthe2−ΔΔCt ing sites of the ADAM10 3󸀠-UTR downstream of the fire-
method. fly luciferase gene, a 2346bp sequence was amplified and
inserted into the XhoI and NotI sites of the psiCHECK-
2 Luciferase vector (Promega, Madison, WI, USA). The
2.5.WesternBlotAnalysisandImmunohistochemistry(IHC). 󸀠
plasmidwiththeADAM103 -UTR,wherethemiR-103target
To determine the level of ADAM10 and other protein
sitewassubjectedtothesite-directedmutagenesis,wasalso
expressions, total cell or tissue extracts were extracted in
constructed. Site-directed Gene Mutagenesis Kit was used
cell lysis buffer (Millipore), followed by SDS-PAGE and
for site-directed mutagenesis of ADMA10 mutated clone
Western blot analysis. Equal quantities of tissue lysates
(BeyotimeNO.D0206).HEK293Tcellswereusedtomeasure
wereresolvedbySDS-PAGE,transferredontoPVDFmem-
luciferase activity. When they grew to 60–70% confluence,
branes (Roche Diagnostics, Indianapolis, IN, USA) using
cells were cotransfected with 100ng luciferase plasmid and
electroblotting, probed with specific primary antibodies,
50ngRenillaluciferaseplasmid(Ambion,Austin,TX,USA)
followedbyhorseradishperoxidase-(HRP-)conjugatedsec-
alongwith650ngmiR-103mimicorNCasdescribedabove.
ondary antibodies, and then analyzed. The primary anti- ∘
Afterincubationfor48hat37 C,theluciferaseactivitywas
bodies used were rabbit polyclonal to ADAM10 ADAM10
quantified with the Dual-Luciferase Reporter Assay System
(1:1,000;Abcam,ab1997,Cambridge,UK),rabbitpolyclonal
(Promega).TheRenillaluciferaseactivitywasusedasinternal
toADAM17(Abcam,ab2051),rabbitpolyclonaltoADAM9
control and the firefly luciferase activity was calculated as
(Abcam, ab186833), rabbit polyclonal to ADAM12 (Abcam, themean±SDafterbeingnormalizedbyRenillaluciferase
ab39155),andmonoclonalrabbitanti-humanGAPDHanti-
activity.
body(1:500;SantaCruzBiotechnology,Inc.,CA,USA)and
were detected using HRP-conjugated goat anti-rabbit IgG
antibody(1:500;Millipore)inblockingsolution.The75kDa 2.7. The Effect of SNP on the Expression Level of miR-103a-1.
band was used to analyze changes in ADAM10 expression SearchtheSNPsiteofmiR-103-1fromMfolgprogramversion
inalltheexperiments.Immunoreactiveproteinbandswere 3.5(http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-
visualized using a Luminata Crescendo Western HRP Sub- Form) to predict pre-miR-103a-1 rs760057865(C) and pre-
strate (Millipore), scanned, and quantified for pixel density miR-103a-1rs760057865(G)secondarystructure.Determine
by the optical density (OD) method, using the Tanon 5200 the effect of SNP on miR-103a in vitro cell model, the first,
automatic chemiluminescence imaging system (Shanghai, celltransfection.Logarithmicgrowthphasecells,according
China). to Invitrogen lipofectamine 2000, were transfected into the
FordetectingCX3CL1,TNF-𝛼,IL-6R,andVE-cadherin plasmid and transfection reagent. After 4 to 6 hours, we
in aorta tissues and cultured cells, the rabbit polyclonal observedthecellconditionthatchangedtheculturemedium.
to CX3CL1 (Abcam, ab25088), rabbit polyclonal to TNF- After 24h, we observed the fluorescent, when the rate of
𝛼 antibodies (Abcam, ab9739), rat monoclonal to IL6R fluorescent was more than 80%, took the picture, and than
(Abcam, ab83053), and rabbit monoclonal to VE-cad- added 500uL medium. The cells were harvested for RNA
herin (Abcam, ab205336) were used as the primary isolation.AndthenwedidtheReal-TimePCRandcollected
antibodies, followed by the procedures as described the cells, total RNAs were extracted with TRIzol, cDNA
above. synthesis was performed using the dNTP, M-MLV-RTase
Immunohistochemicalanalysiswasperformedtodeter- (Promega), and quantitative PCR was performed using the
mine ADAM10 protein localization and expression. The SYBRMasterMixture(Takara).Thestatisticswereanalyzed
primary antibody (rabbit anti-human ADAM10; Abcam) use𝐹=2−ΔΔCt.
was diluted at 1:500 and ADAM10 binding was visualized
usingthestandardavidin/biotinylatedenzymecomplex-HRP 2.8. Bioinformatics and Statistical Analysis. MiRs interact-
staining procedure, with 3,3󸀠-diaminobenzidine as a chro- ing with ADAM10 3󸀠 UTR were predicted by Targetscan
mogenandaHRP-conjugatedgoatanti-rabbitIgGantibody (http://www.targetscan.org), RNAhybrid (http://bibiserv
(1:500; Millipore). The sections were examined using an .techfak.uni-bielefeld.de/rnahybrid/), and miRanda (http://
optical microscope (BX40; Olympus Corporation, Tokyo, www.microrna.org)software.
