research article oncogene-mediated progressive silencing...

Research ArticleRas Oncogene-Mediated Progressive Silencing of ExtracellularSuperoxide Dismutase in Tumorigenesis

Francesca Cammarota1 Gabriella de Vita2 Marco Salvatore1 and Mikko O Laukkanen1

1 IRCCS SDN 80143 Naples Italy2Department of Molecular Medicine and Medical Biotechnologies University of Naples Federico II 80014 Naples Italy

Correspondence should be addressed to Mikko O Laukkanen mlaukkanensdn-napoliit

Received 15 June 2015 Accepted 31 August 2015

Academic Editor Max Costa

Copyright copy 2015 Francesca Cammarota et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Extracellular superoxide dismutase (SOD3) is a secreted enzyme that uses superoxide anion as a substrate in a dismutase reactionthat results in the formation of hydrogen peroxide Both of these reactive oxygen species affect growth signaling in cells AlthoughSOD3 has growth-supporting characteristics the expression of SOD3 is downregulated in epithelial cancer cells In the currentwork we studied the mechanisms regulating SOD3 expression in vitro using thyroid cell models representing different stages ofthyroid cancer We demonstrate that a low level of RAS activation increases SOD3 mRNA synthesis that then gradually decreaseswith increasing levels of RAS activation and the decreasing degree of differentiation of the cancer cells Our data indicate thatSOD3 regulation can be divided into two classes The first class involves RASndashdriven reversible regulation of SOD3 expression thatcan be mediated by the following mechanisms RAS GTPase regulatory genes that are responsible for SOD3 self-regulation RAS-stimulated p38 MAPK activation and RAS-activated increased expression of themir21microRNA which inversely correlates withsod3mRNA expression The second class involves permanent silencing of SOD3mediated by epigenetic DNA methylation in cellsthat represent more advanced cancersTherefore the work suggests that SOD3 belongs to the group of ras oncogene-silenced genes

1 Introduction

The regulation of extracellular superoxide dismutase (SOD3)gene expression has remained challenging Temporal stimu-lation of SOD3 expression could benefit tissue healing afterinjury [1ndash6] while long-term inhibition of overproductionof the enzyme could prevent unwanted hyperplasia [7]According to recent publications SOD3 supports a numberof growth-stimulating events in tumorigenesis Moderatesustained overexpression of SOD3 induces nontransformedmurine primary cell proliferation causing immortalizationand neoplastic transformation [7] In aggressive anaplasticcancer cells moderate sustained overexpression of SOD3stimulates tumorigenesis and growth factor signal trans-duction [7 8] Moreover SOD3 can control small GTPaseactivation and downstream signal transduction bymodifyingthe expression of small GTPase regulatory genes [8 9]

In tumorigenesis genes that are expressed in normalcells but are disadvantageous for cancer development by

inducing apoptosis [10] limiting cell proliferation [11] orcorrecting DNA damage [12] are downregulated throughgenetic events such as deletions [13] and point mutations[14] epigeneticmechanisms such asmethylation and histoneacetylation [15ndash17] and microRNA targeting affecting thegene transcription [18] Intriguingly SOD3 which has severalgrowth-supporting characteristics such as the ability topromote mitogenic and cell survival signaling is frequentlydownregulated in advanced epithelial cancer cells Thusthe role of SOD3 in tumorigenesis requires further studyTo systematically characterize the concomitant mechanismscausing SOD3 downregulation in epithelial cancer cellswe utilized rat and human thyroid cell models harboringdifferent oncogenes and representing variable degrees of dif-ferentiation Based on our observations sod3mRNA expres-sion is progressively altered corresponding to RAS GTPaseactivation and carcinogenesis thus further strengtheningour previous observations suggesting a close cooperativeconnection between SOD3 and RAS

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 780409 13 pageshttpdxdoiorg1011552015780409

