research article overexpression of foxo3, myd88, and gapdh...

Research ArticleOverexpression of FOXO3 MYD88 and GAPDHIdentified by Suppression Subtractive Hybridizationin Esophageal Cancer Is Associated with Autophagy

Mohammad Soltany-Rezaee-Rad12 Negar Mottaghi-Dastjerdi12 Neda Setayesh1

Gholamreza Roshandel3 Farzaneh Ebrahimifard4 and Zargham Sepehrizadeh1

1 Department of Pharmaceutical Biotechnology and Pharmaceutical Biotechnology Research CenterSchool of Pharmacy Tehran University of Medical Sciences Tehran 1417614411 Iran

2 Pharmaceutical Sciences Research Center Sari School of Pharmacy Mazandaran University of Medical SciencesSari 48175-861 Iran

3 Golestan Research Center of Gastroenterology and Hepatology Golestan University of Medical Sciences Golestan 49186-619 Iran4Department of General Surgery School of Medicine Shahid Beheshti University of Medical Sciences Tehran 19857-17443 Iran

Correspondence should be addressed to Zargham Sepehrizadeh zsepehritumsacir

Received 30 September 2013 Revised 12 December 2013 Accepted 16 December 2013 Published 8 January 2014

Academic Editor D Fan

Copyright copy 2014 Mohammad Soltany-Rezaee-Rad 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

To find genes involved in tumorigenesis and the development of esophageal cancer the suppression subtractive hybridization (SSH)method was used to identify genes that are overexpressed in esophageal cancer tissues compared to normal esophageal tissues Inour SSH library the forkhead boxO3 (FOXO3) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) andmyeloid differentiationprimary response 88 (MYD88) genes were the most highly upregulated genes and they were selected for further studies becauseof their potential role in the induction of autophagy Upregulation of these genes was also observed in clinical samples using qRT-PCR In addition coexpression analysis of the autophagy-related genes Beclin1 ATG12 Gabarapl PIK3C3 and LC3 demonstrateda significant correlation between the differentially overexpressed genes and autophagy Autophagy is an important mechanism intumorigenesis and the development of chemoresistance in cancer cellsThe upregulation of FOXO3 GAPDH andMYD88 variantsin esophageal cancer suggests a role for autophagy and provides new insight into the biology of esophageal cancer We propose thatFOXO3 GAPDH and MYD88 are novel targets for combating autophagy in esophageal cancer

1 Introduction

Esophageal cancer is one of the most aggressive and life-threatening types of carcinoma in developing countries andit has a high incidence rate in some geographical regionsparticularly in the ldquoAsian esophageal cancer beltrdquo whichextends from the Caspian Littoral in Iran TurkmenistanUzbekistan and Kazakhstan to the northern provinces ofChina [1] Esophageal cancer is among the top 10 causes ofcancer-related deaths worldwide [2] Although some alteredoncogenes and tumor suppressor genes have been identi-fied in esophageal cancer (eg p53 deletion p21 alteration

and amplification of CCND1 and c-myc) the fundamentalmolecular mechanisms leading to esophageal cancer remainunknown [3ndash6] The identification of genes that are dif-ferentially expressed in esophageal cancer cells allows forthe identification of new biomarkers and therapeutic targetgenes In addition this strategy could lead to an improvedunderstanding of the molecular biology and mechanisms ofcarcinogenesis in esophageal cancer

In contrast to apoptosis autophagy is primarily a cellsurvival process thus autophagy has been considered animportant mechanism in chemoresistance and is knownas a survival factor for tumor cells in the early stages of

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2014 Article ID 185035 8 pageshttpdxdoiorg1011552014185035

2 Gastroenterology Research and Practice

tumorigenesis [7ndash10] In this study suppression subtrac-tive hybridization (SSH) was used to identify genes thatare overexpressed in esophageal cancer cells Among theidentified ESTs from the constructed SSH library potentialautophagy-inducing genes (FOXO3 MYD88 and GAPDH)were selected for further analysisThe incidence of autophagywas also determined in clinical tissue samples by quantifyingautophagy-relatedregulatory genesThis study provides newinformation concerning the genes associated with the devel-opment of esophageal cancer particularly those involved inautophagy which could have a significant influence on thediagnosis and treatment of this type of cancer which typicallyhas a poor prognosis

2 Materials and Methods

21 Tissue Collection The samples used for the SSH weresurgically resected using esophagectomy from a patient priorto chemotherapy Normal tissue was resected 10 cm far fromthe tumor A pathologist dissected the target tissues under themicroscope with special care for minimal contamination ofnonepithelial cells For confirmation of overexpressed genesby qRT-PCR 10 samples from 5 patients (5 tumors and5 normal tissues from the same patients) were collectedusing endoscopy Target tissues were immersed in 10 volumesof an RNAlater solution (Ambion Austin TX USA) Thesamples were stored at minus80∘C freezer The resected tissueswere examined using hematoxylin-eosin (HampE) stainingTheconsent form was approved by the Biologic Sampling EthicsCommittee of the Tehran University of Medical Sciences andobtained from patients prior to sampling

22 Extraction of Total RNA Tissues were lysed using amortar and pestle and liquid nitrogen TwomL of Tripureisolation reagent (Roche Applied Science Indianapolis INUSA) was addedThe lysed tissues were passed through a 20-gauge needle 10 times for homogenizationTheprocedurewasperformed according to the manufacturerrsquos instructions Theconcentration and purity of the total RNA were determinedusing a BioPhotometer (Eppendorf Hamburg Germany)RNA quality was assessed using a 1 denatured agarose gel

23 Isolation of mRNA mRNA was isolated using the Dyn-aBeads mRNA Isolation Kit (Dynal Lake Success NY USA)BrieflyDynaBeads oligo (dT)

25were equilibratedwith 100120583L

of binding buffer (100mM Tris-HCl 500 nM LiCl 10mMEDTA 1 LiDS and 5mM DTT) The extracted total RNAwas diluted to 100 120583L in binding buffer and the equilibratedDynaBeads were added and incubated for 5min at 37∘Cin a shaking incubator The beads were then placed on amagnet the supernatant was removed and the beads werewashed twice using 200 120583L of washing buffer (10mM Tris-HCl 015M LiCl and 1mM of EDTA) Elution buffer (10 120583L)was added to the beads prior to incubation at 65∘C for 2minThe beads were then immediately placed on the magnet andthe eluted mRNA was isolated To improve the reduction ofbackground rRNA this protocol was repeated on the eluted

mRNA The purity of the purified mRNA was assessed usinga 1 denatured agarose gel

24 Construction of the SSH Library When constructing asubtracted library it is routine procedure to use a singlesample pair SSH library construction was achieved using thePCR-Select cDNA subtraction kit (Clontech Palo alto CAUSA) according to the manufacturerrsquos instructions Briefly2 120583g mRNA from normal (as Driver) and tumor (as Tester)tissues was applied for the first and second cDNA synthesisThe resulting cDNA was digested with RsaI and the blunt-ended double-stranded cDNAwas purifiedTheTester cDNAwas divided into two parts which were individually ligated toadaptor 1 and adaptor 2 The adaptor 1- and adaptor 2-ligatedcDNAs were separately hybridized with excess amountsof Driver cDNA for 8 hr at 68∘C The two hybridizationmixtures were combined and hybridizedwith excess amountsof Driver cDNA overnight at 68∘C The final hybridizationmixture was diluted using 200 120583L of dilution buffer for twosubsequent rounds of PCR The constructed SSH librarieswere confirmed using 1 agarose gel electrophoresis

