micb-09-02712 No embe 7, 2018 Time: 21:31 # 1
ORIGINAL RESEARCH
published: 09 No embe 2018
doi: 10.3389/ micb.2018.02712
Edi ed by:
Alain Pie e Gobe ,
Vande bil Uni e si y Medical Cen e ,
Uni ed S a es
Re iewed by:
Lydia E. W oblewski,
Vande bil Uni e si y Medical Cen e ,
Uni ed S a es
Yoshimi Takai,
Kobe Uni e si y G adua e School
o Medicine, Japan
*Co espondence:
Ceu Figuei edo
[email p o ec ed]
†These au ho s ha e sha ed
co-au ho ship
Special y sec ion:
This a icle was submi ed o
In ec ious Diseases,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 04 Augus 2018
Accep ed: 23 Oc obe 2018
Published: 09 No embe 2018
Ci a ion:
Ma ques MS, Melo J, Ca adas B,
Mendes N, Pe ei a L, Ca nei o F,
Figuei edo C and Lei e M (2018)
A adin Down egula ion by
Helicobac e pylo i Induces Epi helial
o Mesenchymal T ansi ion in Gas ic
Cells. F on . Mic obiol. 9:2712.
doi: 10.3389/ micb.2018.02712
A adin Down egula ion by
Helicobac e pylo i Induces Epi helial
o Mesenchymal T ansi ion in Gas ic
Cells
Miguel Sa dinha Ma ques1,2,3, Joana Melo1,2,4, B uno Ca adas1,2,4, Nuno Mendes1,2,
Luísa Pe ei a1,2,3, Fá ima Ca nei o1,2,3,5, Ceu Figuei edo1,2,3*†and Ma ina Lei e1,2,3†
1i3S – Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o, Po o, Po ugal, 2Ipa imup – Ins i u e
o Molecula Pa hology and Immunology, Uni e si y o Po o, Po o, Po ugal, 3Depa men o Pa hology, Facul y
o Medicine, Uni e si y o Po o, Po o, Po ugal, 4Ins i u o de Ciências Biomédicas Abel Salaza , Uni e si y o Po o, Po o,
Po ugal, 5Depa men o Pa hology, Cen o Hospi ala São João, Po o, Po ugal
A adin is a cy oplasmic p o ein o he adhe ens junc ions, which egula es he o ma ion
and s abiliza ion o bo h he adhe ens and he igh junc ions. Abe an exp ession o
A adin has been shown in cance and i s loss has been associa ed wi h epi helial- o-
mesenchymal ansi ion (EMT). EMT is cha ac e ized by he change om an epi helial o
a mesenchymal pheno ype, wi h modi ica ions on he exp ession o adhesion molecules
and acquisi ion o a mig a o y and in asi e cell beha io . While i is known ha
Helicobac e pylo i dis up s he igh and he adhe ens junc ions and induces EMT, he
e ec o he bac e ia on A adin is s ill unknown. The aim o his s udy was o disclose
he e ec o H. pylo i on A adin and i s impac in he induc ion o an EMT pheno ype
in gas ic cells. Using wo di e en cell lines, we obse ed ha H. pylo i in ec ion
dec eased A adin p o ein le els, independen ly o CagA, T4SS, and VacA i ulence
ac o s. H. pylo i in ec ion o cell lines ecapi ula ed se e al EMT ea u es, displacing and
down egula ing mul iple p o eins om cell–cell junc ions, and inc easing he exp ession
o ZEB1, Vimen in, Slug, N-cadhe in, and Snail. Silencing o A adin by RNAi p omo ed
delocaliza ion o junc ional p o eins om he cell–cell con ac s, inc eased pa acellula
pe meabili y, and dec eased ansepi helial elec ical esis ance, all compa ible wi h
impai ed junc ional in eg i y. A adin silencing also led o inc eased exp ession o he
EMT ma ke Snail, and o he o ma ion o ac in s ess ibe s, oge he wi h inc eased
cell mo ili y and in asion. Finally, and in line wi h ou in i o da a, he gas ic mucosa o
indi iduals in ec ed wi h H. pylo i showed dec ease/loss o A adin memb ane s aining a
cell–cell con ac s signi ican ly mo e equen ly han unin ec ed indi iduals. In conclusion,
A adin is down egula ed by H. pylo i in ec ion in i o and in i o, and i s down egula ion
leads o he eme gence o EMT and o he acquisi ion o an agg essi e pheno ype in
gas ic cells, which can con ibu e o gas ic ca cinogenesis.
Keywo ds: Helicobac e pylo i, A adin, epi helial o mesenchymal ansi ion, cell–cell junc ion dis up ion, gas ic
cance
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Ma ques e al. H. pylo i, A adin and EMT
INTRODUCTION
Helicobac e pylo i is he mos p e alen ch onic in ec ion
wo ldwide, wi h almos hal o he human popula ion being
in ec ed by his bac e ium (Zamani e al., 2018). All indi iduals
in ec ed wi h H. pylo i de elop ch onic in lamma ion o he
gas ic mucosa, which in some cases may p og ess h ough a
cascade o al e a ions ha culmina e in gas ic cance (Polk and
Peek, 2010). In ac , H. pylo i is ega ded as he majo isk ac o
o gas ic cance de elopmen , and has been conside ed as a class
I ca cinogen by he Wo ld Heal h O ganiza ion (IARC, 1994,
2011).
