Research Article Differential Expression of MUC12, MUC16...

Research ArticleDifferential Expression of MUC12 MUC16 and MUC20 inPatients with Active and Remission Ulcerative Colitis

Jesuacutes K Yamamoto-Furusho1 Ilse Ascantildeo-Gutieacuterrez1

Janette Furuzawa-Carballeda2 and Gabriela Fonseca-Camarillo1

1 Inflammatory Bowel Disease Clinic Department of Gastroenterology Instituto Nacional deCiencias Medicas y Nutricion Salvador Zubiran 14080 Mexico City Mexico2Department of Immunology and Rheumatology Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran14080 Mexico City Mexico

Correspondence should be addressed to Jesus K Yamamoto-Furusho kazuofurushohotmailcom

Received 24 September 2015 Accepted 22 November 2015

Academic Editor Julio Galvez

Copyright copy 2015 Jesus K Yamamoto-Furusho et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Background Patients with UC have shown an important defect in the secretion and maintenance of the mucosal barrier as partof inadequate expression of mucin genes The aim of the present study was to determine the expression of MUC12 MUC16and MUC20 in colonic tissue from patients with UC in regard to their clinical outcomes Methods We included a total of 40patients with UC and 30 normal controls Mucin gene expression was performed by RT-PCR and protein expression was detectedby immunohistochemistry Results Patients with active UC showed no significant expression of MUC12 gene in mucosa comparedto the group of patients with UC in remission and the normal control group MUC16 gene expression was significantly increasedin the UC active and remission groups compared to the normal control group (119875 = 003) MUC20 gene expression was foundsignificantly decreased in patients with active UC compared to both remission group (119875 = 0001) and normal controls (119875 = 0001)Furthermore an association was found between MUC20 gene expression and the presence of histological remission in patientswith UC (119875 = 0003 OR = 037) Conclusions An increased gene expression of MUC16 and MUC20 was found in patients withremission UC

1 Introduction

Inflammatory bowel disease (IBD) consists of ulcerativecolitis (UC) and Crohnrsquos Disease (CD) progressive andinflammatory disorders of the intestinal tract Ulcerativecolitis (UC) is limited to the colon and involves diffusemucosal inflammation and its etiology remains unknown [1]The UC pathogenesis involves an inappropriate immunolog-ical response (antigen presentation and tolerance) and animbalance of mucin expression Mucins (MUC) are high-molecular-weight glycoproteins and constitute the first lineof innate immunity So far there are at least 21 humanMUC known [2] Mucins are produced by multiple cellsincluding goblet cells epithelial cells enterocytes and restingor activated mononuclear cells in the intestinal tract Mucinfunctional diversity is related to its structureThere are 2 sub-types secretory and membrane-bound which constitute the

main component of the mucous layer lining the GI tract Thesecretory mucins forming the mucus barrier include MUC2MUC5ACMUC5BMUC6 andMUC19 and constitute largepolymers rich of proteoglycans that are highly hygroscopicand hydrophilic Membrane-bound mucins (MUC1 MUC3MUC4 MUC12 MUC13 MUC15 MUC16 MUC17 andMUC20) are anchored to the apical membrane of epithelialcells and form the glycocalyx Nonetheless they are poorlycharacterized [3]

Secretorymembrane-boundmucins in the normal gut areinvolved in cell signaling because they interact with mem-brane receptors and have several functions such as adhesiongrowth and immune modulation [4] Their expression andsecretion are constitutive andor regulated by pathogens IL-1120573 TNF-120572 andTh2 cytokines [5]

Altering of mucus barrier functions composition andlubrication depriving the epithelial gut and favoring the

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 659018 8 pageshttpdxdoiorg1011552015659018

2 Mediators of Inflammation

contact with toxins cytokines and commensal or pathogenicmicroorganisms through adhesins which induces acute andchronic inflammation in the intestine Patients with UC haveshown an aberrantMUC synthesis and degradation as well asalterations ofmucinrsquos glycoprotein structure (o-glycosylationsulfation and silylation) leading to a deficientmucous barrier[1 2] Some groups have described a reduction in MUC1MUC2 and MUC3 and MUC17 gene expression in bothinflamed and nonaffectedmucosae fromUC andCDpatients[2 6ndash8] MUC2 is the predominant sulfated componentof mucin layer in healthy colon In active UC MUC2production and secretion by goblet cells are reduced and thisinduces synthesis of MUC5AC and MUC6 [6] On the otherhand it has been reported that MUC1 is overexpressed insevere UCThis finding correlates with an elegant experimentdemonstrating that MUC1minusminus mice model is less susceptibleto dextran sulfate sodium induction of colitis and that feweractivated T cells were recruited to the inflamed mucosa [5]MUC17 in healthy intestine is one of the major membrane-bound mucins However its expression is reduced in UCpatients [6] MUC13 was increased in active UC but MUC3did not changeTheMUC4was similarly increased especiallywhen associated with the development of neoplasia Anaberrant expression of MUC5AC was found in UC as well asin CD [9 10] Previous studies from Moehle et al describedsignificant downregulation of MUC12 and MUC20 in colonand ileum in patients with Crohnrsquos Disease [6]