Japan).Cellsthatcontainedbrown-yellowstainedgranulesin The software SPSS version 20.0 was used for statistical
themembraneandcytoplasmwereconsideredpositive.Next, analysis. All values are expressed as mean ± SEM and all
fivehundredcellsinfiverandomlyselectedfieldsunderhigh datahavebeenrepeatedatleastthreetimes.Student’s𝑡-tests
magnificationwerecounted.Presenceof<5%ofpositivecells were used to determine the statistical significance of differ-
wasclassifiedas(−),then5–50%as(+),50–75%as(++),and ences between groups. Spearman’s correlation was applied
>75%as(+++). toidentifythecorrelationbetweenmiR-103aexpressionand
For detecting Mac-2 in aorta tissues, the purified ADAM10 expression. Differences with 𝑝 < 0.05 were
monoclonal anti-mouse Mac-2 antibody (BioLegend, consideredsignificant.
4 BioMedResearchInternational
3.Results ComparingmiR-103agroupandnegativecontrol(NC)group,
we found the significantly higher luciferase activity in the
3.1. ADAM10 Was Upregulated in Murine AAA. On day 21 latter (𝑝 = 0.004107), indicating that miR-103a influenced
ofangiotensin(Ang)IIinfusion,theexpressionofADAM10 thefluorescencebybindingADAM103󸀠UTR.Theluciferase
proteininAAAwasmorethantwicethatincontroltissues activity of 103a inhibitor group was substantially higher
(Figure 1(a)), accompanied by the significantly increased than those of blank group and NC inhibitor group (𝑝 =
expression of ADAM9, 12, and 17 (𝑝 < 0.05) (Figures 0.002351and0.011695,resp.),suggestingthattheendogenous
1(b)–1(d)).Immunohistochemistry(IHC)showedabundant miR-103a of 293T cells could inhibit the Renilla luciferase
ADAM10 protein (red color) in the aneurysmal aorta (Fig- of reporter plasmid, and 103a inhibitor suppressed both
ure1(e)).TheADAM10indexofAAAwassignificantlyhigher endogenous and exogenous miR-103a. These data support
than that of normal aorta (𝑝 < 0.05). Intriguingly, the thatmiR-103aboundADAM103󸀠UTR,inhibitedtargetgene
mRNA expression of ADAM10 was not different between expression,andsuppressedtheluciferaseactivity.Moreover,
AAA and control (𝑝 > 0.05) (Figure 1(f)), implying the therecouldbeonlyonebindingsite.
posttranscriptionalregulationofADAM10.
In comparison, transfection of either miR103a/inhibitor
or NC/inhibitor did not elicit any significant change of
3.2.ADAM10WasInvolvedintheFormationofMurineAAA. luciferaseactivity(Figures3(a)and3(b)),ascomparedwith
GI254023X is a specific inhibitor of ADAM10 [18] and was the blank group. These results suggest that the mutated
󸀠
used here to study the correlation between ADAM10 and ADAM10 3 UTR could not bind miR-103a and the latter’s
murine AAA. On day 14 of angiotensin II infusion, the inhibitoryactivityofluciferasewasabolished.