2 BioMed Research International

2 Methods

21 Cells Rat thyroid PC Cl3 PC PTC1 PC E1A andFRLT5 and the related cell clones V13 V21 and V27 stablytransfected with an H-RasV12 expression plasmid [19] werecultured in Hamrsquos F12 medium Coonrsquos modified (Sigma StLouis MO USA) supplemented with 5 calf serum (LifeTechnologies Inc Carlsbad CA) penicillin (100UmL)(Sigma) and streptomycin (100mgL) (Sigma) PC Cl3 andFRLT5 cells (modeling normal thyroid cells) were addition-ally supplemented with 10 nM TSH 10 nM hydrocortisone100 nM insulin 5mgmL transferrin 5 nM somatostatin and20mgmL glycyl-histidyl-lysine Human thyroid NTHY-ori31 (Nthy) and anaplastic thyroid cancer 8505c cells weregrown in RPMI medium (Life Technologies Inc) supple-mented with 10 fetal bovine serum (Sigma) penicillin(100UmL) and streptomycin (100mgL) Human papillarythyroid cancer TPC1 cells were grown in DMEM supple-mented with 10 fetal bovine serum penicillin (100UmL)and streptomycin (100mgL) HEK 293T cells were grownin DMEM supplemented with 10 fetal bovine serumpenicillin (100UmL) and streptomycin (100mgL) To studythe regulation of sod3 gene expression the cells were sup-plemented with 10 120583M 5-azacytidine (Sigma) 100 nM TSA(Sigma) and 10120583M SB202190 p38 MAPK inhibitor (Sigma)for 24 hours or with H

2O2(Sigma) for 6 hours

The human SOD3 expression vector (a gift fromProfessorStefan L Marklund University of Umea Sweden) rabbitsod3 expression vector previously cloned in our group [20]or pcDNA3 control (Invitrogen Paisley UK) or H-RasV12plasmid was transfected with Fugene 6 (Promega WI USA)according to standard protocols

22 Real-TimeReverse Transcription PCR Cells were pelletedfor RNA isolation using an RNeasyMini Kit (Qiagen HildenGermany) and cDNA synthesis using a QuantiTect ReverseTranscription Kit (Qiagen)The PCR reaction was performedusing an iCycler (BioRad Hercules CA USA) and SYBRGreen PCR Master Mix (Applied Biosystems Foster CityCA) The primers were designed with (httpwwwncbinlmnihgovtoolsprimer-blast) Human 120573-actin and rat 120573-actinwere used to normalize human and rat templates respec-tively Relative fluorescence expression values were calculatedas follows ΔCt = Ct (gene of interest)-Ct (120573-actin) ΔΔCt =ΔCt (treated)-ΔCt (control) Relative expression values =2(minusΔΔCt) The primers are listed in Table 1

23 MicroRNA Isolation MicroRNA was isolated using anmiRNeasy Kit (Qiagen) for reverse transcription with amiScript RT II Kit (Qiagen) The miR21 and RNU5 miScriptPrimer Assays (Qiagen) were used for amplification and tonormalize themiR21 expression

24 Western Blot The cells were harvested in lysis buffer(50mM HEPES pH 75 150mM NaCl 10 glycerol 1 Tri-ton X-100 1mM EGTA 15mM MgCl

2 10mM NaF 10mM

sodium pyrophosphate 1mM Na3VO4 10 120583g aprotininmL

and 10 120583g leupeptinmL) separated by sodium dodecyl

Table 1 PCR primer sequences used in the study

Human SOD3 forward cttcgcctctgctgaagtctHuman SOD3 reverse gggtgtttcggtacaaatggRat sod3 forward gacctggagatctggatggaRat sod3 reverse gtggttggaggtgttctgctlowastRabbit sod3 forward gttgcgtgagcggaaagaRabbit sod3 reverse gtgagcgcctgccagatctcHuman SOS1 forward cacctcctcctcaaacacctHuman SOS1 reverse gtgtgtgtgctcccttttgtHuman SOS2 forward ttttgaagaacgggtggcagHuman SOS2 reverse ttttcctttcctgcagtgccHuman 120573-actin forward tgcgtgacattaaggagaagHuman 120573-actin reverse gctcgtagctcttctccaRat 120573-actin forward tcgtgcgtgacattaaggagRat 120573-actin reverse gtcaggcagctcgtagctctlowastPrimer designed against EF1alfa promoter

sulfatendashpolyacrylamide gel electrophoresis and transferredto Hybond C Extra nitrocellulose membranes (GE Health-careWI USA)The following antibodies were used pErk12Erk12 p-p38 MAPK and p38 MAPK (Cell Signaling Tech-nology MA USA)

25 Statistical Analysis The experiments were repeated atleast three times All of the results were expressed as themeanvalues plusmn SDThe 119901 values (lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt0001) were determined using two-tailed independent sample119905-tests