MUC12 is a protein-coding gene whose function isinvolved in the epithelial cell protection [9] The function ofMUC16 is thought to provide a protective lubricating barrieragainst particles and infectious agents at mucosal surfaces[10]

MUC20 is a protein-coding gene that contains at leastfour exons and is located close to MUC4 on chromosome3q29 It has been reported that MUC20 is oversynthesizedin gastric ovarian endometrial and colorectal cancer andpredicts recurrence and poor outcome [11]

Little is known about the MUC12 MUC16 and MUC20expression and their potential role in the physiopathology ofUC Thus the aim of the present study was to characterizethe changes in gene and protein expression of thesemucins incolonic tissue frompatients withUC in regard to their clinicaloutcomes

2 Materials and Methods

21 Study Subjects We studied 20 patients with active UC20 patients in remission UC and 30 normal controls withoutinflammation All individuals belonged to the InflammatoryBowel Disease Clinic at the Instituto Nacional de CienciasMedicas y Nutricion Hospital The diagnosis of UC wasdone by the presence of the following criteria macroscopicappearance by endoscopy and biopsy compatible with UCand history of diarrhea or blood in stool Demographic andclinical variables were collected from personal interview andclinical records The clinical and endoscopic activity wasevaluated by Mayo [12] and histological activity by Rileyscore

The tissue control group consisted of noninflamed con-trols (no documented inflammatory disease) undergoingcolonoscopy for evaluation of anemia and screening ofpolyps All individuals were included as controls if nopathological findings were found

22 Sample Processing and Gene Expression Analysis Thecolonic biopsies for gene expression analysis were taken withcolonoscopy and were immediately placed in preserver ofRNA (Ambion Austin TX USA) and stored at minus70∘C untilprocessing Then total RNA was isolated using High PureRNA Tissue (Roche Diagnostics Mannheim Germany)Colonic biopsies samples were disrupted in 400 120583L LysisBuffer and homogenizedTheRNAwas isolated by binding tothe glass fibers prepacked in theHigh Pure Filter Tube Resid-ual contaminating DNA was digested with DNase I BoundRNA was washed thereby purified from salts proteins andother cellular impurities Finally we added 100120583L of ElutionBuffer for the elution of RNA

Two hundred nanograms of total RNA was reverse-transcribed into cDNA with random hexamer primers(Roche Diagnostics Mannheim Germany)

The MUC gene relative expression was measured byquantitative real-time polymerase chain reaction (RT-PCR)Reference genes RPLP0 ACTB and GAPDH transcriptswere used for relative quantification and quality controlsFor qPCR assays quality control determination of linearityand reproducibility was evaluated (VC lt 10) The mRNArelative quantification of target genes was conducted usingthe LightCycler software 41 according to the 2-delta-delta Ctmethod

We used the following primers MUC12 forward cctg-gaaaccttagcaccag and reverse gacagacgcattgttttccat MUC16forward tggggaccaccaattctatg and reverse atggctgggagtg-gattg MUC20 forward tatgtgccgtggaggattc and reversettgctgtacgtgtctaacttcaatc IL-6 forward tctgctcccacaatgaaacatand reverse gaaggcagcaggcaacac IL-8 forward agacagca-gagcacacaagc and reverse atggttccttccggtggt GADPH for-ward gcccaatacgaccaaatcc and reverse agccacatcgctcagacaRPLP0 forward gaagctctatctcgcctcca and reverse agcaggcaa-caccaggag and ACTB forward caaccgcgagaagatgac andreverse gtccatcacgatgccagt for normalization as also shownin Table 1

The PCR amplification of mucin genes was carried outwith 20 ng of cDNA 200 nM forward and reverse primerand Taqman Master Mix (Roche Diagnostics MannheimGermany) in a final volume of 10 120583L PCR reactions wererun in a Light Cycler 480 (Roche Diagnostics MannheimGermany) for 45 cycles each cycle consists in denaturationfor 15 seconds at 95∘ primer annealing for 15 seconds at55∘ and extension for 30 seconds at 72∘C and cooling for 30seconds at 40∘C

23 Immunohistochemistry All tissues obtained were forma-lin fixed and paraffin embedded Five 120583m tissue sectionswere rehydrated using xylene and graded ethanol series Afterdeparaffinizing and demasking of antigenswith 10mMcitratebuffer pH 60 the slides were then soaked in 1 hydrogenperoxide in methanol for 20min at room temperature to

Mediators of Inflammation 3

Table 1 Primers designs from Universal ProbeLibrary

Gene GenBank Oligo left Oligo right UPL Amplicon size

MUC12 NM 0011644621 cctggaaaccttagcaccag gacagacgcattgttttccat 72cctggaaaccttagcaccagggttgtgccagga-aggacaaatttggaatggaaaacaatgcgtctg-

tc

MUC16 NM 0246902 agtggaccttgggacctca gagagggccagcagatgtag 58 agtggaccttgggacctcagggactccatcctc-cctccccagccctacatctgctggccctctc

MUC20 NM 0010985161 tatgtgccgtggaggattc ttgctgtacgtgtctaacttcaatc 7 tatgtgccgtggaggattcaaatctgtctcttc-tcccagggattgaagttagacacgtacagcaa