administrationofGI254023Xsignificantlyreducedthesizeof AshumanmiR-103aandmiR-107,abundantlyexpressed
theAAA(Figure2(a)),andtheaortadiameterwasdecreased invasculature[13,22](seebelow),areevolutionarilyclosely
from 0.74mm (no inhibitor) to 0.41mm (Figures 2(b) and related and belong to the same miR family [11], we also
2(c)), which was comparable to that of control (0.37mm) transfected miR-107 and reporter plasmid into 293T cells
(esote MyLab-60, probe 6–18hz). On day 21, the inhibitory andfoundthattheexogenousmiR-107ledtothesignificant
effectsofGI254023Xwerestillsignificant.TheMSBP(mean decreaseofluciferaseactivity(61%oftheblank,Figure3(c);
systolic blood pressure) was similar between control group 𝑝 = 0.001753, Figure 3(d)). The reported activity of miR-
andGI254023Xgroup(Figure2(d)),whichwassubstantially 107 group was much lower than that of NC group (𝑝 =
lowerthanthatofangiotensinIIgroup. 0.000443), indicating that miR-107 influenced the fluores-
󸀠
Correspondingly, GI254023X noticeably inhibited the cencebybindingADAM103 UTR.Theluciferaseactivityof
macrophageinfiltrationwithinthemurineaorticwalls,which 107 inhibitor group was substantially higher than those of
is supported by the reduced Mac-2 protein (red color) [19] blank group and NC inhibitor group (𝑝 = 0.020667 and
of the macrophage surface in IHC (Figure 2(e)). Western 0.013883 resp.), suggesting that the endogenous miR-107 of
blot analysis confirmed that the ADAM10 protein level 293T cells could inhibit the Renilla luciferase of reporter
in AngII group was nearly four times that in GI254023X plasmid,and107inhibitorsuppressedbothendogenousand
group (Figure 2(f)). Moreover, GI254023X suppressed the exogenous miR-107. In contrast, miR-107 could not bind
󸀠
protein expression of (C-X3-C motif) ligand 1 (CX3CL1) the mutated ADAM10 3UTR and its inhibitory effect of
(Figure 2(h)), IL-6R (Figure 2(i)), and VE-cadherin (Fig- luciferasewasabolished(Figure3(d)).
ure 2(j)), an endothelial proinflammatory chemokine that Taken together, these data identified ADAM10 as the
bindsmonocyteCX3Cchemokinereceptor1(CX3CR1)[20, directtargetofmiR-103a/107.
21], while it did not decrease the expression of TNF-𝛼
(Figure 2(g)), a substrate of ADAM17. These data suggest
3.4. MiR-103a Suppressed the ADAM10 Expression In Vitro.
that ADAM10 could enhance the macrophage infiltration,
TheoverexpressionandinhibitionofmiR-103ainSMCswere
exacerbatetheinflammatoryresponse,andpromotetheAAA
performedtodetermineitsroleinAAA.Theoverexpression
formationinmice.
of miR-103a apparently inhibited the protein expression of
ADAM10invitro(𝑝 < 0.05) (Figure4(a)). In contrast,the
3.3.ADAM10IstheDirectTargetofmiR-103a/107. Allthree overexpression of miR-107 marginally decreased ADAM10
softwareprogrammesidentifiedtargetingsitesformiR-103a- in SMCs. The anti-miR-103a inhibited miR-103a and subse-
3p and miR-107 in the 3󸀠-untranslated region (3󸀠-UTR) of quentlysignificantlyupregulatedtheADAM10proteinlevel
ADAM10 (Fig. S1A–C, in Supplementary Material available (𝑝 < 0.05) (Figure 4(b)). The anti-miR-107 caused the
online at https://doi.org/10.1155/2017/9645874). The 3󸀠UTR increaseofADAM10toalesserdegree.
sequence of ADAM10 was amplified from human genomic
DNA and cloned into the dual-luciferase reporter vector. 3.5. Temporal Expression of ADAM10 and miR-103a/107