3 Results

31Thyroid Cell Models Representing Different Degrees ofThy-roid Cancer Thyroid stimulating hormone hydrocortisoneinsulin transferrin somatostatin and glycyl-histidyl-lysinesupplementation-dependent FRLT5 cells are a clonal cell linederived from rat normal thyroid FRLT cells by purificationthrough successive isolation of colonies [21] FRLT5 clonesV13 K20 K2 V21 V39 and V27 were created by stabletransfection with H-RasV12 oncogene followed by clonalexpansion of the cell lines derived from a single cellThe cloneV13 harbors 14-fold K20 31-fold K2 63-fold V21 10-foldV39 35-fold and V27 46-fold RAS activity relative to parentalcells This increased RAS activity drives the growth ofthe cells without hormone supplementation thus modelingthe hormone-independent growth characteristics of thyroidcancers [19] Similar to FRLT5 cells PC Cl3 cells representrat normal thyroid cells and are dependent on hormonesupplementation [22] PC PTC1 and PC E1A clones are PCCl3 derivatives that were transformed with PTC1 and E1Aoncogenes [23] PTC1 is a papillary thyroid cancer- (PTC-)associated oncogene derived from a genomic rearrangementof the RET oncogene with the CDC61H4 gene [24] The E1Aoncogene shows structural and functional similarities to theMYC and MYB oncogenes [25] that mechanistically bypass

BioMed Research International 3

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Figure 1 SOD3mRNA expression level in the cell model systems used in this study (a) SOD3 expression analysis in human papillary thyroidTPC1 and anaplastic thyroid 8505c cells showed significantly reducedmRNA expression compared to Nthy cells representing normal thyroid(b) The sod3mRNA expression from rat thyroid PC Cl3 cells modeling normal thyroid PC PTC1 transformed with the PTC1 oncogene andPC E1A cells transformed with the E1A oncogeneThe oncogenic transformation of PC Cl3 cells caused a downregulation of sod3mRNA (c)The expression analysis of sod3 from rat FRLT5 cell clones harboring 14-fold to 46-fold increased RAS activity compared to control FRLT5cells suggested significantly increased sod3 expression in the clones V13 (14-fold RAS activity) K20 (31-fold RAS activity) and K2 (63-foldRAS activity) A significant decrease in sod3 mRNA synthesis was observed in the clone V21 (10-fold RAS activity) that was further reducedin the clones V39 (35-fold RAS activity) and V27 (46-fold RAS activity) (d) The expression analysis of the microRNA mir21 from FRLT5cells Compared to control FRLT5 cells the expression levels of mir21 were similar in the clones V13 (14-fold RAS activity) K20 (31-foldRAS activity) and K2 (63-fold RAS activity) A significant increase in expression was observed in the clones V21 (10-fold RAS activity) V39(35-fold RAS activity) and V27 (46-fold RAS activity) The data are expressed as the mean plusmn SD The 119901 values are represented (lowast119901 lt 005lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001)

the proliferative block of terminally differentiated cells byincreasing the activity of cell cycle-related proteins [26]

To model normal human thyroid we utilized SV40-immortalized Nthy cells [27] which are able to grow withouthormone supplementation TPC1 cells which are sponta-neously transformed thyroid cancer cells isolated from apatient represent well-differentiated papillary thyroid cancerin this studyThe 8505c cell line originates from a patient withundifferentiated highly aggressive anaplastic thyroid cancerharboring mutant p53 [28] and a BRAF V600E mutationresulting in constitutively active MEK-ERK kinase signalingwith consequent uncontrolled cell proliferation and tumori-genesis [29] HEK 293T cells derived from human embryonickidney cells by adenovirus 5 transduction were used in thestudy to corroborate observations from thyroid cell model

systems because of their strong growth and untroublesometransfection characteristics

32 RASActivation Correlates with sod3 andmir21 ExpressionTo validate the cell models for the aims of the currentstudy we investigated the expression of SOD3mRNA SOD3expression was gradually downregulated in papillary thyroidcancer TPC1 cells and anaplastic thyroid cancer 8505c cellscompared to control Nthy cellsmodeling normal thyroid cells(Figure 1(a)) suggesting a correlation between the mRNAexpression and the degree of differentiation of the cancer[7 30]We observed a similar downregulation of sod3mRNAsynthesis in PC Cl3 cells transformed with other oncogenes(Figure 1(b)) confirming the oncogene-dependent regula-tion that has previously been observed [30] According to a


recent report transfection of H-RasV12 b-raf mek and erkinto cells stimulates SOD3 production [9] To investigate themechanism by which increased activation of RAS controlssod3 mRNA production we utilized rat thyroid FRLT5 cellline clones stably transfected with varying amounts of theH-RasV12 oncogene [19] Increased RAS activation of 14-fold to 63-fold stimulated sod3 expression whereas 10-foldRAS activation caused a striking decrease in the mRNAproduction that was further enhanced at 35-fold and 46-foldRAS activation levels (Figure 1(c))