IL-6 NM 0006003 gatgagtacaaaagtcctgatcca ctgcagccactggttctgt 68

gatgagtacaaaagtcctgatccagttcctgca-gaaaaaggcaaagaatctagatgcaataaccac-ccctgacccaaccacaaatgccagcctgctgac-gaagctgcaggcacagaaccagtggctgcag

IL-8 NM 0005842 agacagcagagcacacaagc atggttccttccggtggt 72 agacagcagagcacacaagcttctaggaca-agagccaggaagaaaccaccggaaggaaccat

GAPDH NM 0020463 agccacatcgctcagaca gcccaatacgaccaaatcc 60agccacatcgctcagacaccatggggaagg-tgaaggtcggagtcaacggatttggtcgtattg-

ggc

RPLP0 NM 0532753NM 0010023 agcaggcaacaccaggag gaagctctatctcgcctcca 6

tctacaaccctgaagtgcttgatatcacagagg-aaactctgcattctcgcttcctggagggtgtcc-gcaatgttgccagtgtctgtctgcagattg

ACTB NM 0011013 gtccatcacgatgccagt caaccgcgagaagatgac 64ccaaccgcgagaagatgacccagatcatgtttg-agaccttcaacaccccagccatgtacgttgcta-tccaggctgtgctatccctgtacgcctctgg

block endogenous peroxidases and then washed with PBSSlides were blocked with 10 normal donkey serum andwere incubated with a primary mouse monoclonal anti-humanMUC12mAb (Santa Cruz Biotechnology Santa CruzCA) MUC16 mAb (ABCAM) andMUC20mAb (ABCAM)overnight at 4∘C and then samples were incubated withthe secondary goat anti-mouse IgG (Santa Cruz Biotechnol-ogy) diluted 1 2000 for 1 hour at room temperature Theslides were treated consecutively with streptavidin-horse-radish peroxidase conjugate diluted 1 1000 for 45 minutesat room temperature Next tissues were incubated using6mg 331015840-diaminobenzidine (DAB) substrate and H

2

O2

thecolor reaction was allowed to develop for 5ndash10min Afterthe staining reaction the slides were washed thoroughlyin tap water counterstained with Mayerrsquos hematoxylin for2 minutes and saturated lithium carbonate solution for 10seconds and mounted with cover slides Substitution ofprimary antibodies with PBS was used as the reactive blankNegative control staining was performedwith normal humanserum diluted 1 100 instead of primary antibody Both con-trols excluded nonspecific staining or endogenous enzymaticactivities Scoring of immune-stained sections was done byconventional light microscopy in a blinded manner MUC12- MUC16- and MUC20-producing cells were counted inat least three optical fields from each slide in times320 high-power magnificationsThe average values per slide were usedfor statistical analysis Results are expressed as the mean plusmnstandard error of the mean (SEM) of cells quantified by theprogram Image Pro-Plus version 511

24 Ethical Considerations The protocol was approved bythe ethical medical committee in our institution and it wasaccording to the principles expressed in the Declaration of

Helsinki 1989 Only patients who gave a written informedconsent were recruited for this study

25 Statistical Analysis Descriptive statistics were per-formed and categorical variables were compared using the1205942 test or Fisherrsquos exact test ANOVA on ranks by Holm-

SidakMethod and Dunnrsquos test was performed for all pairwisemultiple comparison procedures Statistical analysiswas doneusing the Sigma Stat 112 program (Aspire Software Inter-national Leesburg VA USA) Data were expressed as themedian range andmeanplusmn standard deviation (SD)standarderror of the mean (SEM) 119875 le 005 was considered assignificant

3 Results

31 Demographic and Clinical Characteristics A total of 40patients with UC (19 men and 21 women with a mean ageof 42 years) and 30 normal controls (16 men and 14 womenwith a mean age of 53 years) were evaluated Regarding thegrade of disease activity 20 had active disease and 20 were inremission according to Mayo scoreThe extent of disease wasevaluated by using total colonoscopy and biopsies were takenfrom different segments of colon in all cases The Montrealclassification was used to define the extent of UC 45 hadpancolitis (E3) 20 had left-sided colitis (E2) and 35 hadproctitis (E1)

Histological analysis of patients with active UC showed20 with mild activity 40 with moderate activity and 40with severe histological inflammationOn the other hand thehistological analysis of UC patients in remission shows anassociation with the gene expression of MUC16 (119875 = 00003


MU

C12

mRN

AG

AD

PH m

RNA

5000

10000

15000

20000

25000

30000

0

ControlrUCaUC

(a)M

UC1

6 m

RNA

GA

DPH

mRN

A

1000

2000

3000

4000

0

0035

ControlrUCaUC

(b)

MU

C20

mRN

AG

AD

PH m

RNA

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0001

0001

ControlrUCaUC

(c)

Figure 1 (a) MUC12 (b) MUC16 and (c) MUC20 mRNA levels in rectal mucosa from patients with ulcerative colitis (119873 = 40) and controls(119873 = 30) RT-qPCR was performed to assess mRNA levels in colonic mucosa biopsies from UC patients bars show means with standarderror of the mean of MUC20 transcript levels with GAPDH as housekeeping gene determined by 2ΔΔCt