The reporter plasmid with the mutated binding site of in Murine AAA. We measured miR-103 level, using qRT-
miR-103a/107 was also constructed. In HEK 293T cells, PCR, in the murine AAA tissues (Figure 5(a)). MiR-103
transfection of human miR-103a-3p led to the significant was downregulated in AAA and closely associated with
decreaseofluciferaseactivity(58%oftheblank,Figure3(a); AAA formation and progression. We reveal that miR-103
𝑝 = 0.000155, Figure 3(b)), while transfection of miR-103a expressionwasinverselycorrelatedwithADAM10inmurine
inhibitordidnotresultinthereductionofluciferaseactivity. AAAsamples.InaortasofapolipoproteinE-deficientmiceon
BioMedResearchInternational 5
Control AngII Control AngII
ADAM10(75 kDa) ADAM17(93kDa)
GAPDH (36kDa) GAPDH (36kDa)
e ADAM10 ADAM17
c
an 2.0 ∗∗ ce 8 ∗∗
d n
n a
u d
ab 1.5 un 6
ative 1.0 ve ab 4
zed rel 0.5 d relati 2
ali ze
orm 0.0 mali 0
N Control AngII Nor Control AngII
(a) (b)
Control AngII Control AngII
ADAM12(42kDa)
ADAM9(91kDa)
GAPDH (36kDa) GAPDH (36kDa)
malized relative abundance1086420 ADA∗M∗ 9 malized relative abundance 3210 Ad∗am∗12
Nor Control AngII Nor Control AngII
(c) (d)
Control AngII
0.04 ∗ n 3
o
dex 0.03 xpressi 2
0 in 0.02 ve e
1M ati
DA 0.01 0 rel 1
A 1
M
0.00 A 0
D
Control AngII A Control AngII
(e) (f)
Figure1:ADAM10wasupregulatedinmurineAAA.Westernblotanalysisof(a)ADAM10(control,0.73±0.15;AngII,1.67±0.07;p,0.000);
(b)ADAM17(control,1.71±0.33;AngII,5.89±0.45;p,0.000);(c)ADAM9(control,2.10±0.49;AngII,7.12±0.60;p,0.000);and(d)ADAM12
onday21ofAngIIinfusion(control,1.88±0.05;AngII,2.40±0.28;p,0.0047).GAPDHisthereferenceprotein.∗𝑝 < 0.05,∗∗𝑝 < 0.01,
controlgroup,𝑛=6,andAngIIgroup,𝑛=6.(e)ExamplesofIHCstainingofADAM10inAngIIinducedAAAandcontrolaorta(scalebar
10𝜇m)(control,0.005±0.004;AngII,0.024±0.007;p,0.033),controlgroup,𝑛=5,andAngIIgroup,𝑛=10;(f)ADAM10geneexpression
determinedbyqRT-PCR(control,1.14±0.52;AngII,1.65±1.05;𝑝,0.56),controlgroup,𝑛=4,andAngIIgroup,𝑛=4.
6 BioMedResearchInternational
AngII Control AngII + GI254023X
0.37 0.74mm 0.43mm
(a) (b)
1.5 Diameter 800 Blood pressure/heart rate
∗∗
m
m 600
AA 1.0 ∗∗ ∗∗ min ∗
of A ∗∗ g, n/ 400 ∗∗
meter 0.5 mmH 200
a
Di
0.0 0
14d 21d BSP AVGHR
AngII AngII
Control Control
AngII + GI254023X AngII + GI254023X
(c) (d)
AngII AngII + GI254023X
ADAM10(75kDa)
IL-6R (55kDa)
VE-cadherin (70kDa)
AngII AngII + GI254023X
훽-Actin (42kDa)
Macrophage
∗ ADAM10
x 0.020 e 1.5 ∗∗
e c
d n
n a
n i 0.015 nd
o u
ati ab 1.0
nfiltr 0.010 ative
age i d rel 0.5
h 0.005 e
crop maliz
Ma 0.000 or 0.0
N
AngII AngII + GI254023X AngII AngII + GI254023X
(e) (f)
AngII AngII + GI254023X AngII AngII + GI254023X
Tnf-
훼(17kDa) Cx3cl1(42kDa)
GAPDH (36kDa) GAPDH (36kDa)
nce 0.6 TNF-
훼 nce 3 CX3CL1
da da
n n
u u
d relative ab 00..42 d relative ab 21
malize malize
Nor 0.0 Nor 0
AngII AngII + GI254023X AngII AngII + GI254023X
(g) (h)
Figure2:Continued.
BioMedResearchInternational 7
IL-6R VE-cadherin
nce 1.5 ∗ nce 600 ∗
da da
n n
u u
ve ab 1.0 ve ab 400
d relati 0.5 d relati 200
malize malize
Nor 0.0 Nor 0
AngII AngII + GI254023X AngII AngII + GI254023X
(i) (j)
Figure2:ADAM10wasinvolvedintheformationofmurineAAA.(a)ComparisonofAAAsizeswith/withoutADAM10inhibitorGI254023X
bynakedeye;(b)therepresentativeultrasonicimagesofAAAdiameterwith/withoutGI254023Xonday14ofAngIIinfusion;(c)comparison
ofAAAdiameterswith/withoutGI254023Xbyultrasonicmeasurementday14(AngII,0.65±0.17;control,0.33±0.03;AngII+GI254023X