25 Cloning and Similarity Searches The constructed SSHlibraries were purified using the PCR Product PurificationKit (Roche Applied Science) and the purified PCR productswere ligated into the pUC19 cloning vector and transformedinto Escherichia coli NovaBlue competent cells (NovagenMadison WI USA) Positive clones were verified usingthe colony PCR method with the adaptor-specific primersN1 (51015840-TCGAGCGGCCGCCCGGGCAGGT-31015840) and N2R(51015840-AGCGTGGTCGCGGCCGAGGT-31015840) and the plasmidswere isolated for sequencing using the High Pure Plas-mid Isolation Kit (Roche Applied Sciences) Single direc-tion DNA sequencing was performed using the BigDyeterminator v31 sequencing kit and a 3730xl automatedsequencer (Applied Biosystems Foster City CA USA)Similarity searches were performed using the BLASTntBLASTn and tBLASTx algorithms in the NCBI GenBankdatabases (httpblastncbinlmnihgovBlastcgi) to analyzethe sequences

26 Analysis of the Subtraction Efficiency The efficiency ofthe subtraction method was analyzed by comparing theabundance of a nondifferentially expressed housekeepinggene (eg beta actin) before and after subtraction Brieflythe subtracted and nonsubtracted samples were diluted 10-fold in H

2O as a template for PCR Real-time PCR reactions

were prepared by adding 10 120583L of SYBR Premix Ex Taq(Takara Kusatsu Japan) 1 120583L of sample 08 pmol of the betaactin primers 04120583L of ROX dye and DEPC-treated waterto a final volume of 20120583L The thermal cycling parametersfor the reaction consisted of an initial heating of 10min at95∘C followed by 40 cycles of 30 sec at 95∘C and 1min at60∘C Melting curve analysis was performed by increasingthe temperature from 65∘C to 95∘C in 01∘Csecond incre-ments for each fluorescence reading using the Step-One-Plusapparatus (Applied Biosystems) The subtraction efficiency

Gastroenterology Research and Practice 3

was determined using the relative expression of the beta actingene in the subtracted and nonsubtracted samples

27 qRT-PCR Analysis of the FOXO3 MYD88 Variants andGAPDH Genes To analyze the overexpression of the genesidentified using the SSH normal and cancerous endoscopictissue samples were collected from patients Total RNAwas extracted using the Tripure Isolation Reagent (RocheApplied Sciences) One 120583g of total RNA was used for cDNAsynthesis with the Expand Reverse Transcriptase (RocheApplied Sciences) and oligo (dT)

18primer Table 1 presents

the primers that were designed to amplify the FOXO3GAPDH and beta actin genes in the qRT-PCR reactionAlternative splicing of MYD88 variants was analyzed usingthe real-time PCR method In this method primers weredesigned based on variant-specific exons from MYD88 Thedesigned primers locations for the specific MyD88 variantsare depicted in Figure 1 The primers were designed usingPrimerSelect software version 710 (DNAstar Madison WIUSA) and synthesized by the TAG Company (TAG Copen-hagen Copenhagen Denmark) The qRT-PCR reaction wasprepared using 10120583L of SYBRPremix ExTaq (Takara) 2120583L ofcDNA 08 pmol of each forward and reverse primer 04 120583L ofROX dye and DEPC-treated water to a final volume of 20 120583LThe thermal cycling program for the reactions was as followsan initial heating for 10min at 95∘C followed by 40 cyclesof 30 sec at 95∘C and 1min at the annealing temperature ofthe selected geneThe annealing temperatures of the designedprimers are presented in Table 1 Melting curve analysiswas performed by increasing the temperature from 65∘Cto 95∘C in 01∘Csecond increments for each fluorescencereading The efficiencies of the reactions amplifying theFOXO3MYD88 variantsGAPDH andbeta actin geneswerecalculated using a standard curve that consisted of 5 pointsfrom 10-fold serial dilutions that were prepared using 100 ngof the synthesized cDNA as the first point Expression levelsof the FOXO3 MYD88 variants and GAPDH genes werenormalized using the beta actin gene The relative expressionlevel for each gene was calculated using REST 2008 softwareV207 (Corbett Research Sydney Australia)

28 Detection of Autophagy Autophagy was detected usingreal-time PCR to obtain a relative quantification of theBeclin1 PIK3C3 ATG12 LC3 and Gabarapl1 genes incancerous and normal tissues The primers were designedusing PrimerSelect software version 710 (Lasergene) andsynthesized by the TAG Company (TAG Copenhagen) Thereal-time PCR reactions were done as described aboveThe expression levels of Beclin1 PIK3C3 ATG12 LC3 andGabarapl1 were normalized using beta actin and relativeexpression was calculated using REST 2008 software V207(Corbett Research)

29 Statistical Analysis The 119905-test was used for statisticalanalysis Spearmanrsquos Rho analysis was applied to assess apossible relationship between the expression levels of theidentified differentially expressed genes (FOXO3 GAPDHand MYD88) and the autophagy-related genes in cancerous

and normal tissues The statistical significance was set at 119875 lt005

3 Results

31 Suppression Subtractive Hybridization and SubtractionEfficiency To identify genes overexpressed in esophagealcancer SSH was performed using cancerous tissue as theTester and normal tissue as theDriver Figure 2 represents theconstructed SSH library in which the subtracted genes canbe seen in the 180ndash1200 bp region Among the significantlyoverexpressed genes (unpublished data) 3 clones represent-ing the FOXO3 MYD88 and GAPDH genes were selectedbecause of their probable role in the induction of autophagySelected clones were annotated as FOXO3 with a length of307 bp and 95 identity with FOXO3 sequences MYD88with a length of 256 bp and 99 identity with MYD88sequences and GAPDH with a length of 758 bp and 98identity with GAPDH sequences from the NCBI databaseReal-time PCR analysis demonstrated that beta actin has a97-fold reduction in the subtracted library comparedwith thenonsubtracted library

32 FOXO3 GAPDH and MYD88 Are Overexpressed inEsophageal Cancer Tissues To confirm the results obtainedfrom the constructed SSH library the expression levels ofthe FOXO3 MYD88 and GAPDH genes were quantified incancerous and normal esophageal tissues using qRT-PCROverexpression of FOXO3 GAPDH andMYD88 variants isdepicted in Figure 3 These results confirmed the efficiencyof the SSH library which indicated that these genes weresignificantly overexpressed in esophageal cancer

33 MYD88v1 and MYD88v3 Are the Dominant Variantsin Esophageal Cancer Tissues The MYD88 variants wereamplified using the Step-One-Plus apparatus The meltingtemperatures of MYD88 v1 v2 v3 and v5 were 8799∘C859∘C 862∘C and 8471∘C respectively (Figure 4) Themean fold induction for each MYD88 variant is depictedin Figure 3 The results demonstrated a significant shift inthe pattern of expression of MYD88 variants 1 and 3 inthe esophageal cancer tissues compared with the normaltissues In normal tissues the expression patterns of theMYD88 variants were as follows MYD88v1 10 MYD88v212 MYD88v3 76 and MYD88v5 2 In contrast thefollowing expression patterns were observed in canceroustissuesMYD88v1 25MYD88v2 13MYD88v3 58 andMYD88v5 4

34 Overexpression of Beclin1 ATG12 Gabarapl1 LC3 andPIK3C3 Indicates Autophagy in Esophageal Cancer TissuesThe transcript level of Beclin1 a gene that is involved inautophagic vacuole formation and acts as a coregulatorof autophagy increased 3825-fold in tumor tissues Thetranscript levels of LC3ATG12 andGabarapl1 genes involvedin autophagic vacuole formation increased 304-fold 2685-fold and 1820-fold in tumor tissues respectively The tran-script level of PIK3C3 a gene that acts as a coregulator of