A recent report has suggested that mir21 targets theSOD3 31015840UTR and results in RNAi-mediated silencing of thegene expression [31] We assessed mir21 expression in theFRLT5 and FRLT5-derived clones and observed increasedmicroRNA production that correlated with increased RASactivity [19] Low 14- to 63-fold increased RAS activationdid not increase mir21 production whereas a significantstrong induction was observed with 10-fold RAS activation(Figure 1(d))These data corroborate the connection betweensod3 andmir21 expression

33 RAS-Induced p38 MAPK Phosphorylation Inhibits sod3mRNA Expression A previous study has suggested thatactivation of p38 MAPK one of the RAS downstreamtarget cellular kinases contributes to the regulation of SOD3production [32] We therefore investigated the effect of thep38 MAPK inhibitor SB202190 on SOD3mRNA synthesis inthe thyroid cell models

Inhibition of the kinase had a minor impact on the SOD3gene expression in human Nthy cells (Figure 2(a)) whereasSOD3 expression was significantly increased in human TPC1papillary thyroid cancer cells and in human 8505c anaplasticthyroid cancer cells (Figures 2(b) and 2(c)) These data sug-gest that p38MAPKdownregulates SOD3mRNAsynthesis inadvanced thyroid cancer cells The expression data from PCCl3 cells PC PTC1 cells transformed with the papillary thy-roid cancer PTC oncogene and PC E1A cells immortalizedwith the aggressive adenovirus E1A oncogene (Figures 2(d)ndash2(f)) treated with the p38 inhibitor indicated similar upreg-ulation of sod3 expression suggesting oncogene-dependentsilencing of sod3 via p38 MAPK activation The oncogene-derived effect on p38 MAPK activation and sod3 downreg-ulation was then studied in FRLT5 cells In accord withthe other cell models used the kinase inhibitor minimallyaffected FRLT5 cells whereas a significant increase in mRNAproduction was observed in FRLT5 clones V13 V21 and V39harboring 14-fold 10-fold and 35-fold RAS activity (Figures2(g)ndash2(j)) Thus the data corroborate previous observations[32] suggesting that activation of RAS through p38 MAPKdownregulates sod3 expression whereas inhibition of p38MAPK phosphorylation increases sod3mRNA production

We then analyzed the p38 MAPK phosphorylation statusin FRLT5 cells and related clones The analysis showedsignificantly increased phosphorylation levels in the clonesV13 V21 and V39 corresponding to RAS activation hencecorroborating p38 MAPK inhibitor results and suggest-ing that the RAS-p38 MAPK signal transduction pathwaycontrols sod3 mRNA synthesis (Figures 2(k) and 2(m))

The involvement of the RAS-p38 MAPK signaling pathwaywas further confirmed by transient transfection of HEK293T cells with increasing concentrations of the H-RasV12oncogene the increase in H-RasV12 levels correlated withincreased p38 MAPK phosphorylation (Figures 2(l) and2(n))

34 Cancer-Induced Hypermethylation Downregulates sod3mRNA Expression The coding sequence of the sod3 gene islocated in a hypermethylated CpG island [33] which suggeststhat methylation is involved in the regulation of the geneexpression Treatment with the hypomethylation agent 5-azacytidine did not affect SOD3mRNA expression in humanNthy cells rat PC Cl3 cells rat FRLT5 cells or FRLT5 cloneV13 containing a modest 14-fold increased RAS activationlevel (Figures 3(a) 3(d) 3(g) and 3(h)) these results areconsistent with our previous data showing no impact ofhypomethylation on sod3 expression in aortic smoothmusclecells representing normal vascular tissue [33] However the5-azacytidine treatment significantly increased SOD3 expres-sion in the human papillary thyroid cancer TPC1 and humananaplastic thyroid cancer 8505c cells (Figures 3(b) and 3(c))Similarly rat thyroid PC PTC1 and PC E1A cells (Figures3(e) and 3(f)) and FRLT5 cell clones V21 and V39 with10-fold and 35-fold increased RAS activation respectivelydemonstrated increased sod3mRNA expression (Figures 3(i)and 3(j)) The results thus suggest that methylation-mediatedSOD3 downregulation in cancer corresponds to the degree ofdifferentiation of the cancer and to the activation level of theRAS small GTPase