OR = 037) As part of the clinical features 21 of our UCpatients (52) had extraintestinal manifestations

Regarding the medical treatment all patients were takingsulfasalazine or 5-aminosalicylic acid (5-ASA) 475 usedoral or systemic steroids 30 were taking azathioprine and25 had anti-TNF therapy

The demographic and clinical characteristics from all UCpatients are shown in Table 2

32 Mucin 12 Mucin 16 and Mucin 20 Gene and ProteinExpression in Colonic Tissue of UC Patients Patients withactive UC showed no significant expression of MUC12 genein mucosa compared to the group of patients with UC inremission and the normal control group (Figure 1(a)) Noassociation was found between MUC12 gene expression andthe clinical features of UC In the immunohistochemistrycolonic tissue of UC patients had abundant inflammatory


Imm

unor

eact

ive c

ells

()

0004

lt0001

0

5

10

15

20

25

Mucosa MuscularSubmucosa Adventitia

ControlaUC(a) Membrane-bound mucin 12


lt0001

0024

005

Imm

unor

eact

ive c

ell (

)

0

5

10

15

20

25

30

ControlaUC(b) Membrane-bound mucin 16


Imm

unor

eact

ive c

ell (

) lt0001

lt0001

0

5

10

15

20

25

30

ControlaUC

(c) Membrane-bound mucin 20

Figure 2 (a) Percentage of MUC12 (b) MUC16 and (c) MUC20mdashexpressing cells in active UC patients (119873 = 10) Results are expressed asmedian (black line)

infiltrates predominantlymononuclear cells which extendedfrom the serosa to mucosa being more abundant in theepithelium and in the serosa In all cases a significantdestruction of the mucosa with decreased production ofMUC12 protein in epithelial cells and mononuclear infiltratein submucosa (119875 = 0004) and adventitia (lt0001) of colonspecimen of active UC (Figures 2(a) and 3) was shown

MUC16 gene expressionwas significantly increased in theUC remission group compared to the normal control groupand active UC group (119875 = 003) as shown in Figure 1(b)No association was found between MUC16 gene expression

and the clinical features of UC In order to evaluate the geneexpression findings of differential mucin regulation in theintestine MUC16 was identified by immunohistochemistryThe protein expression of MUC16 was higher in epithelialcells and mononuclear infiltrate from mucosa (119875 = 005)submucosa (119875 lt 0001) and muscular layer (119875 = 0024) incolonic specimen from active UC patients compared to thecontrol group (Figures 2(b) and 3)

Finally MUC20 gene expression was found significantlyincreased in patients with remission UC compared to activeUC (119875 = 0001) and normal controls (119875 = 0001)


Muc

in 1

2M

ucin

16

Muc

in 2

0

Control Ulcerative colitis

Figure 3 MUC12 MUC16 and MUC20mdashexpressing cells in colonic tissue from patients with ulcerative colitis Representativeimmunoperoxidase analysis in noninflamed colonic tissue (119873 = 10) (left panel) and active ulcerative colitis (119873 = 10) (right panel) Arrowsdepict immunoreactive cells in mucosa submucosa muscular and adventitia layers Original magnification was times320

Furthermore an association was found between MUC20gene expression and the presence of histological remissionin patients with UC (119875 = 0003 OR = 037) as shownin Figure 1(c) In this instance the protein expression ofMUC20 showed a decreased production in epithelial andmononuclear cells from mucosa (119875 lt 0001) and submucosa(119875 lt 0001) in patients with active UC compared to thecontrol group (Figures 2(c) and 3)

33 IL-6 and IL-8 as Intestinal Inflammatory Markers in UCPatients To further evaluate the relation of mucins mRNAlevels in UC and intestinal inflammation in UC patients wealso measure IL6 and IL-8 mRNA levels The IL6 mRNAlevels showed an important capacity to differentiate betweenremission and active disease The IL6 and IL-8 mRNA levelswere higher in the active group compared to remission of UCFigures 4(a) and 4(b)

4 Discussion

The findings of this study showed a significant increase of theproduction ofmucins 16 and 20mainly produced by epithelialcells and mononuclear infiltrate No increase was found inthe production of mucin 12 Interestingly the production ofmucin 20 was found to be associated with the presence ofhistological remission in patients with UC

In normal conditions the gastrointestinal tract is pro-tected by a mucus barrier with both secreted and cell-surfacemucins contributing to the exclusion of luminalmicrobes andtoxins Alterations in the structure andor quantity of mucinsmodify the barrier function of mucus and could play a rolein initiating and maintaining mucosal inflammation in IBD[13 14] In the IBD a loss of tight junctions has been lead anincrease in the intestinal permeability it could be explainedby a loss of cell polarity and hence overexpression of cellsurface mucins [6 14]


0

2

4

6

8

10

12

14

16

18

20IL

-6 m

RNA

GA

PDH

mRN

A

Active UC Remission UC Control

P = 0004

P = 0001

(a)

0

10

20

30

40

50

60

70

80


IL-8

mRN

AG

APD

H m

RNA

P = 0003

P = 0010

P = 0010

(b)