Table1Designedprim

erssequences

used

toqu

antifygene

expressio

nby

real-timeP

CR

Application

Prim

ername

Accessionnu

mber

Leng

thSequ

ence

(51015840

to31015840)

Ann

ealin

gtemperature

Locatio

nProd

uctsize

Overexpressed

genes

FOXO

3-F

NM

0014553

20AC

GGTG

TTCG

GAC

CTTC

ATC

60∘

C1613ndash1632

196b

pFO

XO3-R

NM

0014553

20TG

CTGGCC

TGAG

ACAT

CAAG

1789ndash1808

GAPD

H-F

NM

002046

420

CGAC

AGTC

AGCC

GCA

TCTT

C61∘

C118

ndash137

250b

pGAPD

H-R

NM

002046

420

CGTT

CTCA

GCC

TTGAC

GGTG

348ndash367

MYD

88-v1-F

NM

00117

25671

20TC

ATCG

AAAAG

AGGTT

GGCT

57∘

C855ndash875

158b

pMYD

88-v1-R

NM

00117

25671

20GAT

GGGGAT

CAGTC

GCT

TCT

993ndash1012

MYD

88-v2-F

NM

0024684

20CC

ACAC

TTGAT

GAC

CCCC

TG61∘

C66

6ndash685

210b

pMYD

88-v2-R

NM

0024684

20GGCG

GCA

CCTC

TTTT

CGAT

G856ndash

875

MYD

88-v3-F

NM

00117

25681

20GGGAC

CCAG

CATT

GGGCA

TA61∘

C538ndash557

289b

pMYD

88-v3-R

NM

00117

25681

20AC

CTGGAG

AGAG

GCT

GAG

TG807ndash826

MYD

88-v4-F

NM

00117

25691

20CT

TGAT

GAC

CCCC

TGGGTG

C62∘

C671ndash690

207b

pMYD

88-v4-R

NM

00117

25691

20GGTT

GGTG

TAGTC

GCA

GAC

A858ndash877

MYD

88-v5-F

NM

00117

25661

17AC

CCAG

CATT

GGTG

CCG

60∘

C541ndash557

202b

pMYD

88-v5-R

NM

00117

2566

120

GGTT

GGTG

TAGTC

GCA

GAC

A723ndash742

Con

trolgene

ACTB

-FNM

0011013

21AT

GGCC

ACGGCT

GCT

TCCA

GC

60∘

C763ndash783

322b

pAC

TB-R

NM

0011013

21CA

GGAG

GAG

CAAT

GAT

CTTG

A1064

ndash1084

Autoph

agy-relatedgenes

Gabarapl1-F

NM

0314122

20CA

GGGTC

CCCG

TGAT

TGTA

G61∘

C321ndash340

179b

pGabarapl1-R

NM

0314122

20TG

GGAG

GGAT

GGTG

TTGTT

G480ndash

499

Beclin1-F

NM

003766

320

GCT

GAAG

ACAG

AGCG

ATGGT

595∘

C30ndash4

9169b

pBe

clin1-R

NM

003766

320

CATG

GTG

CTGTT

GTT

GGAC

G179ndash

198

PIK3

C3-F

NM

0026472

20GGCA

CACA

GAG

TGAG

CAGTA

595∘

C2423ndash244

2204b

pPIK3

C3-R

NM

0026472

20CA

CAGCC

TCTT

CATC

CGAC

A2607ndash2626

ATG12-F

NM

0047073

20TG

CTGGAG

GGGAAG

GAC

TTA

595∘

C347ndash366

186b

pAT

G12-R

NM

0047073

20GTT

CGCT

CTAC

TGCC

CACT

T513ndash532

MAP1LC

3B-F

NM

0228184

20GCC

GCC

TTTT

TGGGTA

GAAG

595∘

C1012ndash1031

225b

pMAP1LC

3B-F

NM

0228184

20TA

CCCC

TGCA

AGAG

TGAG

GA

1217ndash1236


Exon 1 Exon 2Exon location 1

Exon size 550 bp 134 bp 204 bp 91 bp 1890 bp

551 552

MYD88-v1-F MYD88-v1-R

686 687 891 892 983 984 2874Exon 3 Exon 4 Exon 5

Exon 1Exon location 1

Exon size 550 bp 180 bp 91 bp 1890 bp

551


552 732 733 824 825 2715Exon 3 Exon 4 Exon 5



551 552


686 687 778 779 2669Exon 4 Exon 5


Exon size 550 bp 91 bp 1890 bp

551


552 643 644 2534Exon 4 Exon 5



551 552


686 687 867 868 959 960 2850Exon 3a Exon 4 Exon 5

MYD88 variant 1

MYD88 variant 2

MYD88 variant 3

MYD88 variant 4

MYD88 variant 5

NM 0011725671

NM 0024684

NM 0011725681

NM 0011725691

NM 0011725661

Figure 1 Alternative splicing variants of the MYD88 gene and designed primers locations

DN

Ala

dder

Forw

ard

libra

ry

1000 bp

250bp

(a)

Nonsubtracted library Subtracted library00

02

04

06

08

10

12

Fold

chan

ge

1

0103

(b)

Figure 2 Construction of the SSH library (a) Forward constructed library (Lane 2) which ranges in length from 180 to 1200 bp (b) Evaluationof the subtraction efficiency of the constructed library The relative expression levels of the beta actin gene in the two samples indicate thatthere is a 97-fold decrease in beta actin expression in the subtracted cDNAs

autophagy and is involved in the induction of autophagy inresponse to intracellular signals was 2704-fold higher intumor tissues than in normal tissues (Figure 3) Correlationof FOXO3 MYD88 isoforms and GAPDH with autophagy-related genes is represented in Table 2

4 Discussion

Autophagy is categorized as a protein degradation systemwhich participates in the cellular homeostasis that is typicallyobserved in nutrient-deprived cells In this process excess orunnecessary proteins and organelles are fused to lysosomesand digested by lysosomal enzymes [11 12] In the earlystages of the tumorigenesis and during cancer treatmentautophagy induction fortifies cancer cell growth in thetumor microenvironment by protecting cells from nutrient

deprivation and providing an alternative energy source forcancer cells [13 14]

In this study suppression subtractive hybridization wasused to identify the overexpressed genes FOXO3 MYD88and GAPDH which have potential roles in the induction ofautophagy in esophageal cancer cells Upregulation of thesegenes was confirmed in esophageal cancer samples usingqRT-PCR

The work presented here identified the FOXO3 geneas being overexpressed in esophageal cancer This genebelongs to a family of transcription proteins that is involvedin the regulation of autophagy Autophagy-related genes(eg LC3 and BNIP3) are directly upregulated by FOXO3[15 16] In addition previous studies revealed that FOXO3overexpression induced autophagy in the HEK293T cell line[17] Our results indicated that FOXO3 was upregulated


0

5

10

15

20

SSH library overexpressed genes

Fold

chan

ge (t

arge

t gen

eAC

TB)

FOXO

3

GA

PDH

MYD

88

v1

MYD

88

v2

MYD

88

v3

MYD

88

v5

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

Fold

chan

ge (t

arge

t gen

eAC

TB)

0

1

2

3

4

5

Autophagy-related genes

Becli

n1

ATG12

Gab

arap

l1

PIK3

C3 LC3

lowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

(b)