0.41±0.09;p,AngII/control,0.0000;AngII+GI254023X/control,0.0010;AngII/AngII+GI254023X,0.0000),AngIIgroup,𝑛=21,control
group,𝑛 = 3,andAngII+GI254023Xgroup,𝑛 = 8;day21(AngII,0.95±0.18;control,0.39±0.03;AngII+GI254023X,0.56±0.07;p,
AngII/control,0.0000;AngII+GI254023X/control,0.0000;AngII/AngII+GI254023X,0.0000),AngIIgroup,𝑛=19,controlgroup,𝑛=3,
andAngII+GI254023Xgroup,𝑛=7;(d)comparisonofbloodpressureandheartratewith/withoutGI254023X.SBPissystolicbloodpressure
(AngII,218.58±83.52;control,149.21±66.91;AngII+GI254023X,162.95±43.97;p,AngII/control,0.0030;AngII+GI254023X/control,
0.4585;AngII/AngII+GI254023X,0.0111),AVGHRisaverageheartrate(AngII,351.37±92.53;control,414.53±183.88;AngII+GI254023X,
420.47±138.96;p,AngII/control,0.1917;AngII+GI254023X/control,0.8999;AngII/AngII+GI254023X,0.1023),AngIIgroup,𝑛=19,control
group,𝑛=18,andAngII+GI254023Xgroup,𝑛=6;(e)examplesofIHCstainingofMac-2inAAAmacrophageswith/withoutGI254023X
(scalebar10𝜇m)(AngII,0.017±0.002;AngII+GI254023X,0.004±0.003;p,0.0151),AngIIgroup,𝑛=4,andAngII+GI254023Xgroup,𝑛=8;
(f)comparisonofADAM10proteinexpressionswith/withoutGI254023XbyWesternblotanalysis(AngII,0.98±0.44;AngII+GI254023X,
0.26±0.12;p,0.002),AngIIgroup,𝑛=5,andAngII+GI254023Xgroup,𝑛=4;(g)comparisonofTNF-𝛼proteinexpressionswith/without
GI254023XbyWesternblotanalysis(AngII,0.08±0.03;AngII+GI254023X,0.38±0.11;p,0.06),AngIIgroup,𝑛=6,andAngII+GI254023X
group,𝑛 = 6;(h)comparisonofCX3CL1proteinexpressionswith/withoutGI254023XbyWesternblotanalysis(AngII,2.40±0.41;AngII
+GI254023X,1.62±0.22;p,0.02),AngIIgroup,𝑛=6,andAngII+GI254023Xgroup,𝑛=6;(i)comparisonofIL-6Rproteinexpressions
with/withoutGI254023XbyWesternblotanalysis(AngII,0.99±0.33;AngII+GI254023X,0.32±0.07;p,0.04),AngIIgroup,𝑛=5,andAngII
+GI254023Xgroup,𝑛 = 4;(j)comparisonofVE-cadherinproteinexpressionswith/withoutGI254023XbyWesternblotanalysis(AngII,
421.18±110.00;AngII+GI254023X,230.48±62.72;p,0.04).GAPDHisthereferenceprotein;∗𝑝<0.05,∗∗𝑝<0.01,AngIIgroup,𝑛=5,
andAngII+GI254023Xgroup,𝑛=4.
angiotensin II infusion, the mRNA expression of miR103a- theprecursormicroRNA(pre-miR).Theexpressionlevelof
3p was significantly decreased on day 14 as compared with pre-miR-103a-1inWT103+WT67was4792513.1timesthat
control group (𝑝 < 0.01). MiR103a-3p expression on day ofNC(Figure6(b)),andMU103+WT67furtherincreased
28 was still substantially lower than control (𝑝 < 0.01), its expression (𝑝 < 0.01, compared to WT103 + WT67
althoughitwasalittlehigherthanthatonday14.ThemRNA or NC). Consequently, the transfection of mutant pri-miR-
expressionofmiR107-3pwasalsodramaticallydecreasedon 103a-1,containingtheSNPrs760057865(C>T),significantly
day14ascomparedwithcontrol(𝑝<0.05)(Figure5(b)),but enhancedtheexpressionofhumanmiR-103a-1inHEK293T
itsexpressiononday28wasnotsubstantiallydifferentfrom (𝑝<0.01,Figure6(c)).TheseresultssuggestthatSNPofthe
thatofcontrol.TheproteinexpressionofADAM10gradually miRNAmightinfluenceitsexpressionlevel,followedbythe
increased between day 0 and day 14 (Figure 5(c)), and the alteredmodulationofADAM10expression.
expression level on day 14 was noticeably higher than that
ofcontrol(𝑝 < 0.05).AlthoughtheADAM10expressionon
4.DiscussionandConclusion
day28decreased,itwasstillconsiderablyhigherthanthatof
controlgroup(𝑝 < 0.05).Theseresultssuggestthenegative
Several studies have addressed the involvement of miRs in
modulationofADAM10bymiR-103a-3p.