Gas ic mucosal in lamma ion and he de elopmen o mo e
se e e clinical ou comes o H. pylo i in ec ion ha e been
a ibu ed o a ia ion o i ulence ac o s be ween di e en
s ains. Among hem, he ype 4 sec e ion sys em (T4SS)-
ansloca ed CagA oncop o ein and he VacA cy o oxin a e he
bes ecognized, and in ec ion wi h s ains ha bo ing he mos
pa hogenic a ian s o hese ac o s a e associa ed wi h g ea e
in ensi ies o gas ic in lamma ion, and wi h inc eased isk
o de eloping gas ic p emalignan lesions, and gas ic cance
(A he on e al., 1995;Figuei edo e al., 2002;Gonzalez e al.,
2011).
In he s omach, H. pylo i can be ound in he mucus and
in close con ac wi h he epi helium, wi h a opism o cell–
cell junc ions (Tan e al., 2009;Bugay so a e al., 2017). This
p oximi y o H. pylo i o in e cellula con ac s, leads o dis up ion
o he epi helial apical junc ional complex (AJC), which includes
he igh junc ions (TJs) and he adhe ens junc ions (AJs)
(Amie a e al., 2003;W oblewski e al., 2009, 2015;Hoy e al.,
2010).
The TJs con ibu e o he egula ion o epi helial pa acellula
pe meabili y and o main enance o cell pola i y, and a e
cons i u ed by ansmemb ane p o eins, such as occludin,
claudins, and junc ional adhesion molecules (JAMs), and by
cy oplasmic-associa ed p o eins, like zonula occludens 1 (ZO-
1) (Zihni e al., 2016). The AJs a e loca ed below he TJs,
unc ion mainly in cell–cell adhesion, and a e composed by he
E-cadhe in-ca enins and by he nec in-A adin complexes (Takai
e al., 2008a;Zihni e al., 2016).
A adin (AFDN, AF6 o MLLT4) is an ac in-binding p o ein
ha associa es wi h nec ins a AJs, and ansien ly wi h ZO-1,
and ha egula e he o ma ion and s abiliza ion o he junc ional
complexes (Ikeda e al., 1999;Zhadano e al., 1999;Yokoyama
e al., 2001;Fukuha a e al., 2002;Lo ge and Moelling, 2006;
Takai e al., 2008b). A g owing body o e idence sugges s ha
A adin is in ol ed in ca cinogenesis. In addi ion o epo s o
loss o A adin exp ession in epi helial-de i ed b eas , colon, and
panc eas umo s (Le essie e al., 2007;Sun e al., 2014;Xu e al.,
2015), i s down egula ion led o inc eased cell in asion in i o
and o accele a ed umo g ow h in mice (Fou nie e al., 2011).
Fu he mo e, A adin was shown o be a nega i e egula o o
he epi helial- o-mesenchymal ansi ion (EMT) ma ke Snail in
panc ea ic cance (Xu e al., 2015).
Epi helial- o-mesenchymal ansi ion desc ibes he
di e en ia ion o epi helial cells in o mesenchymal cells,
and is an impo an p ocess du ing emb yogenesis, o gan
de elopmen , issue egene a ion, and cance p og ession
(Kallu i and Weinbe g, 2009). EMT is cha ac e ized by loss o he
AJC, whe e junc ional p o eins a e deg aded o delocalized, he
co ical ac in cy oskele on is eo ganized wi h he o ma ion o
lamellipodia and ilopodia, and he e is ep ession o cy oke a in
in e media e ilamen s and exp ession o imen in ilamen s
(Lamouille e al., 2014). Accompanying hese mo phological
changes, he e is ep og amming o gene exp ession h ough
ac i a ion o he mesenchymal pheno ype egula o s, such as
Snail, Slug, and zinc- inge E-box-binding homeobox 1 (ZEB1)
ansc ip ion ac o s, concomi an ly wi h down egula ion o
epi helial ma ke s (Lamouille e al., 2014).
Along wi h he changes in exp ession and localiza ion o
p o eins o he AJC, H. pylo i in ec ion is able o inc ease cell
in asi e p ope ies (Oli ei a e al., 2006;Cos a e al., 2016),
and o ac i a e se e al signaling pa hways ha induce an EMT
pheno ype in he in ec ed cells (Sai o e al., 2010;Yin e al., 2010;
Baud e al., 2013;Bessede e al., 2014;Lee e al., 2014;Yu e al.,
2014;W oblewski e al., 2015). The e o e, we aimed o de e mine
he e ec o H. pylo i in ec ion on A adin and i s impac in he
induc ion o an EMT pheno ype in gas ic cells.
MATERIALS AND METHODS
Cell Cul u e and T ans ec ions
The human gas ic cance cell lines MKN74 (a kind gi om
Ca la Oli ei a, Uni e si y o Po o) and NCI-N87 (ATCCR
CRL-
5822TM), we e cul u ed in RPMI 1640 (Biowes ), supplemen ed
wi h 10% e al bo ine se um (HyCloneTM, GE Heal hca e Li e
Sciences) and wi h 100 U-100 µg/mL penicillin-s ep omycin
sul a e (GibcoR
), a 37◦C, unde a 5% CO2humidi ied
a mosphe e.
Cell ans ec ions we e pe o med using he Lipo ec amineR

2000 ans ec ion eagen (In i ogenTM Li e Technologies),
acco ding o he manu ac u e ’s p o ocol. An siRNA silencing
A adin exp ession (sc-43007, San a C uz) and a nega i e con ol
siRNA (All S a s Nega i e Con ol siRNA; QIAGEN, Ge many),
we e used a a inal concen a ion o 75 nM in se um- and
an ibio ic- ee Op i-MEM medium (In i ogen). The e iciency
o ans ec ion was e alua ed by Wes e n blo .