Figure 3 Relative expression of genes identified from the SSH library MYD88 isoforms- and autophagy-related genes in esophageal cancertissues assessed using real-time PCR (a) Relative expression levels of genes that are overexpressed in esophageal tumor (FOXO3 GAPDHand MYD88 variants) Expression of MYD88v4 was not detectable in samples (b) Detection of autophagy in esophageal tumors Relativeexpression of autophagy-related genes (Beclin1 ATG12 Gabarapl1 LC3 and PIK3C3) in esophageal tumor tissues (e) represent the meanand whiskers the SEM of expression in samples (119899 = 10) lowast

119875

lt 005 lowastlowast119875

lt 001

Table 2 Correlations of FOXO3 MYD88 isoforms and GAPDH with autophagy-related genes

LC3 Gabarapl1 ATG12 PIK3C3 Beclin1FOXO3 0000 0007 0007 0000 0000MYD88v1 0000 0500 0007 0007 0000MYD88v2 0000 0000 0000 0000 0000MYD88v3 0000 0062 0000 0007 0000MYD88v5 0062 0062 0062 0062 0229GAPDH 0062 0500 0000 0000 0062Correlations were considered significant when 119875 lt 001

950900850800750700650

200000

150000

100000

50000

Der

ivat

ive r

epor

ter (minus

Rn998400)

Temperature (∘C)

MYD88v5Tm 8471∘C

MYD88v3Tm 8799∘C

MYD88v2

Tm 862∘C

MYD88v1

Tm 859∘C

MYD88v4Tm unknown

Figure 4 Melting curve analysis of MYD88 variants Peaks showeach variant and its melting temperature in ∘C Expression ofMYD88v4 was not detectable

5187-fold in esophageal cancer tissues FOXO3 overexpres-sion in cancer cells could be related to nutrient deprivationstress and other stimulatory factors that lead to autophagyinduction Elevated expression of the transcription factorFOXO3 induces autophagy protein expression and promotesthe induction of autophagy

MYD88 is another overexpressed gene in the con-structed library MYD88 is a cytosolic adaptor protein in theinterleukin-1 (IL-1) and Toll-like receptor (TLR) signalingpathways which have critical roles in innate and adaptiveimmunity [18] Previous studies demonstrated that MYD88has an important function in tumorigenesis and high levelsof MYD88 correlate with poor prognosis in patients withcolorectal cancers [19] Lipopolysaccharides a TLR receptorligand upregulates 120573-1 integrin which leads to tumor cell-endothelial cell adhesion tumor cell-extracellular matrixadhesion and tumor cell invasion of the extracellular matrix[20] In addition MYD88 promotes tumor cell proliferationvia NF-120581B activation Previous studies demonstrated a directinteraction between MYD88 and Beclin1 Interaction of


MYD88 with Beclin1 reduces Beclin1 binding to Bcl-2 whichleads to autophagy induction in cancer cells [21] Our resultsrevealed for the first time that MYD88 variants are upreg-ulated in esophageal cancer tissues compared with normaltissues Our findings indicated that MYD88 overexpressioncould lead to autophagy in esophageal cancer The cause ofMYD88 overexpression in esophageal cancer tissues likelyoriginates from the extracellular tumor matrix where thepresence of mucin could trigger prolonged activation of TLRreceptors and subsequent upregulation of MYD88 Com-pared with normal esophageal tissues there was a significantreduction in MYD88v3 levels and a significant increase inMYD88v1 levels in esophageal tumor tissues Interaction ofMYD88 with Beclin1 has been previously demonstrated butno information is available concerning the participation ofindividual exons in this interaction thus the role of each ofthe MYD88 variants in the interaction with Beclin1 and thesubsequent induction of autophagy remains to be elucidated

GAPDH is a key protein in the production of energyOverexpression of GAPDH has been reported in cancersand the primary role for GAPDH was proposed to beits pivotal function in glycolysis However cooperation ofGAPDH with ATG12 could induce autophagy instead ofcaspase-independent cell death (CICD) [22] After apoptoticcytochrome c release without caspase activation whichleads to CICD GAPDH elevates ATP levels in the cells byinducing glycolysis and cooperates with ATG12 to shift thecells away from CICD and toward autophagy In our SSHlibrary GAPDH was identified as an overexpressed genewith a 4659-fold induction Nutrient deprivation hypoxiaand high levels of cell division demand additional energyresources and GAPDH overexpression could alleviate thesedemands by inducing both glycolysis and autophagy inesophageal cancer cells

Because this is the first report of the overexpression of theFOXO3 GAPDH and MYD88 genes in esophageal cancercells the precise role of these genes in esophageal cancerremains to be elucidated Significant correlations betweenthe expression levels of these genes and those of autophagy-related genes point to a potential role for these genes in theinduction of autophagy Prolonged autophagy induction intumor tissues and degradation of autophagy-related proteinsvia autophagosome formation leads to the upregulation ofautophagy-related genes in esophageal cancer tissues FOXO3overexpression correlated with expression of the Gabarapl1ATG12 PIK3C3 LC3 and Beclin1 genes in cancer tissues Inlight of its transcriptional activity and regulatory effects onthe process of autophagy in skeletal muscle FOXO3 couldincrease the expression level of autophagy-related genesin esophageal cancer tissues [15] A significant correlationwas observed between the expression of GAPDH and theautophagy-related genes ATG12 and PIK3C3 in the tissuesamples This correlation could be related to the autophagy-induction activity of GAPDH which causes a subsequentupregulation of autophagy-related genes Overexpression ofMYD88v1 MYD88v2 and MYD88v3 correlated with upreg-ulation of the autophagy-related genes Beclin1 ATG12LC3 and PIK3C3 This correlation could be related to the

induction of autophagy via TLR receptors which leads toMYD88 upregulation

The results of this study suggest that the induction ofautophagy in cancerous tissues is an important event inthe molecular biology and tumorigenesis of esophageal can-cer Tumor cells experience metabolic stress and starvationcaused by increased proliferation and cell metabolism andhypoxia caused by inadequate angiogenesis in which ATPproduction is inefficientThese environmental stresses induceautophagy in tumor cells located far from the blood vessels[23] Autophagy decreases the energy consumption of tumorcells by reducing metabolic stress and eliminating cellularorganelles that limit damage Autophagy has previously beenstudied as a target for the development of new therapiesfor esophageal cancer because inhibition of autophagy hasthe potential to sensitize tumor cells including chemore-sistant cells to therapeutic agents [24] To date autophagyhas not been considered a fundamental molecular eventin esophageal cancer and the role of autophagy in thetumorigenesis of this cancer has not been addressed There-fore future studies that focus on autophagy as a target forcancer therapeutics are necessary Because of intraepithelialneoplasia importance in tumorigenesis of esophageal canceroccurrence of autophagy and expression of FOXO3 MYD88and GAPDH genes should be studied in this stage Thiscould reveal the role of the above genes in the early stage oftumorigenesis

In conclusion using the SSH method our study revealedfor the first time that the FOXO3 MYD88 and GAPDHgenes are overexpressed in esophageal cancer In additionthere was a significant correlation between overexpressionof these genes and upregulation of autophagy-related genesin esophageal cancer Considering the fundamental role ofautophagy in tumorigenesis and the high expression levels ofFOXO3 MYD88 and GAPDH in esophageal cancer furtherstudies are needed to evaluate the roles of these genes andautophagy in the development of esophageal cancer In addi-tion autophagy could be responsible for the poor prognosisand chemoresistance that are observed in esophageal cancerpatients The overexpression of the FOXO3 MYD88 andGAPDHgenes could lead to the induction of autophagy genesin esophageal cancer Thus these genes are candidates fortargeting autophagy and tumorigenesis in esophageal cancer