Figure 4 IL-6 and IL-8 mRNA levels in colonic mucosa from patients with ulcerative colitis and controls RT-qPCR was performed to assessmRNA levels in colonicmucosa biopsies fromUCpatients bars showmeans with standard error of themean of IL-6 and IL-8 transcript levelswithGAPDH RPLP0 andACTB as housekeeping gene determined by 2ΔΔCt and differences among groups were assessed byKruskal-Wallistest

Table 2 Clinical and demographic characteristics of ulcerativecolitis patients

Characteristics Number of patientsGender (MF) 1921Current age (mean years range) 40 (18ndash65)Age at diagnosis (mean years range) 24 (18ndash55)Disease duration 4 plusmn 2 yearsDisease activity (activeremission) 2020Disease extension distal colitispancolitis 2218Endoscopic activity(inactivemildmoderatesevere) 20488

Histological activity(inactivemildmoderatesevere) 20488

Current therapy5-Aminosalicylate 40Corticosteroids 12Azathioprine 15

Extraintestinal manifestations(absentpresent) 1921

Control groupNumber of patients sex (MF) 921Mean age (years range) 53 (19ndash73)

Patients with UC showed a significant decrease in thegene and protein expression and production of mucin 20in epithelial cells from mucosa as well as the submucosa inpatients with active disease compared to both in remissiongroup and in normal controls This gene was associated withhistological remission in patients with UC where expressionof MUC20 might have a protective role in patients with UCThis study is the first depiction of the association of highmucin 20 expression associated with histological remissionin UC patients On the other hand the overexpression ofMUC20 was associated with recurrence and poor outcomein patients with colorectal cancer [15]

In contrast MUC16 gene and protein expression weresignificantly increased in active UC compared to the controlgroup at different levels such as mucosa and submucosaNevertheless no association was found between MUC16gene expression and clinical characteristics of UC MUC16is synthesized in several layers of the colon such as mucosasubmucosa andmuscular in activeUCpatients In contrast aprevious study demonstrated that overexpression of MUC16was associated with lower survival rate in patients withendocervical adenocarcinoma [16]

MUC12 gene and protein expression were decreased insubmucosa and adventitia of colonic tissue from active UCpatients as compared to normal controls However no associ-ation was found between decreased MUC12 gene expressionand clinical features of UC The production of MUC12 ismainly by epithelial cells from ocular and respiratory tractand no production of MUC12 in goblet cells [17]

Finally it is important to note that this study providesevidence about the production of MUC20 MUC16 andMUC12 from enterocytes and confirms that the MUC12 andMUC20 are produced by epithelial cells from colon and rectalareas compared to goblet cells that secretes MUC1 MUC2MUC3 and MUC5AC [3 18]

Previously it has been reported that low expression ofMUC9 gene in UC patients (active and remission) comparedto control individuals suggests that MUC9 has an aberrantexpression contributing to a defect in the mucus layer andaltered physical barrier between luminal contents and themucosal surface According to other studies in pediatric andadult patients with IBD mucins have been involved in thedevelopment of IBD such as MUC1 MUC2 MUC5AC andMUC12 [14 19ndash21]

Since this is an observational cross section study it shouldfurthermore be stated that long-term follow-up studies areneeded in order to make prognostic predictions in a givenpatient population


In conclusion MUC16 and 20 showed an increasedexpression in the colonic mucosa from patients with remis-sion UCThis might be explained as a protection mechanismin patients with quiescent UC

Abbreviations

IBD Inflammatory bowel diseaseUC Ulcerative colitisRT-PCR Real-time polymerase chain reactionMUC Mucin

Conflict of Interests

The authors declared that they have no conflict of interests

Authorsrsquo Contribution

Jesus K Yamamoto-Furusho designed and provided theresearch idea directed and reassessed both clinical andhistological diagnostic from all UC patients and controlsand prepared and coordinated the paper editing as well asproviding the financial resources for performing the studyIlse Ascano-Gutierrez participated in the sample collectingand performed RT-qPCR analysis for mucin transcript levelquantification Janette Furuzawa-Carballeda participated inimmunohistochemistry data analysis and critical reviewingand data discussion and analysis and bibliographic analysisGabriela Fonseca-Camarillo participated in the sample pro-cessing performed RT-qPCR analysis for mucin transcriptlevel quantification immunohistochemistry and data discus-sion analysis and prepared the paper

References

[1] C W Png S K Linden K S Gilshenan et al ldquoMucolyticbacteria with increased prevalence in IBD mucosa augment invitro utilization of mucin by other bacteriardquo American Journalof Gastroenterology vol 105 no 11 pp 2420ndash2428 2010

[2] C Moehle N Ackermann T Langmann et al ldquoAberrantintestinal expression and allelic variants of mucin genes asso-ciated with inflammatory bowel diseaserdquo Journal of MolecularMedicine vol 84 no 12 pp 1055ndash1066 2006

[3] M Andrianifahanana NMoniaux and S K Batra ldquoRegulationof mucin expression mechanistic aspects and implications forcancer and inflammatory diseasesrdquo Biochimica et BiophysicaActa vol 1765 no 2 pp 189ndash222 2006

[4] D Boltin T T Perets A Vilkin and Y Niv ldquoMucin functionin inflammatory bowel disease an updaterdquo Journal of ClinicalGastroenterology vol 47 no 2 pp 106ndash111 2013