Bac e ial S ains and G ow h Condi ions
H. pylo i s ain 26695 (ATCC 700392, cagPAI+, acA s1/m1)
was ob ained om ATCC (LGC S anda ds, Uni ed Kingdom),
and H. pylo i s ain 60190 (ATCC 49503 cagPAI+, acA
s1/m1) and i s espec i e 60190CagA−, 60190CagE−, and
60190VacA−mu an s we e a kind gi om P o esso John
A he on (Uni e si y o No ingham). S ains we e cul u ed
in T yp icaseTM Soy Aga wi h 5% Sheep Blood (TSAII;
Bec on, Dickinson and Company) a 37◦C unde mic oae ophilic
a mosphe e (GENbox mic oae ; bioMé ieux S.A.) o 48 h, as
p e iously desc ibed (Cos a e al., 2016). Fo he p oduc ion o
condi ioned media, bac e ia we e g own in F12 medium (Gibco)
supplemen ed wi h 1x choles e ol (Gibco) unde mic oae ophilic
condi ions a 37◦C wi h cons an o a ion (150 pm) o e
24 h. Subsequen ly, bac e ial suspensions we e cen i uged a
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Ma ques e al. H. pylo i, A adin and EMT
15,000 ×g, o 15 min, and he supe na an s we e il e ed h ough
a 0.2-µm s e ile il e , and concen a ed by ul a il a ion using
a 10 kDa po e size Amicon (EMD Millipo e). The esul ing
concen a ed bac e ial condi ioned media was used as media o
gas ic cell cul u es in compa ison wi h F12 medium plus 1x
choles e ol alone wi hou bac e ia, as con ol.
In ec ion o Gas ic Cells
Gas ic cell lines we e g own in an ibio ic- ee medium a 100%
con luence o 5 days in 6-well pla es (TPPR
Plas ic P oduc s
AG). Medium changes we e ca ied ou e e y day. Fo in ec ion
expe imen s, bac e ia g own o 48 h we e collec ed in phospha e
bu e saline (PBS, pH 7.4) and added o gas ic cell monolaye s,
a a mul iplici y o in ec ion (MOI) o 100 bac e ia pe cell. Co-
cul u es we e main ained o 16 h a 37◦C, unde a 5% CO2
humidi ied a mosphe e. Unin ec ed con ol cell cul u es we e
p ocessed simila ly, wi h he addi ion o PBS ins ead o bac e ia.
Fo s udies wi h bac e ial condi ioned media, F12 medium
supplemen ed wi h 1x choles e ol (Gibco) alone o a e 24 h o
bac e ial g ow h was used as cul u e medium.
Wes e n Blo and An ibodies
Cells we e collec ed and lysed in 1% T i on X-100, 1% NP-
40 in PBS, pH 7.4, con aining a cock ail o inhibi o s o
p o eases (Roche Applied Science, Mannheim, Ge many) and o
phospha ases (Sigma-Ald ich). A e supe na an eco e y, he
p o ein concen a ion was assessed using he Bio-Rad p o ein
assay ki (Bio-Rad), acco ding o he manu ac u e ’s p o ocol.
Iden ical amoun s o p o ein we e subjec ed o SDS-PAGE
and ans e ed on o ni ocellulose memb anes (Ame sham).
Memb anes we e blocked wi h 5% a - ee milk o 1 h, a oom
empe a u e, incuba ed o e nigh wi h p ima y an ibodies a
4◦C, and incuba ed wi h seconda y an ibodies o 1 h, a oom
empe a u e. An ibodies we e as ollows: an i-AF6 (610732)
and an i-β-Ca enin (610153) we e om BD T ansduc ion
Labo a o iesTM; an i-Vimen in (5741), an i-N-Cadhe in (13116),
an i-Snail (3879), an i-Slug (9585), an i-TCF8/ZEB1 (3396), and
an i-E-Cadhe in (3195) we e om Cell Signaling Technology;
an i-GAPDH (sc-47724) was om San a C uz Bio echnology;
an i-E-cad HECD1 (13-1700), an i-ZO-1 (61-7300), and an i-
Occludin (71-1500) we e om The mo Fishe Scien i ic; an i-
αTubulin (T9026) was om Sigma-Ald ich; and ho se adish
pe oxidase-conjuga ed seconda y an i- abbi (NA934) and an i-
mouse (NA931) we e om GE Heal hca e Li e Sciences.
T answell Ma igel In asion Assay
Ma igel-coa ed 24-well in asion inse s wi h 8 µm po es
(Co ningTM, BD Biosciences) we e used o he in i o in asion
assay. Upon hyd a ion wi h RPMI medium, cells we e seeded a
a densi y o 5 ×104cells in RPMI wi h 10% FBS, on op o he
Ma igel in he uppe side o he inse ; he bo om side was illed
wi h cul u e medium. Fo in ec ion expe imen s, H. pylo i was
added a a MOI o 100. A e 24 h incuba ion, non-in ading cells
we e emo ed wi h a co on swab, he emaining cells we e ixed
in me hanol o 10 min on ice, and he il e was moun ed wi h
Vec ashieldR
wi h DAPI (Vec o Labo a o ies). The whole il e
was coun ed, using a 20×magni ica ion.