aneurysm formation and complications [4]. For instance,
miR-195targetsacadreofextracellularmatrixproteinsand
3.6.TheEffectofrs760057865(C>T)ontheExpressionLevel displays significant inverse correlation with aortic diameter
of miR-103a-1. SNP site of miR-103-1 was shown in Fig. S2. [23].MiR-24limitsaorticvascularinflammationandmurine
In HEK293Tcells,theexpression levelofpri-miR-103a-1in abdominal aneurysm development [7]. Downregulating
NC + WT67 was 45.4% of NC group (Figure 6(a)). The miR-98increasedthechemotaxisofTHP-1macrophagesand
expression level of pri-miR-103a-1 in WT103 + WT67 was MCP-1inducedIL-6expressioninTHP-1cells[6].Notwith-
150501.5 times that of NC, and MU103 + WT67 expressed standing,ADAMfamilymembersarenotthetargetofthese
much higher level of pri-miR-103a-1 (𝑝 < 0.01, compared antianeurysm miRs, and whether miR103a/107 is involved
toWT103+WT67orNC).Thepri-miRisprocessedtoform in the pathogenesis of AAA has never been revealed. We
8 BioMedResearchInternational
psiCHECK-ADAM103㰀UTR
∗∗ ∗∗ psiCHECK-mutADAM10 3㰀UTR
5 6
∗∗
4
fly fly
Fire Fire 4
nilla/ 3 nilla/
Re Re
Luciferase 21 Luciferase 2
0 0
Blank NC Blank NC
has-mir-103a-3p NC inhibitor has-mir-103a-3p NC inhibitor
has-mir-103a-3p inhibitor has-mir-103a-3p inhibitor
(a) (b)
psiCHECK-ADAM103㰀UTR psiCHECK-mutADAM10 3㰀UTR
5 ∗ ∗ 6
∗∗ ∗∗
4
y y
fl fl
e e
Fir Fir 4
a/ 3 a/
nill nill
e e
R R
e 2 e
eras eras 2
cif cif
Lu 1 Lu
0 0
Blank NC Blank NC
has-mir-107 NC inhibitor has-mir-107 NC inhibitor
has-mir-107inhibitor has-mir-107inhibitor
(c) (d)
Figure 3: The dual-luciferase reporter assay indicates that ADAM10 is the direct target of miR-103a/107. (a) Luciferase activities on the
cotransfectionofthereporterplasmidwithnormalADAM103󸀠UTR(blank,3.503±0.0697;hsa-mir-103a-3p,2.048±0.0587;hsa-mir-103a-3p
inhibitor,4.196±0.0924;NC,3.453±0.1123;NCinhibitor,3.453±0.0513;p,3󸀠UTRADAM10+hsa-mir-103a-3p/3󸀠UTRADAM10,0.000155;
󸀠 󸀠 󸀠 󸀠
3UTR ADAM10 + hsa-mir-103a-3p/3UTR ADAM10 + NC, 0.004107; 3UTR ADAM10 + hsa-mir-103a-3p inhibitor/3UTR ADAM10,
󸀠 󸀠 󸀠
0.002351;3UTRADAM10+hsa-mir-103a-3pinhibitor/3UTRADAM10+NCinhibitor,0.011695);(b)theeffectofmutatedADAM103UTR
ontheluciferaseactivitywhencotransfectingblank,humanmiR-103a-3p,103ainhibitor,NC,orNCinhibitor(blank,5.071±0.0938;hsa-mir-
103a-3p,5.061±0.0910;hsa-mir-103a-3pinhibitor,5.146±0.1471;NC,5.164±0.0577;NCinhibitor,5.135±0.0961;p,3󸀠UTRmutADAM10
󸀠 󸀠 󸀠 󸀠
+hsa-mir-103a-3p/3UTRmutADAM10,0.933656;3UTRmutADAM10+hsa-mir-103a-3p/3UTRmutADAM10+NC,0.346206;3UTR
󸀠 󸀠 󸀠
mutADAM10+hsa-mir-103a-3pinhibitor/3UTRmutADAM10,0.177698;3UTRmutADAM10+hsa-mir-103a-3pinhibitor/3UTRmut
󸀠
ADAM10+NCinhibitor,0.811131);(c)luciferaseactivitiesonthecotransfectionofthereporterplasmidwithnormalADAM103UTR(blank,
3.503±0.0697;hsa-mir-107,2.139±0.0671;hsa-mir-107inhibitor,3.892±0.0292;NC,3.453±0.1123;NCinhibitor,3.425±0.0982;p,3󸀠UTR
󸀠 󸀠 󸀠 󸀠
ADAM10+hsa-mir-107/3UTRADAM10,0.001753;3UTRADAM10+hsa-mir-107/3UTRADAM10+NC,0.000443;3UTRADAM10+
󸀠 󸀠 󸀠
hsa-mir-107inhibitor/3UTRADAM10,0.020667;3UTRADAM10+hsa-mir-107inhibitor/3UTRADAM10+NCinhibitor,0.013883);(d)
󸀠
theeffectofmutatedADAM103UTRontheluciferaseactivitywhencotransfectingblank,humanmiR-107,107inhibitor,NC,orNCinhibitor
(blank,5.071±0.0938;hsa-mir-107,5.194±0.0986;hsa-mir-107inhibitor,5.233±0.2665;NC,5.164±0.0577;NCinhibitor,5.135±0.0961;
󸀠 󸀠 󸀠 󸀠
p,3UTRmutADAM10+hsa-mir-107/3UTRmutADAM10,0.020979;3UTRmutADAM10+hsa-mir-107/3UTRmutADAM10+NC,
󸀠 󸀠 󸀠 󸀠
0.544506;3UTRmutADAM10+hsa-mir-107inhibitor/3UTRmutADAM10,0.516319;3UTRmutADAM10+hsa-mir-107inhibitor/3UTR
mutADAM10+NCinhibitor,0.677649).∗𝑝<0.05;∗∗𝑝<0.01.