[5] S Cornick A Tawiah and K Chadee ldquoRoles and regulationof the mucus barrier in the gutrdquo Tissue Barriers vol 3 no 1-2Article ID e982426 2015

[6] S Senapati S B Ho P Sharma et al ldquoExpression of intestinalMUC17 membrane-bound mucin in inflammatory and neo-plastic diseases of the colonrdquo Journal of Clinical Pathology vol63 no 8 pp 702ndash707 2010

[7] K Kyo T Muto H Nagawa G M Lathrop and Y NakamuraldquoAssociations of distinct variants of the intestinal mucin gene

MUC3A with ulcerative colitis and Crohnrsquos diseaserdquo Journal ofHuman Genetics vol 46 no 1 pp 5ndash20 2001

[8] S J Williams M A McGuckin D C Gotley H J Eyre G RSutherland and T M Antalis ldquoTwo novel mucin genes down-regulated in colorectal cancer identified by differential displayrdquoCancer Research vol 59 no 16 pp 4083ndash4089 1999

[9] B W T Yin and K O Lloyd ldquoMolecular cloning of the CA125ovarian cancer antigen identification as a newmucin MUC16rdquoThe Journal of Biological Chemistry vol 276 no 29 pp 27371ndash27375 2001

[10] T Higuchi T Orita S Nakanishi et al ldquoMolecular Cloninggenomic structure and expression analysis of MUC20 a novelmucin protein up-regulated in injured kidneyrdquo The Journal ofBiological Chemistry vol 279 no 3 pp 1968ndash1979 2004

[11] S A Riley V Mani M J Goodman M E Herd S Dutt and LA Turnberg ldquoComparison of delayed release 5 aminosalicyclicacid (mesazaline) and sulphalazine in the treatment of mild tomoderate ulcerative colitis relapserdquo Gut vol 29 no 5 pp 669ndash674 1988

[12] KW SchroederW J Tremaine andDM Ilstrup ldquoCoated oral5-aminosalicylic acid therapy for mildly to moderately activeulcerative colitis a randomized studyrdquoTheNewEngland Journalof Medicine vol 317 no 26 pp 1625ndash1629 1987

[13] Y H Sheng S Z Hasnain T H J Florin andM AMcGuckinldquoMucins in inflammatory bowel diseases and colorectal cancerrdquoJournal of Gastroenterology andHepatology vol 27 no 1 pp 28ndash38 2012

[14] J K Yamamoto-Furusho E J Mendivil-Rangel and GFonseca-Camarillo ldquoReduced expression of mucin 9 (MUC9)in patients with ulcerative colitisrdquo Inflammatory Bowel Diseasesvol 18 no 3 p E601 2012

[15] X Xiao L Wang P Wei et al ldquoRole of MUC20 overexpressionas a predictor of recurrence and poor outcome in colorectalcancerrdquo Journal of Translational Medicine vol 11 article 1512013

[16] S Togami M Nomoto M Higashi et al ldquoExpression ofmucin antigens (MUC1 and MUC16) as a prognostic factorfor mucinous adenocarcinoma of the uterine cervixrdquo Journal ofObstetrics and Gynaecology Research vol 36 no 3 pp 588ndash5972010

[17] T Pelaseyed J H Bergstrom J K Gustafsson et al ldquoThemucusand mucins of the goblet cells and enterocytes provide the firstdefense line of the gastrointestinal tract and interact with theimmune systemrdquo Immunological Reviews vol 260 no 1 pp 8ndash20 2014

[18] M E V Johansson H Sjovall and G C Hansson ldquoThegastrointestinal mucus system in health and diseaserdquo NatureReviews Gastroenterology andHepatology vol 10 no 6 pp 352ndash361 2013

[19] A E Furr S Ranganathan and O J Finn ldquoAberrant expressionof MUC1 mucin in pediatric inflammatory bowel diseaserdquoPediatric and Developmental Pathology vol 13 no 1 pp 24ndash312010

[20] R Shaoul Y Okada E Cutz and M A Marcon ldquoColonicexpression of MUC2 MUC5AC and TFF1 in inflammatorybowel disease in childrenrdquo Journal of Pediatric Gastroenterologyand Nutrition vol 38 no 5 pp 488ndash493 2004

[21] K O Hensel V Boland J Postberg et al ldquoDifferentialexpression of mucosal trefoil factors and mucins in pediatricinflammatory bowel diseasesrdquo Scientific Reports vol 4 article7343 2014

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Evidence-Based Complementary and Alternative Medicine