Immuno luo escence
Cells we e g own on glass co e slips (Ma ien eld, Ge many),
ixed wi h 4% pa a o maldehyde o ice-cold me hanol. Upon
pe meabiliza ion and blocking wi h 5% goa se um, 0.3% T i on
X-100 in PBS, o 1 h, a oom empe a u e, p ima y an ibody
was added o 2 h a oom empe a u e. Co e slips we e
hen washed se e al imes wi h PBS pH 7.4, be o e incuba ion
wi h he espec i e Alexa Fluo -conjuga ed seconda y an ibodies
(In i ogen). Co e slips we e moun ed wi h Vec ashieldR
wi h
DAPI, and luo escence was moni o ed in a Zeiss Axio Image
Z1 Apo ome mic oscope.
T ansepi helial Elec ical Resis ance
(TER) and Fluo escein Iso hiocyana e
(FITC)-Dex an Pe meabili y Assay
Cells we e seeded a 100% con luence in 6.5 mm T answellR

wi h 3.0 µm Po e Polyes e Memb ane Inse (Co ning).
In eg i y o he epi helial monolaye was de e mined using a
Millicell ERS Vol ohmme e (Millipo e). P ocedu es o elec ode
equilib a ion and decon amina ion we e pe o med acco ding o
he manu ac u e ’s ins uc ions.
T ansepi helial elec ical esis ance (TER) was measu ed e e y
24 h, o six consecu i e days. Fo in ec ion expe imen s, H. pylo i
was added a MOI o 100, and TER was measu ed e e y 24 h,
o wo consecu i e days. To a oid empe a u e in luence, pla es
we e allowed o each oom empe a u e o 15 min, and medium
was changed a e each measu emen . TER alues we e calcula ed
as ohms/cm2. On he six h day pos A adin silencing, and he
second day pos in ec ion pa acellula pe meabili y was assessed
by measu ing he pe meabili y o he cell monolaye o FITC-
dex an (FD4, Sigma). A e measu ing he TER, he medium
was changed in he inne and bo om chambe , and allowed o
equilib a e o a leas 1 h. One mg/mL o FITC-dex an was
added o he inne chambe and incuba ed a 37◦C o 1 h.
A e incuba ion, luo escence was measu ed a an exci a ion o
485 nm and emission o 544 nm, on a Syne gy Mx mic opla e
eade . The amoun o di used dex an was de e mined using
calib a ion cu es es ablished wi h he s ock solu ion. Medium
wi hou FITC-dex an was used as blank.
Single Cell Mo ili y
Fo e alua ion o single cell mo ili y, 12-well pla es (TPPR

Plas ic P oduc s AG) we e coa ed wi h 5 µg/mL o Fib onec in
(Bioch om), o e nigh a 4◦C. Pla es we e washed wi h s e ile
PBS pH 7.4, and 1.5 ×103cells we e added o each well,
allowing hem o adhe e o 16 h. Adhe en cells we e labeled wi h
5µg/mL o Vyb an R
CM-Dil Cell-Labeling Solu ion (The mo
Fishe Scien i ic) o e 20 min, ollowed by h ee washes wi h
wa med RPMI wi hou phenol ed media. Ten di e en a eas
we e selec ed pe well, and li e images we e cap u ed e e y
15 min on a Leica DMI 6000 ime-lapse mic oscope (Leica
Mic osys ems, We zla , Ge many). The mig a o y pa hs and
eloci y o each cell we e analyzed wi h he Manual T acking
plugin unc ion a ailable a he Fiji so wa e package1.
1h p://imagej.ne /
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Ma ques e al. H. pylo i, A adin and EMT
AFDN Gene Exp ession in No mal
H. pylo i-In ec ed and Non-in ec ed
S omachs
RNA-seq eads sequenced in s omach samples om 181
indi iduals wi hou disease we e ob ained om he Geno ype-
Tissue Exp ession (GTEx) da abase (Conso ium, 2015). Non-
human unmapped eads we e aligned agains a da ase o
197 bac e ial whole genomes collec ed om NCBI, comp ising
species iden i ied in he gas oin es inal ac by he Human
Mic obiome P ojec and addi ional species appea ing in disease
condi ions (Tu nbaugh e al., 2007). Bac e ial quan i ica ion
and no maliza ion we e es ima ed using he QmihR pipeline
(Ca adas e al., 2017). A sample was conside ed as in ec ed
by H. pylo i when coun s o his species eached a log2cu o
alue o 5. mRNA exp ession da a o he AFDN gene, in eads
pe kilobase pe million (RPKM), was ex ac ed om GTEx
elease 6.
Pa ien Ma e ials and His opa hology
Fo y- wo o malin- ixed and pa a in-embedded gas ic issue
specimens we e e ospec i ely e ie ed om he Depa men o
Pa hology o Cen o Hospi ala S. João (CHSJ; Supplemen a y
Table 1), om pa ien s unde going ba ia ic su ge y ha had
specimens ob ained o his opa hologic examina ion (mean age
[±SD], 40.4 ±12.6 yea s; emale o male a io, 6:1). Samples
ha e been used e ospec i ely, and ha e been delinked and
uniden i ied om hei dono s. The s udy was app o ed by he
e hics commi ee o CHSJ.
Specimens we e s ained wi h hema oxylin/eosin and modi ied
Giemsa, he la e o H. pylo i de ec ion. His ological assessmen
e alua ed he ollowing pa ame e s: H. pylo i in ec ion, ch onic
in lamma ion, polymo phonuclea ac i i y, glandula a ophy,
and in es inal me aplasia. His ological slides we e examined by
an expe ienced pa hologis , blinded o he clinical in o ma ion
o he pa ien s.