Conflict of Interests

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

Acknowledgment

This study was supported by Grant no 24141 from ResearchCouncil of the Tehran University of Medical Sciences(TUMS)

References

[1] J Kmet and E Mahboubi ldquoEsophageal cancer in the Caspianlittoral of Iran initial studiesrdquo Science vol 175 no 4024 pp846ndash853 1972


[2] F Bray J-S Ren E Masuyer and J Ferlay ldquoGlobal estimates ofcancer prevalence for 27 sites in the adult population in 2008rdquoInternational Journal of Cancer vol 132 pp 1133ndash1145 2013

[3] T Anayama M Furihata T Takeuchi et al ldquoInsufficient effectof p27(KIP1) to inhibit cyclin D1 in human esophageal cancer invitrordquo International Journal of Oncology vol 18 no 1 pp 151ndash155 2001

[4] U Ribeiro S D Finkelstein A V Safatle-Ribeiro et al ldquop53sequence analysis predicts treatment response and outcome ofpatients with esophageal carcinomardquo Cancer vol 83 pp 7ndash181998

[5] R Ralhan S Arora T K Chattopadhyay N K Shukla and MMathur ldquoCirculating p53 antibodies p53 gene mutational pro-file and product accumulation in esophageal squamous-cell car-cinoma in Indiardquo International Journal of Cancer vol 85 pp791ndash795 2000

[6] M E Nita H Nagawa O Tominaga et al ldquop21Waf1Cip1 ex-pression is a prognostic marker in curatively resected eso-phageal squamous cell carcinoma but not p27Kip1 p53 or RbrdquoAnnals of Surgical Oncology vol 6 no 5 pp 481ndash488 1999

[7] P Maycotte and A Thorburn ldquoAutophagy and cancer therapyrdquoCancer Biology andTherapy vol 11 no 2 pp 127ndash137 2011

[8] L Moretti E S Yang K W Kim and B Lu ldquoAutophagy sig-naling in cancer and its potential as novel target to improveanticancer therapyrdquoDrug ResistanceUpdates vol 10 no 4-5 pp135ndash143 2007

[9] N Chen and J Debnath ldquoAutophagy and tumorigenesisrdquo FEBSLetters vol 584 no 7 pp 1427ndash1435 2010

[10] L Yang Y Yu R Kang et al ldquoUp-regulated autophagy by en-dogenous highmobility group box-1 promotes chemoresistancein leukemia cellsrdquo Leukemia and Lymphoma vol 53 no 2 pp315ndash322 2012

[11] D J Klionsky and S D Emr ldquoAutophagy as a regulated pathwayof cellular degradationrdquo Science vol 290 no 5497 pp 1717ndash17212000

[12] D Glick S Barth and K F Macleod ldquoAutophagy cellular andmolecular mechanismsrdquo Journal of Pathology vol 221 no 1 pp3ndash12 2010

[13] Y Kondo T Kanzawa R Sawaya and S Kondo ldquoThe roleof autophagy in cancer development and response to therapyrdquoNature Reviews Cancer vol 5 no 9 pp 726ndash734 2005

[14] Y Kondo and S Kondo ldquoAutophagy and cancer therapyrdquoAutophagy vol 2 no 2 pp 85ndash90 2006

[15] C Mammucari G Milan V Romanello et al ldquoFoxO3 controlsautophagy in skeletal muscle in vivordquo Cell Metabolism vol 6no 6 pp 458ndash471 2007

[16] J Zhao J J Brault A Schild et al ldquoFoxO3 coordinately activatesprotein degradation by the autophagiclysosomal and proteaso-mal pathways in atrophying muscle cellsrdquo Cell Metabolism vol6 no 6 pp 472ndash483 2007

[17] J Zhou W Liao J Yang et al ldquoFOXO3 induces FOXO1-de-pendent autophagy by activating the AKT1 signaling pathwayrdquoAutophagy vol 8 pp 1712ndash1723 2012

[18] R Medzhitov P Preston-Hurlburt E Kopp et al ldquoMyD88 isan adaptor protein in the hTollIL-1 receptor family signalingpathwaysrdquoMolecular Cell vol 2 no 2 pp 253ndash258 1998

[19] E L Wang Z R Qian M Nakasono et al ldquoHigh expressionof Toll-like receptor 4myeloid differentiation factor 88 signalscorrelates with poor prognosis in colorectal cancerrdquo BritishJournal of Cancer vol 102 no 5 pp 908ndash915 2010

[20] J H Wang B J Manning Q D Wu S Blankson D Bouchier-Hayes and H P Redmond ldquoEndotoxinlipopolysaccharideactivates NF-120581b and enhances tumor cell adhesion and inva-sion through a 1205731 integrin-dependent mechanismrdquo Journal ofImmunology vol 170 no 2 pp 795ndash804 2003

[21] C-S Shi and J H Kehrl ldquoMyD88 and Trif target Beclin 1 totrigger autophagy in macrophagesrdquo Journal of Biological Chem-istry vol 283 no 48 pp 33175ndash33182 2008

[22] A Colell J-E Ricci S Tait et al ldquoGAPDH and autophagy pre-serve survival after apoptotic cytochrome c release in theabsence of caspase activationrdquo Cell vol 129 no 5 pp 983ndash9972007

[23] S Jin and E White ldquoRole of autophagy in cancer managementof metabolic stressrdquo Autophagy vol 3 no 1 pp 28ndash31 2007

[24] T R OrsquoDonovan G C OrsquoSullivan and S L McKenna ldquoInduc-tion of autophagy by drug-resistant esophageal cancer cellspromotes their survival and recovery following treatment withchemotherapeuticsrdquo Autophagy vol 7 no 5 pp 509ndash524 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


tumorigenesis [7ndash10] In this study suppression subtrac-tive hybridization (SSH) was used to identify genes thatare overexpressed in esophageal cancer cells Among theidentified ESTs from the constructed SSH library potentialautophagy-inducing genes (FOXO3 MYD88 and GAPDH)were selected for further analysisThe incidence of autophagywas also determined in clinical tissue samples by quantifyingautophagy-relatedregulatory genesThis study provides newinformation concerning the genes associated with the devel-opment of esophageal cancer particularly those involved inautophagy which could have a significant influence on thediagnosis and treatment of this type of cancer which typicallyhas a poor prognosis

2 Materials and Methods

21 Tissue Collection The samples used for the SSH weresurgically resected using esophagectomy from a patient priorto chemotherapy Normal tissue was resected 10 cm far fromthe tumor A pathologist dissected the target tissues under themicroscope with special care for minimal contamination ofnonepithelial cells For confirmation of overexpressed genesby qRT-PCR 10 samples from 5 patients (5 tumors and5 normal tissues from the same patients) were collectedusing endoscopy Target tissues were immersed in 10 volumesof an RNAlater solution (Ambion Austin TX USA) Thesamples were stored at minus80∘C freezer The resected tissueswere examined using hematoxylin-eosin (HampE) stainingTheconsent form was approved by the Biologic Sampling EthicsCommittee of the Tehran University of Medical Sciences andobtained from patients prior to sampling

22 Extraction of Total RNA Tissues were lysed using amortar and pestle and liquid nitrogen TwomL of Tripureisolation reagent (Roche Applied Science Indianapolis INUSA) was addedThe lysed tissues were passed through a 20-gauge needle 10 times for homogenizationTheprocedurewasperformed according to the manufacturerrsquos instructions Theconcentration and purity of the total RNA were determinedusing a BioPhotometer (Eppendorf Hamburg Germany)RNA quality was assessed using a 1 denatured agarose gel

23 Isolation of mRNA mRNA was isolated using the Dyn-aBeads mRNA Isolation Kit (Dynal Lake Success NY USA)BrieflyDynaBeads oligo (dT)