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contact with toxins cytokines and commensal or pathogenicmicroorganisms through adhesins which induces acute andchronic inflammation in the intestine Patients with UC haveshown an aberrantMUC synthesis and degradation as well asalterations ofmucinrsquos glycoprotein structure (o-glycosylationsulfation and silylation) leading to a deficientmucous barrier[1 2] Some groups have described a reduction in MUC1MUC2 and MUC3 and MUC17 gene expression in bothinflamed and nonaffectedmucosae fromUC andCDpatients[2 6ndash8] MUC2 is the predominant sulfated componentof mucin layer in healthy colon In active UC MUC2production and secretion by goblet cells are reduced and thisinduces synthesis of MUC5AC and MUC6 [6] On the otherhand it has been reported that MUC1 is overexpressed insevere UCThis finding correlates with an elegant experimentdemonstrating that MUC1minusminus mice model is less susceptibleto dextran sulfate sodium induction of colitis and that feweractivated T cells were recruited to the inflamed mucosa [5]MUC17 in healthy intestine is one of the major membrane-bound mucins However its expression is reduced in UCpatients [6] MUC13 was increased in active UC but MUC3did not changeTheMUC4was similarly increased especiallywhen associated with the development of neoplasia Anaberrant expression of MUC5AC was found in UC as well asin CD [9 10] Previous studies from Moehle et al describedsignificant downregulation of MUC12 and MUC20 in colonand ileum in patients with Crohnrsquos Disease [6]

MUC12 is a protein-coding gene whose function isinvolved in the epithelial cell protection [9] The function ofMUC16 is thought to provide a protective lubricating barrieragainst particles and infectious agents at mucosal surfaces[10]

MUC20 is a protein-coding gene that contains at leastfour exons and is located close to MUC4 on chromosome3q29 It has been reported that MUC20 is oversynthesizedin gastric ovarian endometrial and colorectal cancer andpredicts recurrence and poor outcome [11]

Little is known about the MUC12 MUC16 and MUC20expression and their potential role in the physiopathology ofUC Thus the aim of the present study was to characterizethe changes in gene and protein expression of thesemucins incolonic tissue frompatients withUC in regard to their clinicaloutcomes

2 Materials and Methods

21 Study Subjects We studied 20 patients with active UC20 patients in remission UC and 30 normal controls withoutinflammation All individuals belonged to the InflammatoryBowel Disease Clinic at the Instituto Nacional de CienciasMedicas y Nutricion Hospital The diagnosis of UC wasdone by the presence of the following criteria macroscopicappearance by endoscopy and biopsy compatible with UCand history of diarrhea or blood in stool Demographic andclinical variables were collected from personal interview andclinical records The clinical and endoscopic activity wasevaluated by Mayo [12] and histological activity by Rileyscore

The tissue control group consisted of noninflamed con-trols (no documented inflammatory disease) undergoingcolonoscopy for evaluation of anemia and screening ofpolyps All individuals were included as controls if nopathological findings were found

22 Sample Processing and Gene Expression Analysis Thecolonic biopsies for gene expression analysis were taken withcolonoscopy and were immediately placed in preserver ofRNA (Ambion Austin TX USA) and stored at minus70∘C untilprocessing Then total RNA was isolated using High PureRNA Tissue (Roche Diagnostics Mannheim Germany)Colonic biopsies samples were disrupted in 400 120583L LysisBuffer and homogenizedTheRNAwas isolated by binding tothe glass fibers prepacked in theHigh Pure Filter Tube Resid-ual contaminating DNA was digested with DNase I BoundRNA was washed thereby purified from salts proteins andother cellular impurities Finally we added 100120583L of ElutionBuffer for the elution of RNA

Two hundred nanograms of total RNA was reverse-transcribed into cDNA with random hexamer primers(Roche Diagnostics Mannheim Germany)

The MUC gene relative expression was measured byquantitative real-time polymerase chain reaction (RT-PCR)Reference genes RPLP0 ACTB and GAPDH transcriptswere used for relative quantification and quality controlsFor qPCR assays quality control determination of linearityand reproducibility was evaluated (VC lt 10) The mRNArelative quantification of target genes was conducted usingthe LightCycler software 41 according to the 2-delta-delta Ctmethod

We used the following primers MUC12 forward cctg-gaaaccttagcaccag and reverse gacagacgcattgttttccat MUC16forward tggggaccaccaattctatg and reverse atggctgggagtg-gattg MUC20 forward tatgtgccgtggaggattc and reversettgctgtacgtgtctaacttcaatc IL-6 forward tctgctcccacaatgaaacatand reverse gaaggcagcaggcaacac IL-8 forward agacagca-gagcacacaagc and reverse atggttccttccggtggt GADPH for-ward gcccaatacgaccaaatcc and reverse agccacatcgctcagacaRPLP0 forward gaagctctatctcgcctcca and reverse agcaggcaa-caccaggag and ACTB forward caaccgcgagaagatgac andreverse gtccatcacgatgccagt for normalization as also shownin Table 1

The PCR amplification of mucin genes was carried outwith 20 ng of cDNA 200 nM forward and reverse primerand Taqman Master Mix (Roche Diagnostics MannheimGermany) in a final volume of 10 120583L PCR reactions wererun in a Light Cycler 480 (Roche Diagnostics MannheimGermany) for 45 cycles each cycle consists in denaturationfor 15 seconds at 95∘ primer annealing for 15 seconds at55∘ and extension for 30 seconds at 72∘C and cooling for 30seconds at 40∘C