Immunohis ochemis y
Immunohis ochemis y was pe o med in a LabVision
Au os aine 480S (The mo Scien i ic). B ie ly, slides we e
depa a inized in xylene and hyd a ed h ough passages
in 100%, 95%, 70% e hanol, and insed in wa e . An igen
unmasking was pe o med o 35 min using 1x an igen
e ie al solu ion om he Enzo Poly iewR
Plus HRP-DAB
(an i-Rabbi ) ki . Slides we e cooled o 20 min and washed
wi h TBS-Tween-20, 0.01%. Endogenous pe oxidase ac i i y
was blocked o 10 min, and slides we e washed and placed
in he Au os aine . A e blocking o endogenous pe oxidase
ac i i y, slides we e incuba ed wi h An ibody blocke /diluen
(p o ided in he ki ) o 10 min o p e en unspeci ic binding,
ollowed by 1 h incuba ion wi h p ima y polyclonal an ibody
An i-AFDN (HPA030213, Sigma), 1:1250 dilu ed in an ibody
diluen (The mo Scien i ic, Lab ision). To p e en non-
speci ic binding, slides we e again incuba ed wi h an ibody
blocke /diluen o 5 min. A e wash, he Poly iew Plus
HRP (an i-Rabbi ) was added and incuba ed o 1 h. The
eac ion was e ealed wi h 3,30-diaminobenzidine (DAB) 5 min.
Coun e s ain was pe o med wi h HIGHDEFR
hema oxylin
(ENZO).
RNA Ex ac ion and Quan i ica ion o
A adin Exp ession by Quan i a i e
Real-Time PCR
To al RNA was ex ac ed using mi VanaTM miRNA Isola ion Ki
(The mo Fishe Scien i ic) ollowing manu ac u e ’s ins uc ions
om unin ec ed and 24 h-in ec ed MKN74 cells. RNA was
e e sed- ansc ibed using M-MuLV Re e se T ansc ip ase
and andom-hexame s (NZYTech). Quan i ica ion o A adin
exp ession was pe o med by quan i a i e eal- ime PCR (RT-
qPCR) using he TaqMan Gene Exp ession Assays (Applied
Biosys ems), Hs00984486_m1 o A adin and Hs99999905_m1
o GAPDH, as endogenous con ol gene, on an ABI P ism
7000 Sequence De ec ion Sys em (Applied Biosys ems), in
ou independen expe imen s. Da a we e analyzed by he
compa a i e 2(−11CT)me hod (Li ak and Schmi gen, 2001)
and Wilcoxon signed- ank es applied.
S a is ical Analysis
The unpai ed S uden ’s - es was used o compa isons be ween
wo independen g oups, and he one-way ANOVA wi h pos hoc
Tukey’s es was used o compa isons be ween h ee independen
g oups. K uskal–Wallis es was applied o GTEx da a ha
compa ed AFDN mRNA exp ession be ween in ec ed and non-
in ec ed samples. Wilcoxon signed- ank es was applied o
quan i a i e eal- ime PCR analysis. The ela ionship be ween he
p esence o H. pylo i and al e a ions o he immunohis ochemical
pa e n o A adin was assessed using he Fishe ’s exac es .
Da a analysis was pe o med using G aphPad P ism Ve sion
6.0. S a is ically signi icance was se a P≤0.05 (∗∗∗∗p≤0.0001,
∗∗∗p≤0.001, ∗∗p≤0.01, ∗p≤0.05). Da a in g aphs ep esen
mean ±s anda d e o o he mean (SEM) o a leas h ee
expe imen s.
RESULTS
H. pylo i In ec ion Dec eases A adin
Exp ession
Al e a ions o se e al TJ and AJ p o eins induced by H. pylo i
in ec ion ha e been epo ed (Amie a e al., 2003;Hoy e al.,
2010), bu he in luence o H. pylo i on A adin is so a unknown.
To in es iga e he e ec o H. pylo i on A adin, MKN74
and NCI-N87 cell lines ha es ablish compe en junc ional
complexes, we e in ec ed wi h s ain 26695 o 24 h.
Immuno luo escence analysis showed A adin p esen bo h
a he memb ane and in he nucleus in unin ec ed condi ions.
Upon in ec ion wi h H. pylo i he e was a signi ican dec ease
in A adin s aining in bo h cell lines, and a bo h memb ane
and nucleus subcellula localiza ions, as de e mined by imaging
quan i ica ion (Figu e 1A). Wi hin he nucleus, a signi ican
dec ease in he numbe o A adin nuclea do s was also
obse ed in in ec ed cells compa ed o unin ec ed gas ic cells
(Supplemen a y Figu e 1A).
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Ma ques e al. H. pylo i, A adin and EMT
FIGURE 1 | Impac o H. pylo i in ec ion on A adin exp ession. MKN74 and NCI-N87 gas ic cell lines we e le un ea ed o in ec ed wi h H. pylo i 26695 (A,B) o
in ec ed wi h H. pylo i 60190, o wi h he espec i e CagA-, CagE-and VacA-mu an s (C), o 24 h a a MOI o 100. A adin de ec ion by immuno luo escence ( ed)
and quan i ica ion o he luo escence in ensi y a he memb ane and he nucleus (A), and by Wes e n blo and i s quan i ica ion, using GAPDH o ubulin as loading
con ols (B,C). Scale ba , 10 µm. Da a co espond o he mean alue ±SEM and a e ep esen a i e o a leas h ee independen expe imen s. S a is ical
signi icance was e alua ed wi h he S uden ’s - es o wi h one-way ANOVA.