35 Histone Acetylation Affects sod3mRNAExpression in CellsThat Model Normal Thyroid Tissue Histone deacetylasesregulate the expression of genes involved in the initiation oftumorigenesis Deacetylation creates a tight nonpermissivechromatin conformation that inhibits transcription The his-tone deacetylation inhibitor trichostatin A (TSA) interfereswith the histone deacetylase function of removing the acetylgroups from the histones therefore increasing the histoneacetylation level and resulting in the formation of an openeuchromatin structure Treatment with TSA significantly(119901 lt 0001) decreased sod3 mRNA expression in all ofthe thyroid cells that model normal thyroid (Figures 4(a)4(d) and 4(g)) while only a tendency towards increasedexpression was observed in transformed and cancer cells(Figure 4)

36 Self-Regulation of sod3 Expression Is Controlled by RASSmall GTPase Regulatory Genes Our previous results havedemonstrated SOD3-driven increased RAS GTP loading andRAS-ERK12-derived sod3mRNA expression therefore sug-gesting a self-regulatory loop for SOD3 [9] This observationwas further corroborated by data indicating that SOD3 causesincreased growth signaling at moderate expression levels [7]and inhibits activation of the small GTPases RAS RACCDC42 and RHO at high enzyme levels [8] To evaluatethe concentration-dependent self-regulation of SOD3 wetransfected 025 120583g and 10 120583g of human SOD3 into the rat


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Figure 2 Effect of the p38 MAPK inhibitor SB202190 on SOD3 mRNA expression (andashc) The inhibition of p38 MAPK phosphorylationincreased the SOD3 expression in human TPC1 and 8505c cells while SB202190 treatment did not alter the SOD3 mRNA synthesis in Nthycells modeling normal thyroid (dndashf) The inhibitor treatment increased sod3 mRNA expression in PC Cl3-derived PC PTC1 and PC E1Acells while the treatment had no effect on PC Cl3 control cells modeling normal thyroid (gndashj) FRLT5 cell clones V13 V21 and V39 stablytransfected with RAS showed a significant increase in sod3 mRNA expression after SB202190 treatment whereas SB202190 had no effecton FRLT5 control cells which is consistent with the results using the other cell models (k m) The Western blot analysis for p38 MAPKactivation in FRLT5 control cells clone V13 clone V21 and clone V39 The histogram (panel (m)) suggested significantly increased p38MAPK phosphorylation in FRLT5-derived clones compared to control cells Total p38 MAPK was used to normalize the phosphorylationlevel (l n) The transient transfection of an H-RasV12 expression plasmid into HEK 293T cells showed a gradual increase in p38 MAPKphosphorylation that corresponded to the amount of plasmid transfected Total p38MAPK was used to normalize the phosphorylation levelThe data are expressed as the mean plusmn SD The 119901 values are represented (lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001)

thyroid PC E1A cell line which expresses low levels ofendogenous sod3 mRNA Transfection with low amounts ofhuman SOD3 significantly increased the rat sod3 expression(Figure 5(a)) whereas transfection with high amounts ofSOD3had the opposite effect significantly decreasing rat sod3mRNA synthesis (Figure 5(b)) These data were supportedby Western blot analysis suggesting a SOD3 dose-dependentactivation of ERK12 kinase in PC E1A cells (Figures 5(c)and 5(d)) We have previously demonstrated that ERK12stimulates endogenous SOD3 expression [9] To corroboratethe dose-dependent self-regulation we transfected rabbitsod3 into human HEK 293T cells and analyzed species-specific mRNA production on consecutive days While therabbit sod3 transgene expression decreased with increasingtime from transfection the endogenous human SOD3mRNAproduction increased to maintain the total rabbithumansod3 mRNA expression at similar levels on different days(Figure 5(e)) Because the only known physiological functionof SOD3 is to dismutase superoxide to hydrogen peroxide weinvestigated the effect of hydrogen peroxide on SOD3mRNAsynthesis Treatment with low levels of H