BioMedResearchInternational 9
ADAM10
1
a-
ADAM10 3 7
0 0
1 1
ntrol R-103a-1 R-107 ontrol nti-miR- nti-miR-
Co mi mi C A A
ADAM10(75kDa)
ADAM10(75kDa)
GAPDH (36kDa)
GAPDH (36kDa)
∗∗
0.5 ∗∗ 0.8
nce 0.4 nce
da da 0.6
n n
u u
ve ab 0.3 ve ab
d relati 0.2 d relati 0.4
malize 0.1 malize 0.2
or or
N N
0.0 0.0
Control mir-103a-1 mir-107 Control anti-mir-103a-1 anti-mir-107
(a) (b)
Figure4:TheeffectsofmiR-103a-1,miR-107,andtheirantisenseRNAsontheADAM10proteinexpression.(a)Westernblotofcontrol,miR-
103a-1,andmiR-107(control,0.36±0.05;miR-103a-1,0.20±0.15;miR-107,0.27±0.12;p,miR-103a-1/control,0.000;miR-107/control,0.000;
miR-107/miR-103a-1,0.000).(b)WesternblotofcontrolandantisenseRNAs(control,0.29±0.15;anti-miR-103a-1,0.61±0.11;anti-miR-107,
0.47±0.20;p,anti-miR-103a-1/control,0.000;anti-miR-107/control,0.000;anti-miR-107/anti-miR-103a-1,0.000).∗∗𝑝<0.01.
thusinvestigatedthemechanismsunderlyingtheroleofmiR- bladder, lung, and giant cell tumors of bone [27–30]. It
103inAAAformationandprogressionandprovidethefirst has been found that, besides ADAM12, several ADAMs,
batch of evidence that miR-103a/107 may contribute to the including ADAM9, 10, 17, and 19, are also involved in the
pathogenesisofaorticaneurysmviasuppressingADAM10. tumorigenesis, invasion, and metastasis of several types of
ADAMs are a family of transmembrane and secreted solid tumors [26, 28, 29, 31–35]. In addition, ADAM12 is
metalloendopeptidases. All ADAMs are characterized by a strongly expressed in the cardiovascular system, erythroid
particular domain organization featuring a prodomain, a progenitors,brain,andjawcartilageduringzebrafishdevel-
metalloprotease,adisintegrin,acysteine-rich,anepidermal- opment[36].ThesignificantincreaseofADAM10inarteries
growthfactorlike,andatransmembranedomain,aswellasa maycontributetotheprogressionofatherosclerosis[37].The
C-terminalcytoplasmictail[24].Nonetheless,notallhuman neutrophilsoftheintraluminalthrombusexposedtotobacco
ADAMshaveafunctionalproteasedomain.ThoseADAMs smoke extract showed increased ADAM10 and 17 content,
whichareactiveproteasesareclassifiedassheddasesbecause cleavage of these molecules into active forms, and release
theycutofforshedextracellularportionsoftransmembrane of membrane microvesicles carrying mature ADAM10 and
proteins[25].AmongtheseADAMs,ADAM17wasthefirst detectableADAM17,whichcontributedtothedamageofthe
sheddase to be identified and plays a crucial role in cell- underlyingaorticwall[38].Asmightbeexpected,wefound
cell communication, being able to release not only TNF- thatbothADAM10andADAM17proteinswereupregulated
𝛼 but alsoseveral othertransmembraneproteins,including inthemurineAAAinthecurrentstudy,whichissimilarto
cytokines, adhesion molecules, receptors, and growth fac- thepreviousobservationthatbothtransmembraneproteases
tors [8]. ADAM10 is another important membrane-bound arealsoelevatedintheratthoracicaorticaneurysm[39].The
sheddaseofADAMfamily,whichisrequiredfortheproper dataalsooffernewexperimentalsupportfortheinvolvement
functioningofNotch,Eph/ephrin,HER2receptor,orclassic of ADAM10 in the development of AAA, consistent with
cadherins[24,26].Duringthelastdecade,ADAM12emerged previousstudy[14].