A adin has wo majo iso o ms, he l-A adin iso o m ha
con ains an F-ac in-binding domain, and he less exp essed
s-A adin iso o m ha lacks he F-ac in-binding domain (Buche
e al., 2007). Wes e n blo analysis wi h an an ibody ha
ecognizes he wo A adin iso o ms e ealed he p esence
in bo h cell lines o a highly exp essed 220 kDa p o ein,
co esponding o l-A adin, and ain bands abo e he 150 kDa
ma ke , co esponding o s-A adin. Silencing o A adin by
siRNA con i med he loss o he wo iso o ms (Supplemen a y
Figu e 1B). Upon in ec ion wi h H. pylo i 26695, he e was a
signi ican dec ease o he p o ein le els o A adin (Figu e 1B),
wi hou changes in he mRNA exp ession le els (Supplemen a y
Figu e 1C).
To e alua e he impac o he majo bac e ia i ulence ac o s
on H. pylo i-media ed A adin down egula ion, cells we e in ec ed
wi h H. pylo i s ain 60190 and i s espec i e CagA−, CagE−
( equi ed o a unc ional T4SS), and VacA−mu an s. As
p e iously obse ed in in ec ions wi h s ain 26695, H. pylo i
60190 was also able o signi ican ly educe A adin p o ein le els
in he wo cell lines (Figu e 1C). In ec ions wi h all o he
H. pylo i mu an s ains also led o signi ican dec eases in A adin
exp ession meaning ha loss o A adin is independen o CagA,
VacA, and any ac o deli e ed ia he T4SS (Figu e 1C). To
de e mine i sec e ed soluble bac e ial ac o s o iable bac e ia
a e in ol ed in loss o A adin, ei he condi ioned medium o
H. pylo i liquid cul u es o hea -killed H. pylo i we e incuba ed
wi h epi helial cells. The esul s e ealed ha loss o A adin
p o ein exp ession was dependen on he p esence o iable
bac e ia bu ha was no igge ed by soluble bac e ial ac o s
sec e ed by H. pylo i (Supplemen a y Figu e 1D).
H. pylo i Induces an EMT Pheno ype in
Gas ic Cells
Epi helial- o-mesenchymal ansi ion is cha ac e ized by he
ansi ion om an epi helial o a mesenchymal pheno ype, wi h
he modi ica ion o he adhesion molecules exp essed by he
cell, allowing i o adop a mig a o y and in asi e beha io
(Nie o e al., 2016). I has been shown ha H. pylo i is
able o modi y se e al cell–cell adhesion molecules (Fedwick
e al., 2005;Fio en ino e al., 2013;Schmid e al., 2016;
Teg meye e al., 2017), o induce an elonga ion in he cell
shape (Moese e al., 2004;Bou zac e al., 2007), o inc ease cell
in asion (Oli ei a e al., 2006;Cos a e al., 2016), and o up-
egula e EMT gene exp ession (Yin e al., 2010;Baud e al.,
2013). H. pylo i was also shown o al e Snail localiza ion in
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Ma ques e al. H. pylo i, A adin and EMT
bo h MKN28 cells, which o m unc ional TJs, and in human
gas ic specimens (W oblewski e al., 2015). S ill, he g ea
majo i y o hese esul s we e ob ained using gas ic cance
cell lines ha ha e de icien AJs due o mu a ions in he
E-cadhe in-encoding gene, as is he case o AGS cells. The e o e,
we wan ed o know i H. pylo i could ecapi ula e some o
hese p ope ies in ou gas ic cell line models ha es ablish
p o icien AJs.
E alua ion o he e ec s o H. pylo i on cell monolaye s,
showed ha a e 24 h o in ec ion, he e was a dec ease in
he exp ession and/o memb ane displacemen o E-cadhe in,
occludin, and ZO-1 (Figu e 2A). Concu en ly, H. pylo i
in ec ion signi ican ly educed E-cadhe in, β-ca enin, occludin,
and ZO-1 p o ein le els, bo h in MKN74 and NCI-N87 cell
lines (Figu e 2B). Fu he mo e, H. pylo i in ec ion signi ican ly
inc eased he p o ein le els o ZEB1, Vimen in, Slug, N-cadhe in,
and Snail in bo h cell lines (Figu e 2C).
These esul s con i m ha in addi ion o A adin, H. pylo i
in ec ion is able o al e junc ional complexes, dampening
epi helial cell p ope ies. These esul s u he show ha
H. pylo i-media ed inc ease in he exp ession o EMT-associa ed
ma ke s also occu s in cell lines ha es ablish AJs.
FIGURE 2 | H. pylo i induces an EMT pheno ype in epi helial gas ic cells. MKN74 and NCI-N87 gas ic cell lines we e le un ea ed o in ec ed wi h H. pylo i 26695
o a pe iod o 24 h a a MOI o 100. (A) Immuno luo escence o E-cadhe in, occludin, and ZO-1. Scale ba , 10 µm. (B) Wes e n blo and espec i e quan i ica ions
o he le els o apical junc ional complex p o eins E-cadhe in, β-ca enin, ZO-1, and occludin. Tubulin was used as loading con ol. (C) Wes e n blo and espec i e
quan i ica ions o he le els o mesenchymal ma ke s Snail, N-cadhe in, ZEB1, Slug, and Vimen in. GAPDH was used as loading con ol. Da a co espond o he
mean alue ±SEM and a e ep esen a i e o h ee independen expe imen s. S a is ical signi icance was e alua ed wi h he S uden ’s - es .