23 Immunohistochemistry All tissues obtained were forma-lin fixed and paraffin embedded Five 120583m tissue sectionswere rehydrated using xylene and graded ethanol series Afterdeparaffinizing and demasking of antigenswith 10mMcitratebuffer pH 60 the slides were then soaked in 1 hydrogenperoxide in methanol for 20min at room temperature to





tc







ggc





2

O2






3 Results




MU

C12

mRN

AG

AD

PH m

RNA

5000

10000

15000

20000

25000

30000

0

ControlrUCaUC

(a)M

UC1

6 m

RNA

GA

DPH

mRN

A

1000

2000

3000

4000

0

0035

ControlrUCaUC

(b)

MU

C20

mRN

AG

AD

PH m

RNA

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0001

0001

ControlrUCaUC

(c)







Imm

unor

eact

ive c

ells

()

0004

lt0001

0

5

10

15

20

25




lt0001

0024

005

Imm

unor

eact

ive c

ell (

)

0

5

10

15

20

25

30



Imm

unor

eact

ive c

ell (

) lt0001

lt0001

0

5

10

15

20

25

30

ControlaUC








Muc

in 1

2M

ucin

16

Muc

in 2

0





4 Discussion




0

2

4

6

8

10

12

14

16

18

20IL

-6 m

RNA

GA

PDH

mRN

A


P = 0004

P = 0001

(a)

0

10

20

30

40

50

60

70

80


IL-8

mRN

AG

APD

H m

RNA

P = 0003

P = 0010

P = 0010

(b)
















Abbreviations






References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















tc







ggc





2

O2






3 Results




MU

C12

mRN

AG

AD

PH m

RNA

5000

10000

15000

20000

25000

30000

0

ControlrUCaUC

(a)M

UC1

6 m

RNA

GA

DPH

mRN

A

1000

2000

3000

4000

0

0035

ControlrUCaUC

(b)

MU

C20

mRN

AG

AD

PH m

RNA

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0001

0001

ControlrUCaUC

(c)







Imm

unor

eact

ive c

ells

()

0004

lt0001

0

5

10

15

20

25




lt0001

0024

005

Imm

unor

eact

ive c

ell (

)

0

5

10

15

20

25

30



Imm

unor

eact

ive c

ell (

) lt0001

lt0001

0

5

10

15

20

25

30

ControlaUC








Muc

in 1

2M

ucin

16

Muc

in 2

0





4 Discussion




0

2

4

6

8

10

12

14

16

18

20IL

-6 m

RNA

GA

PDH

mRN

A


P = 0004

P = 0001

(a)

0

10

20

30

40

50

60

70

80


IL-8

mRN

AG

APD

H m

RNA

P = 0003

P = 0010

P = 0010

(b)
















Abbreviations






References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MU

C12

mRN

AG

AD

PH m

RNA

5000

10000

15000

20000

25000

30000

0

ControlrUCaUC

(a)M

UC1

6 m

RNA

GA

DPH

mRN

A

1000

2000

3000

4000

0

0035

ControlrUCaUC

(b)

MU

C20

mRN

AG

AD

PH m

RNA

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0001

0001

ControlrUCaUC

(c)







Imm

unor

eact

ive c

ells

()

0004

lt0001

0

5

10

15

20

25




lt0001

0024

005

Imm

unor

eact

ive c

ell (

)

0

5

10

15

20

25

30



Imm

unor

eact

ive c

ell (

) lt0001

lt0001

0

5

10

15

20

25

30

ControlaUC








Muc

in 1

2M

ucin

16

Muc

in 2

0





4 Discussion




0

2

4

6

8

10

12

14

16

18

20IL

-6 m

RNA

GA

PDH

mRN

A


P = 0004

P = 0001

(a)

0

10

20

30

40

50

60

70

80


IL-8

mRN

AG

APD

H m

RNA

P = 0003

P = 0010

P = 0010

(b)
















Abbreviations






References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Imm

unor

eact

ive c

ells

()

0004

lt0001

0

5

10

15

20

25




lt0001

0024

005

Imm

unor

eact

ive c

ell (

)

0

5

10

15

20

25

30



Imm

unor

eact

ive c

ell (

) lt0001

lt0001

0

5

10

15

20

25

30

ControlaUC








Muc

in 1

2M

ucin

16

Muc

in 2

0





4 Discussion




0

2

4

6

8

10

12

14

16

18

20IL

-6 m

RNA

GA

PDH

mRN

A


P = 0004

P = 0001

(a)

0

10

20

30

40

50

60

70

80


IL-8

mRN

AG

APD

H m

RNA

P = 0003

P = 0010

P = 0010

(b)
















Abbreviations






References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Muc

in 1

2M

ucin

16

Muc

in 2

0





4 Discussion




0

2

4

6

8

10

12

14

16

18

20IL

-6 m

RNA

GA

PDH

mRN

A


P = 0004

P = 0001

(a)

0

10

20

30

40

50

60

70

80


IL-8

mRN

AG

APD

H m

RNA

P = 0003

P = 0010

P = 0010

(b)
















Abbreviations






References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

12

14

16

18

20IL

-6 m

RNA

GA

PDH

mRN

A


P = 0004

P = 0001

(a)

0

10

20

30

40

50

60

70

80


IL-8

mRN

AG

APD

H m

RNA

P = 0003

P = 0010

P = 0010

(b)
















Abbreviations






References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Abbreviations






References




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Differential Expression of MUC12, MUC16...

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