25were equilibratedwith 100120583L

of binding buffer (100mM Tris-HCl 500 nM LiCl 10mMEDTA 1 LiDS and 5mM DTT) The extracted total RNAwas diluted to 100 120583L in binding buffer and the equilibratedDynaBeads were added and incubated for 5min at 37∘Cin a shaking incubator The beads were then placed on amagnet the supernatant was removed and the beads werewashed twice using 200 120583L of washing buffer (10mM Tris-HCl 015M LiCl and 1mM of EDTA) Elution buffer (10 120583L)was added to the beads prior to incubation at 65∘C for 2minThe beads were then immediately placed on the magnet andthe eluted mRNA was isolated To improve the reduction ofbackground rRNA this protocol was repeated on the eluted

mRNA The purity of the purified mRNA was assessed usinga 1 denatured agarose gel

24 Construction of the SSH Library When constructing asubtracted library it is routine procedure to use a singlesample pair SSH library construction was achieved using thePCR-Select cDNA subtraction kit (Clontech Palo alto CAUSA) according to the manufacturerrsquos instructions Briefly2 120583g mRNA from normal (as Driver) and tumor (as Tester)tissues was applied for the first and second cDNA synthesisThe resulting cDNA was digested with RsaI and the blunt-ended double-stranded cDNAwas purifiedTheTester cDNAwas divided into two parts which were individually ligated toadaptor 1 and adaptor 2 The adaptor 1- and adaptor 2-ligatedcDNAs were separately hybridized with excess amountsof Driver cDNA for 8 hr at 68∘C The two hybridizationmixtures were combined and hybridizedwith excess amountsof Driver cDNA overnight at 68∘C The final hybridizationmixture was diluted using 200 120583L of dilution buffer for twosubsequent rounds of PCR The constructed SSH librarieswere confirmed using 1 agarose gel electrophoresis

25 Cloning and Similarity Searches The constructed SSHlibraries were purified using the PCR Product PurificationKit (Roche Applied Science) and the purified PCR productswere ligated into the pUC19 cloning vector and transformedinto Escherichia coli NovaBlue competent cells (NovagenMadison WI USA) Positive clones were verified usingthe colony PCR method with the adaptor-specific primersN1 (51015840-TCGAGCGGCCGCCCGGGCAGGT-31015840) and N2R(51015840-AGCGTGGTCGCGGCCGAGGT-31015840) and the plasmidswere isolated for sequencing using the High Pure Plas-mid Isolation Kit (Roche Applied Sciences) Single direc-tion DNA sequencing was performed using the BigDyeterminator v31 sequencing kit and a 3730xl automatedsequencer (Applied Biosystems Foster City CA USA)Similarity searches were performed using the BLASTntBLASTn and tBLASTx algorithms in the NCBI GenBankdatabases (httpblastncbinlmnihgovBlastcgi) to analyzethe sequences

26 Analysis of the Subtraction Efficiency The efficiency ofthe subtraction method was analyzed by comparing theabundance of a nondifferentially expressed housekeepinggene (eg beta actin) before and after subtraction Brieflythe subtracted and nonsubtracted samples were diluted 10-fold in H

2O as a template for PCR Real-time PCR reactions

were prepared by adding 10 120583L of SYBR Premix Ex Taq(Takara Kusatsu Japan) 1 120583L of sample 08 pmol of the betaactin primers 04120583L of ROX dye and DEPC-treated waterto a final volume of 20120583L The thermal cycling parametersfor the reaction consisted of an initial heating of 10min at95∘C followed by 40 cycles of 30 sec at 95∘C and 1min at60∘C Melting curve analysis was performed by increasingthe temperature from 65∘C to 95∘C in 01∘Csecond incre-ments for each fluorescence reading using the Step-One-Plusapparatus (Applied Biosystems) The subtraction efficiency









3 Results






Table1Designedprim

erssequences

used

toqu

antifygene

expressio

nby

real-timeP

CR

Application

Prim

ername

Accessionnu

mber

Leng

thSequ

ence

(51015840

to31015840)

Ann

ealin

gtemperature

Locatio

nProd

uctsize

Overexpressed

genes

FOXO

3-F

NM

0014553

20AC

GGTG

TTCG

GAC

CTTC

ATC

60∘

C1613ndash1632

196b

pFO

XO3-R

NM

0014553

20TG

CTGGCC

TGAG

ACAT

CAAG

1789ndash1808

GAPD

H-F

NM

002046

420

CGAC

AGTC

AGCC

GCA

TCTT

C61∘

C118

ndash137

250b

pGAPD

H-R

NM

002046

420

CGTT

CTCA

GCC

TTGAC

GGTG

348ndash367

MYD

88-v1-F

NM

00117

25671

20TC

ATCG

AAAAG

AGGTT

GGCT

57∘

C855ndash875

158b

pMYD

88-v1-R

NM

00117

25671

20GAT

GGGGAT

CAGTC

GCT

TCT

993ndash1012

MYD

88-v2-F

NM

0024684

20CC

ACAC

TTGAT

GAC

CCCC

TG61∘

C66

6ndash685

210b

pMYD

88-v2-R

NM

0024684

20GGCG

GCA

CCTC

TTTT

CGAT

G856ndash

875

MYD

88-v3-F

NM

00117

25681

20GGGAC

CCAG

CATT

GGGCA

TA61∘

C538ndash557

289b

pMYD

88-v3-R

NM

00117

25681

20AC

CTGGAG

AGAG

GCT

GAG

TG807ndash826

MYD

88-v4-F

NM

00117

25691

20CT

TGAT

GAC

CCCC

TGGGTG

C62∘

C671ndash690

207b

pMYD

88-v4-R

NM

00117

25691

20GGTT

GGTG

TAGTC

GCA

GAC

A858ndash877

MYD

88-v5-F

NM

00117

25661

17AC

CCAG

CATT

GGTG

CCG

60∘

C541ndash557

202b

pMYD

88-v5-R

NM

00117

2566

120

GGTT

GGTG

TAGTC

GCA

GAC

A723ndash742

Con

trolgene

ACTB

-FNM

0011013

21AT

GGCC

ACGGCT

GCT

TCCA

GC

60∘

C763ndash783

322b

pAC

TB-R

NM

0011013

21CA

GGAG

GAG

CAAT

GAT

CTTG

A1064

ndash1084

Autoph

agy-relatedgenes

Gabarapl1-F

NM

0314122

20CA

GGGTC

CCCG

TGAT

TGTA

G61∘

C321ndash340

179b

pGabarapl1-R

NM

0314122

20TG

GGAG

GGAT

GGTG

TTGTT

G480ndash

499

Beclin1-F

NM

003766

320

GCT

GAAG

ACAG

AGCG

ATGGT

595∘

C30ndash4

9169b

pBe

clin1-R

NM

003766

320

CATG

GTG

CTGTT

GTT

GGAC

G179ndash

198

PIK3

C3-F

NM

0026472

20GGCA

CACA

GAG

TGAG

CAGTA

595∘

C2423ndash244

2204b

pPIK3

C3-R

NM

0026472

20CA

CAGCC

TCTT

CATC

CGAC

A2607ndash2626

ATG12-F

NM

0047073

20TG

CTGGAG

GGGAAG

GAC

TTA

595∘

C347ndash366

186b

pAT

G12-R

NM

0047073

20GTT

CGCT

CTAC

TGCC

CACT

T513ndash532

MAP1LC

3B-F

NM

0228184

20GCC

GCC

TTTT

TGGGTA

GAAG

595∘

C1012ndash1031

225b

pMAP1LC

3B-F

NM

0228184

20TA

CCCC

TGCA

AGAG

TGAG

GA

1217ndash1236




551 552


686 687 891 892 983 984 2874Exon 3 Exon 4 Exon 5



551


552 732 733 824 825 2715Exon 3 Exon 4 Exon 5



551 552


686 687 778 779 2669Exon 4 Exon 5



551


552 643 644 2534Exon 4 Exon 5



551 552


686 687 867 868 959 960 2850Exon 3a Exon 4 Exon 5

MYD88 variant 1

MYD88 variant 2

MYD88 variant 3

MYD88 variant 4

MYD88 variant 5

NM 0011725671

NM 0024684

NM 0011725681

NM 0011725691

NM 0011725661


DN

Ala

dder

Forw

ard

libra

ry

1000 bp

250bp

(a)