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Ma ques e al. H. pylo i, A adin and EMT
A adin Down egula ion Delocalizes AJ
and TJ P o eins, P omo es he Fo ma ion
o Ac in S ess Fibe s, and Up egula es
he EMT Ma ke Snail
To mimic he e ec o H. pylo i in ec ion on A adin
down egula ion and o e alua e he e ec o he loss o A adin in
gas ic cells, he nex expe imen s we e pe o med wi h cells ha
we e ansien ly ans ec ed wi h a siRNA o A adin o wi h a
non-silencing siRNA con ol. E icien knockdown was achie ed
in MKN74 (87%) and in NCI-N87 (54%) cells.
Since A adin is an AJC p o ein, we i s e alua ed he e ec
o A adin down egula ion on he localiza ion o AJs and TJs
p o eins in MKN74 cells. In cells ans ec ed wi h he non-
silencing siRNA, he AJs p o eins E-cadhe in and β-ca enin,
and he TJs p o eins occludin and ZO-1 p e e en ially localized
a he cell memb ane. In con as , in cells whe e A adin was
silenced, he e was an al e a ion o he memb ane localiza ion o
E-cadhe in, β-ca enin, occludin, and ZO-1 (Figu e 3A), wi hou
changes in he espec i e p o ein exp ession le els (Figu e 4A).
Al e a ions in he cellula localiza ion o hese p o eins in cells
whe e A adin was silenced we e accompanied by a s iking
change in he epi helial cell mo phology, wi h loss o he
ac in bel , o ma ion o ac in s ess ibe s, and o ma ion o
lamellipodia and ilopodia (Figu e 3A). In e es ingly, a si es
whe e A adin was no e icien ly silenced by he siRNA, cells
e ained he polyhed al epi helial mo phology wi h igh cell-
o-cell con ac s, con as ing wi h si es whe e A adin exp ession
was ully abolished by siRNA, and in which cell–cell adhesion
was los and he AJC p o eins we e disloca ed o he cy osol
(Figu e 3A). Quan i ica ion o A adin luo escence in ensi y a
he memb ane and he nucleus in bo h A adin silenced and non-
silenced cells is depic ed in Figu e 3B. A signi ican dec ease in
A adin exp ession was obse ed in bo h localiza ions in A adin
silenced cells, ein o cing he speci ici y o he A adin siRNA and
an ibody.
FIGURE 3 | A adin down egula ion displaces apical junc ional complex p o eins and p omo es he o ma ion o ac in s ess ibe s. Double immuno luo escence o
A adin ( ed) wi h AJs p o eins E-cadhe in and β-ca enin (g een), and wi h TJs p o eins ZO-1 and occludin (g een) in MKN74 cells ans ec ed wi h a non-silencing
siRNA (siNS) o wi h a siRNA o A adin (siAFDN). Immuno luo escence o ac in (g een) and o Snail ( ed) a e also shown. Nuclei we e coun e s ained wi h DAPI. Scale
ba , 10 µm. Whi e a ows ep esen cells wi h A adin no e icien ly silenced by he siRNA and ha e ain he epi helial mo phology; Yellow a ows, lamellipodia; cyan
a ows, ilopodia (A). Quan i ica ion o A adin luo escence in ensi y in MKN74 cells in bo h memb ane and he nucleus upon ea men wi h non-silencing siRNA o
wi h a siRNA o A adin (B).
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Ma ques e al. H. pylo i, A adin and EMT
FIGURE 4 | A adin down egula ion does no al e he exp ession o apical junc ional complex p o eins, bu up egula es he EMT ma ke Snail. Wes e n blo analyses
o MKN74 and NCI-N87 cells, cells ans ec ed wi h a non-silencing siRNA (siNS), o wi h a siRNA o A adin (siAFDN), e alua ing (A) apical junc ional complex
p o eins E-cadhe in, β-ca enin, ZO-1, and occludin, and (B) mesenchymal ma ke s Snail, N-cadhe in, ZEB1, Slug, and Vimen in. GAPDH was used as loading
con ol. Numbe s below he bands co espond o quan i ica ions. Da a a e ep esen a i e o h ee independen expe imen s. S a is ical signi icance was e alua ed
wi h he S uden ’s - es .
Ha ing shown ha A adin down egula ion al e s he s uc u e
o gas ic cell–cell junc ions and cell mo phology, and knowing
ha A adin nega i ely egula es he ansc ip ion o Snail o
ac i a e EMT in panc ea ic cance cells (Xu e al., 2015), we
sough o in es iga e he in ol emen o A adin in he EMT
pheno ype in he gas ic con ex . In bo h MKN74 and NCI-N87
cell lines, silencing o A adin signi ican ly inc eased he le els
o he EMT-associa ed ansc ip ion ac o Snail (Figu es 3A,
4B), bu no o he EMT ma ke s Slug, ZEB1, Vimen in, and
N-Cadhe in (Figu e 4B). O e all, hese esul s a e sugges i e ha
A adin down egula ion is implica ed in he eme gence o he
EMT pheno ype in he gas ic se ing.
A adin Down egula ion Al e s he TJ
Ba ie Func ion and Inc eases Cell
Mo ili y and In asion
Ha ing shown ha A adin silencing esul ed in delocaliza ion o
TJ and AJ p o eins om he memb ane o he cy oplasm, we nex
add essed he unc ional consequences o A adin down egula ion
in e ms o TJ in eg i y, cell mo ili y/mig a ion, and in asion.