as the most strongly functional ADAM in human tumor ADAM10 is involved in the shedding of dozens of sub-
development.UpregulationofADAM12hasbeendescribed strates,suchasCX3CL1[40],IL6-receptor(IL-6R)[40–42],
innumerouscancers,includingbreast,colon,hepatocellular TNF-𝛼, and vascular endothelial cadherin (VE-cadherin)
carcinomas, glioblastomas, stomach, oral cavity, esophagus, [43, 44]. Among these substrates, Notch, CD44, IL-6R,
10 BioMedResearchInternational
1.5 mir-103a-3p 1.5 ∗∗mir-107-3p
∗∗
n
xpressio 1.0 ∗ ∗ pression 1.0
e x
ve e e
3a-p relati 0.5 3-p relativ 0.5
3 7
0 0
1 1
mir- mir-
0.0 0.0
Control 7d 14d 28d Control 7d 14d 28d
(a) (b)
ADAM10(75kDa)
GAPDH (36kDa)
Control 7d 14d 28d
2.0 ∗
e
c
n
a 1.5
d
n
u
b
a
e
v
ati 1.0
el
d r
e
z
ali 0.5
m
or
N
0.0
Control 7d 14d 28d
(c)
Figure5:TemporalexpressionofADAM10andmiR-103a/107inAngIIinducedmurineAAA.Controlgroup,𝑛=3;AngII7dgroup,𝑛=6;
AngII14dgroup,𝑛=6;AngII28dgroup,𝑛=6.(a),miR103a-3pexpressiondeterminedbyqRT-PCR(control,0.94±0.07;7d,0.67±0.10;
14d,0.31±0.03;28d,0.49±0.07;p,7d/control,0.029;14d/control,0.000;28d/control,0.006;14d/7d,0.014;28d/7d,0.099;28d/14d,0.055);
(b),miR107-3pexpressiondeterminedbyqRT-PCR(control,1.08±0.220;7d,0.61±0.32;14d,0.56±0.01;28d,0.81±0.13;p,7d/control,0.169;
14d/control,0.000;28d/control,0.125;14d/7d,0.950;28d/7d,0.293;28d/14d,0.099);(c)ADAM10proteinexpressionshownbyWestern
blotanalysis(control,0.64±0.07;7d,0.90±0.48;14d,1.29±0.32;28d,1.10±0.05;p,7d/control,0.416;14d/control,0.046;28d/control,
0.014;14d/7d,0.105;28d/7d,0.528;28d/14d,0.391).∗𝑝<0.05;∗∗𝑝<0.01.
CX3CL1, CXCL16, VEGFR2, and VE-cadherin are of spe- constitutivereleaseofendogenousIL-6Rislargelymediated
cial importance for vascular biology [45]. The cleavage byADAM10[42].IL-6Rconcentrationsaretypicallyelevated
events mediated by ADAM10 play a critical role in various inavarietyofinflammatorydisorders[47].Thechemokine
inflammation-relateddiseases[46],aswellasthepathogen- CX3CL1, expressed by endothelial cells [21], could interact
esis of vascular diseases [43]. ADAM10 modulates vascular with the monocyte C-X3-C motif receptor 1 (CX3CR1) and
permeability by cleaving endothelial adhesion molecules attract monocyte into the inflammatory site of aortic walls.
including VE-cadherin [43, 44]. And it is also the main Duringliverinjury,theCX3CL1/CX3CR1signalingpathway
protease responsible for the ectodomain shedding of var- wasactivatedbyNF-𝜅BandCD4+,CD8+,andNKTcellinfil-
ious cell surface molecules including the IL-6R and the trationincreasedintheliver[20].GI254023Xpotentlyblocks
transmembrane chemokines CX3CL1 and CXCL16 [40, 41]. the constitutive release of IL-6R, CX3CL1, and CXCL16,
Interestingly,endogenousIL-6Rofbothhumanandmurine whichisinlinewiththereportedinvolvementofADAM10
origin is shed by ADAM17 in an induced manner, whereas but not ADAM17 in this process [41]. Thus, GI254023X