02

04

06

08

10

12

Fold

chan

ge

1

0103

(b)



4 Discussion






0

5

10

15

20


Fold

chan

ge (t

arge

t gen

eAC

TB)

FOXO

3

GA

PDH

MYD

88

v1

MYD

88

v2

MYD

88

v3

MYD

88

v5

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

Fold

chan

ge (t

arge

t gen

eAC

TB)

0

1

2

3

4

5


Becli

n1

ATG12

Gab

arap

l1

PIK3

C3 LC3

lowast

lowastlowast


lowastlowast

(b)


119875


lt 001



950900850800750700650

200000

150000

100000

50000

Der

ivat

ive r

epor

ter (minus

Rn998400)

Temperature (∘C)

MYD88v5Tm 8471∘C

MYD88v3Tm 8799∘C

MYD88v2

Tm 862∘C

MYD88v1

Tm 859∘C

MYD88v4Tm unknown













Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results






Table1Designedprim

erssequences

used

toqu

antifygene

expressio

nby

real-timeP

CR

Application

Prim

ername

Accessionnu

mber

Leng

thSequ

ence

(51015840

to31015840)

Ann

ealin

gtemperature

Locatio

nProd

uctsize

Overexpressed

genes

FOXO

3-F

NM

0014553

20AC

GGTG

TTCG

GAC

CTTC

ATC

60∘

C1613ndash1632

196b

pFO

XO3-R

NM

0014553

20TG

CTGGCC

TGAG

ACAT

CAAG

1789ndash1808

GAPD

H-F

NM

002046

420

CGAC

AGTC

AGCC

GCA

TCTT

C61∘

C118

ndash137

250b

pGAPD

H-R

NM

002046

420

CGTT

CTCA

GCC

TTGAC

GGTG

348ndash367

MYD

88-v1-F

NM

00117

25671

20TC

ATCG

AAAAG

AGGTT

GGCT

57∘

C855ndash875

158b

pMYD

88-v1-R

NM

00117

25671

20GAT

GGGGAT

CAGTC

GCT

TCT

993ndash1012

MYD

88-v2-F

NM

0024684

20CC

ACAC

TTGAT

GAC

CCCC

TG61∘

C66

6ndash685

210b

pMYD

88-v2-R

NM

0024684

20GGCG

GCA

CCTC

TTTT

CGAT

G856ndash

875

MYD

88-v3-F

NM

00117

25681

20GGGAC

CCAG

CATT

GGGCA

TA61∘

C538ndash557

289b

pMYD

88-v3-R

NM

00117

25681

20AC

CTGGAG

AGAG

GCT

GAG

TG807ndash826

MYD

88-v4-F

NM

00117

25691

20CT

TGAT

GAC

CCCC

TGGGTG

C62∘

C671ndash690

207b

pMYD

88-v4-R

NM

00117

25691

20GGTT

GGTG

TAGTC

GCA

GAC

A858ndash877

MYD

88-v5-F

NM

00117

25661

17AC

CCAG

CATT

GGTG

CCG

60∘

C541ndash557

202b

pMYD

88-v5-R

NM

00117

2566

120

GGTT

GGTG

TAGTC

GCA

GAC

A723ndash742

Con

trolgene

ACTB

-FNM

0011013

21AT

GGCC

ACGGCT

GCT

TCCA

GC

60∘

C763ndash783

322b

pAC

TB-R

NM

0011013

21CA

GGAG

GAG

CAAT

GAT

CTTG

A1064

ndash1084

Autoph

agy-relatedgenes

Gabarapl1-F

NM

0314122

20CA

GGGTC

CCCG

TGAT

TGTA

G61∘

C321ndash340

179b

pGabarapl1-R

NM

0314122

20TG

GGAG

GGAT

GGTG

TTGTT

G480ndash

499

Beclin1-F

NM

003766

320

GCT

GAAG

ACAG

AGCG

ATGGT

595∘

C30ndash4

9169b

pBe

clin1-R

NM

003766

320

CATG

GTG

CTGTT

GTT

GGAC

G179ndash

198

PIK3

C3-F

NM

0026472

20GGCA

CACA

GAG

TGAG

CAGTA

595∘

C2423ndash244

2204b

pPIK3

C3-R

NM

0026472

20CA

CAGCC

TCTT

CATC

CGAC

A2607ndash2626

ATG12-F

NM

0047073

20TG

CTGGAG

GGGAAG

GAC

TTA

595∘

C347ndash366

186b

pAT

G12-R

NM

0047073

20GTT

CGCT

CTAC

TGCC

CACT

T513ndash532

MAP1LC

3B-F

NM

0228184

20GCC

GCC

TTTT

TGGGTA

GAAG

595∘

C1012ndash1031

225b

pMAP1LC

3B-F

NM

0228184

20TA

CCCC

TGCA

AGAG

TGAG

GA

1217ndash1236




551 552


686 687 891 892 983 984 2874Exon 3 Exon 4 Exon 5



551


552 732 733 824 825 2715Exon 3 Exon 4 Exon 5



551 552


686 687 778 779 2669Exon 4 Exon 5



551


552 643 644 2534Exon 4 Exon 5



551 552


686 687 867 868 959 960 2850Exon 3a Exon 4 Exon 5

MYD88 variant 1

MYD88 variant 2

MYD88 variant 3

MYD88 variant 4

MYD88 variant 5

NM 0011725671

NM 0024684

NM 0011725681

NM 0011725691

NM 0011725661


DN

Ala

dder

Forw

ard

libra

ry

1000 bp

250bp

(a)


02

04

06

08

10

12

Fold

chan

ge

1

0103

(b)



4 Discussion






0

5

10

15

20


Fold

chan

ge (t

arge

t gen

eAC

TB)

FOXO

3

GA

PDH

MYD

88

v1

MYD

88

v2

MYD

88

v3

MYD

88

v5

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

Fold

chan

ge (t

arge

t gen

eAC

TB)

0

1

2

3

4

5


Becli

n1

ATG12

Gab

arap

l1

PIK3

C3 LC3

lowast

lowastlowast


lowastlowast

(b)


119875


lt 001



950900850800750700650

200000

150000

100000

50000

Der

ivat

ive r

epor

ter (minus

Rn998400)

Temperature (∘C)

MYD88v5Tm 8471∘C

MYD88v3Tm 8799∘C

MYD88v2

Tm 862∘C

MYD88v1

Tm 859∘C

MYD88v4Tm unknown













Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Designedprim

erssequences

used

toqu

antifygene

expressio

nby

real-timeP

CR

Application

Prim

ername

Accessionnu

mber

Leng

thSequ

ence

(51015840

to31015840)