To e alua e TJ in eg i y, cells we e seeded in answells
and allowed o each 100% con luence, a e which TER was
measu ed o e six consecu i e days, allowing cells o pola ize.
Cells whe e A adin was down egula ed had signi ican ly lowe
elec ical esis ance in compa ison o cells ans ec ed wi h he
non-silencing siRNA o o un ea ed cells, an e ec ha s a ed
o be obse ed om day 3 (Figu e 5A). Pe meabili y assays
we e pe o med on day 6, by measu ing he pe meabili y o he
gas ic monolaye s o he high molecula weigh 4 kDa dex an.
Monolaye s es ablished wi h cells whe e A adin exp ession was
silenced we e signi ican ly mo e pe meable han con ol cell
monolaye s (Figu e 5B). Simila esul s ega ding TER and
pe meabili y o FITC-4 kDa Dex an we e ound o MKN74
(Figu es 5A,B) and NCI-N87 gas ic cells (Supplemen a y
Figu es 2A,B) upon in ec ion wi h H. pylo i s ain 26695.
To analyze whe he he e was a di ec link be ween loss o
A adin and cell mo ili y/mig a ion, cells we e seeded a low
densi y, s ained wi h he lipophilic dye CM-Dil, and acked
o 24 h wi h ime-lapse mic oscopy. Analysis o he ime-lapse
mic oscopy ideos e ealed ha , e en when small g oups o cells
we e seeded, cells wi h down egula ion o A adin we e unable
o main ain cell–cell adhesion, as opposed o un ea ed cells,
o cells ans ec ed wi h he non-silencing siRNA (Figu e 5C).
Coo dina es o each indi idual cell pa h o e he 24 h pe iod we e
ep esen ed in wo-dimensional g aphs (Figu e 5D). Silencing o
A adin signi ican ly inc eased he mig a ion eloci y (Figu e 5E)
and he dis ance (Figu e 5F) co e ed by each indi idual cell,
compa ed o non-silenced o o un ea ed con ol cells. No
signi ican di e ences on di ec ionali y we e obse ed, wi h each
cell displaying a andom mig a ion pa e n.
We nex add essed he in ol emen o A adin in gas ic
cell in asion in Ma igel in asion assays. Silencing o A adin
esul ed in a wo old inc ease in he cell in asi e capaci y
compa ed o con ol cells ans ec ed wi h he non-silencing
siRNA (Figu e 5G). Inc eased cell in asion was also obse ed
upon in ec ion wi h H. pylo i s ain 26695 in bo h MKN74 cells
(Figu e 5H) and NCI-N87 cells (Supplemen a y Figu e 2C).
O e all, hese esul s sugges ha A adin plays a ole in gas ic
epi helial in eg i y by main aining he s uc u e and unc ions o
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Ma ques e al. H. pylo i, A adin and EMT
FIGURE 5 | A adin down egula ion al e s he TJ ba ie unc ion and inc eases cell mo ili y and in asion. (A) T ansepi helial elec ical esis ance (TER) measu emen s
o MKN74 gas ic cells du ing a pe iod o 6 days pos A adin silencing (siAFDN) in compa ison wi h non-silenced (siNS) and wi h un ea ed cells, and du ing 2 days
a e H. pylo i in ec ion in compa ison wi h unin ec ed cells. (B) Cell monolaye pe meabili y o 4 kDa FITC-Dex an on day 6 pos A adin silencing and on day 2 a e
H. pylo i in ec ion. (C–F) Single cell mo ili y analysis: (C) Time-lapse mic oscopy pho os o cells du ing he 24 h pe iod. (D) Rep esen a i e g aphs o he X and Y
coo dina es (in pixels, px) o each cell pa h, on a ib onec in coa ed su ace. (E) Cell eloci y in µm/minu e and (F) o al dis ance co e ed by and indi idual cell in µm.
(G,H) Cell in asion capaci y on Ma igel-coa ed answells o MKN74 cells ans ec ed wi h siNS con ol o ans ec ed wi h siAFDN (G), and o MKN74 cells
in ec ed o no wi h H. pylo i 26695 (H). Da a a e p esen ed as mean ±SEM. S a is ical signi icance was e alua ed wi h he one- and wo-way ANOVA and wi h
S uden ’s - es .
he TJs and AJs, namely p ese ing he epi helial ba ie and cell–
cell adhesion, and supp essing cell mo ili y and in asion.
H. pylo i In ec ion A ec s he Exp ession
and Localiza ion o A adin in he Human
Gas ic Mucosa
To de e mine he impac o H. pylo i in ec ion on A adin
in i o, we i s e alua ed AFDN gene exp ession using he
da a a ailable a he GTEx da abase. Da a was e ie ed
om gas ic samples o 181 indi iduals wi hou disease,
and 67 indi iduals (37%) we e iden i ied o be in ec ed
wi h H. pylo i. No s a is ically signi ican di e ences
(p= 0.30) we e obse ed be ween AFDN gene exp ession
le els in unin ec ed and H. pylo i-in ec ed pa ien s
(Figu e 6A).
The exp ession o A adin was also e alua ed by
immunohis ochemis y in a se ies o 42 issue specimens
ha ha e been cha ac e ized ega ding he his opa hological
ea u es o he gas ic mucosa and he p esence o
F on ie s in Mic obiology | www. on ie sin.o g 9No embe 2018 | Volume 9 | A icle 2712