Ann

ealin

gtemperature

Locatio

nProd

uctsize

Overexpressed

genes

FOXO

3-F

NM

0014553

20AC

GGTG

TTCG

GAC

CTTC

ATC

60∘

C1613ndash1632

196b

pFO

XO3-R

NM

0014553

20TG

CTGGCC

TGAG

ACAT

CAAG

1789ndash1808

GAPD

H-F

NM

002046

420

CGAC

AGTC

AGCC

GCA

TCTT

C61∘

C118

ndash137

250b

pGAPD

H-R

NM

002046

420

CGTT

CTCA

GCC

TTGAC

GGTG

348ndash367

MYD

88-v1-F

NM

00117

25671

20TC

ATCG

AAAAG

AGGTT

GGCT

57∘

C855ndash875

158b

pMYD

88-v1-R

NM

00117

25671

20GAT

GGGGAT

CAGTC

GCT

TCT

993ndash1012

MYD

88-v2-F

NM

0024684

20CC

ACAC

TTGAT

GAC

CCCC

TG61∘

C66

6ndash685

210b

pMYD

88-v2-R

NM

0024684

20GGCG

GCA

CCTC

TTTT

CGAT

G856ndash

875

MYD

88-v3-F

NM

00117

25681

20GGGAC

CCAG

CATT

GGGCA

TA61∘

C538ndash557

289b

pMYD

88-v3-R

NM

00117

25681

20AC

CTGGAG

AGAG

GCT

GAG

TG807ndash826

MYD

88-v4-F

NM

00117

25691

20CT

TGAT

GAC

CCCC

TGGGTG

C62∘

C671ndash690

207b

pMYD

88-v4-R

NM

00117

25691

20GGTT

GGTG

TAGTC

GCA

GAC

A858ndash877

MYD

88-v5-F

NM

00117

25661

17AC

CCAG

CATT

GGTG

CCG

60∘

C541ndash557

202b

pMYD

88-v5-R

NM

00117

2566

120

GGTT

GGTG

TAGTC

GCA

GAC

A723ndash742

Con

trolgene

ACTB

-FNM

0011013

21AT

GGCC

ACGGCT

GCT

TCCA

GC

60∘

C763ndash783

322b

pAC

TB-R

NM

0011013

21CA

GGAG

GAG

CAAT

GAT

CTTG

A1064

ndash1084

Autoph

agy-relatedgenes

Gabarapl1-F

NM

0314122

20CA

GGGTC

CCCG

TGAT

TGTA

G61∘

C321ndash340

179b

pGabarapl1-R

NM

0314122

20TG

GGAG

GGAT

GGTG

TTGTT

G480ndash

499

Beclin1-F

NM

003766

320

GCT

GAAG

ACAG

AGCG

ATGGT

595∘

C30ndash4

9169b

pBe

clin1-R

NM

003766

320

CATG

GTG

CTGTT

GTT

GGAC

G179ndash

198

PIK3

C3-F

NM

0026472

20GGCA

CACA

GAG

TGAG

CAGTA

595∘

C2423ndash244

2204b

pPIK3

C3-R

NM

0026472

20CA

CAGCC

TCTT

CATC

CGAC

A2607ndash2626

ATG12-F

NM

0047073

20TG

CTGGAG

GGGAAG

GAC

TTA

595∘

C347ndash366

186b

pAT

G12-R

NM

0047073

20GTT

CGCT

CTAC

TGCC

CACT

T513ndash532

MAP1LC

3B-F

NM

0228184

20GCC

GCC

TTTT

TGGGTA

GAAG

595∘

C1012ndash1031

225b

pMAP1LC

3B-F

NM

0228184

20TA

CCCC

TGCA

AGAG

TGAG

GA

1217ndash1236




551 552


686 687 891 892 983 984 2874Exon 3 Exon 4 Exon 5



551


552 732 733 824 825 2715Exon 3 Exon 4 Exon 5



551 552


686 687 778 779 2669Exon 4 Exon 5



551


552 643 644 2534Exon 4 Exon 5



551 552


686 687 867 868 959 960 2850Exon 3a Exon 4 Exon 5

MYD88 variant 1

MYD88 variant 2

MYD88 variant 3

MYD88 variant 4

MYD88 variant 5

NM 0011725671

NM 0024684

NM 0011725681

NM 0011725691

NM 0011725661


DN

Ala

dder

Forw

ard

libra

ry

1000 bp

250bp

(a)


02

04

06

08

10

12

Fold

chan

ge

1

0103

(b)



4 Discussion






0

5

10

15

20


Fold

chan

ge (t

arge

t gen

eAC

TB)

FOXO

3

GA

PDH

MYD

88

v1

MYD

88

v2

MYD

88

v3

MYD

88

v5

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

Fold

chan

ge (t

arge

t gen

eAC

TB)

0

1

2

3

4

5


Becli

n1

ATG12

Gab

arap

l1

PIK3

C3 LC3

lowast

lowastlowast


lowastlowast

(b)


119875


lt 001



950900850800750700650

200000

150000

100000

50000

Der

ivat

ive r

epor

ter (minus

Rn998400)

Temperature (∘C)

MYD88v5Tm 8471∘C

MYD88v3Tm 8799∘C

MYD88v2

Tm 862∘C

MYD88v1

Tm 859∘C

MYD88v4Tm unknown













Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















551 552


686 687 891 892 983 984 2874Exon 3 Exon 4 Exon 5



551


552 732 733 824 825 2715Exon 3 Exon 4 Exon 5



551 552


686 687 778 779 2669Exon 4 Exon 5



551


552 643 644 2534Exon 4 Exon 5



551 552


686 687 867 868 959 960 2850Exon 3a Exon 4 Exon 5

MYD88 variant 1

MYD88 variant 2

MYD88 variant 3

MYD88 variant 4

MYD88 variant 5

NM 0011725671

NM 0024684

NM 0011725681

NM 0011725691

NM 0011725661


DN

Ala

dder

Forw

ard

libra

ry

1000 bp

250bp

(a)


02

04

06

08

10

12

Fold

chan

ge

1

0103

(b)



4 Discussion






0

5

10

15

20


Fold

chan

ge (t

arge

t gen

eAC

TB)

FOXO

3

GA

PDH

MYD

88

v1

MYD

88

v2

MYD

88

v3

MYD

88

v5

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

Fold

chan

ge (t

arge

t gen

eAC

TB)

0

1

2

3

4

5


Becli

n1

ATG12

Gab

arap

l1

PIK3

C3 LC3

lowast

lowastlowast


lowastlowast

(b)


119875


lt 001



950900850800750700650

200000

150000

100000

50000

Der

ivat

ive r

epor

ter (minus

Rn998400)

Temperature (∘C)

MYD88v5Tm 8471∘C

MYD88v3Tm 8799∘C

MYD88v2

Tm 862∘C

MYD88v1

Tm 859∘C

MYD88v4Tm unknown













Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20


Fold

chan

ge (t

arge

t gen

eAC

TB)

FOXO

3

GA

PDH

MYD

88

v1

MYD

88

v2

MYD

88

v3

MYD

88

v5

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

Fold

chan

ge (t

arge

t gen

eAC

TB)

0

1

2

3

4

5


Becli

n1

ATG12

Gab

arap

l1

PIK3

C3 LC3

lowast

lowastlowast


lowastlowast

(b)


119875


lt 001



950900850800750700650

200000

150000

100000

50000

Der

ivat

ive r

epor

ter (minus

Rn998400)

Temperature (∘C)

MYD88v5Tm 8471∘C

MYD88v3Tm 8799∘C

MYD88v2

Tm 862∘C

MYD88v1

Tm 859∘C

MYD88v4Tm unknown













Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgment


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 overexpression of foxo3, myd88, and gapdh...

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