2O2increased the

endogenous SOD3 expression while treatment with highlevels of H

2O2(1mM and 10mM) significantly decreased the

mRNAproduction (Figure 5(f)) these data support the dose-dependent self-regulation data

We have recently demonstrated that SOD3 activatesa number of tyrosine kinase receptors cell membrane-associated nontyrosine kinases and cellular kinases [8 9 34]Based on our recent data SOD3 regulates mitogen signaltransduction by controlling the expression of small GTPaseregulatory genes including guanine nucleotide exchange

factors (GEF) GTPase activating proteins (GAP) and guano-sine nucleotide disassociation inhibitors (GDI) thus regu-lating the activation level of RAS [8] In the present workwe investigated in 8505c cells the effects of low and highSOD3 expression levels on the guanine nucleotide exchangefactor genes SOS1 and SOS2 which are responsible for RASGTP loadingThe data suggested that SOS1 and SOS2mRNAexpression is higher in cells that harbor low SOD3 mRNAcontent compared to cells with high SOD3 mRNA synthesis(Figures 5(g) and 5(h)) The data are in agreement with ourprevious results in which we have shown increased growthand increased RhoGEF16 and RalGEF RGL1 expression withmoderately increased SOD3 mRNA levels whereas highSOD3 expression led to decreased growth and RhoGEF16and RalGEF RGL1 expression [8] Hence the results suggestthat SOD3 self-regulation is coordinated through guaninenucleotide exchange factors that are responsible for RASactivation

4 Discussion

The signal transduction-related events that stimulate orinhibit SOD3 expression have previously been described andsuggest the involvement of cytokines [35 36] epigeneticregulation [37 38] MAP kinases [9 32] and the microRNAmir21 [31] While the expression of the SOD3 enzyme isrelatively stable in normal tissues a significant increase insod3 mRNA levels has been observed in rat thyroid benigntumors [30] with sequential downregulation of expression incarcinogenesis that corresponds to the degree of differentia-tion of the cells [30] To characterize the cellular mechanisms


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Figure 3 Effect of the demethylation reagent 5-azacytidine on SOD3 mRNA expression (andashc) Nthy cells modeling normal human thyroidcells showed similar SOD3 mRNA expression in nontreated and treated control cells whereas the mRNA production was significantlyincreased in 5-azacytidine-treated papillary TPC1 and anaplastic 8505c thyroid cancer cells (dndashf) Similar to the human cells no alterationin sod3 mRNA was observed in rat PC Cl3 cells modeling normal rat thyroid cells after hypomethylation treatment whereas the treatmentinduced increased mRNA synthesis in PTC1 and E1A oncogene-transfected cells (gndashj)The hypomethylation treatment did not affect normalrat thyroid FRLT5 cells or FRLT5 clone V13 cells harboring low 14-fold RAS activity whereas a significant increase in the sod3 mRNAproduction was observed in clones V21 and V39 harboring 10-fold and 35-fold RAS activity respectively The data are expressed as the meanplusmn SD The 119901 values are represented (lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001)


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(j)

Figure 4 Effect of the histone deacetylation inhibitor trichostatin A (TSA) on SOD3mRNA expression (andashc) Human thyroid cells showedreduced SOD3mRNAproduction inNthy cells modeling normal thyroid cells and no alteration in papillary thyroid cancer TPC1 or anaplasticthyroid cancer 8505c cells (dndashf)The expression analysis from PCCl3 cells supported the data obtained from the human cells sod3 expressionwas significantly reduced in control PCCl3 cells and not altered in E1A transfected cells (gndashj) TSA treatment led to decreased sod3 expressionin FRLT5 cells modeling normal thyroid but did not affect expression in clone V13 or V21 stably transfected with the H-RasV12 oncogeneTSA treatment increased sod3mRNA synthesis only in PC PTC1 and FRLT5 V39 cellsThe data are expressed as the mean plusmn SDThe 119901 valuesare represented (lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001)


Control Endogenous sod3 Transgene SOD3

Low transgene expression

0

3

6

9

12

15Re

lativ

e exp

ress

ion

lowastlowastlowast

(a)

High transgene expression

lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n


(b)

Erk12

Control

p-Erk12

SOD3 low SOD3 high

(c)

Control SOD3low

SOD3high

lowastlowast

lowastlowast

0

2

4

6

8

10

12

Rela

tive i

nten

sity

(d)

Day 1 Day 2 Day 3 Day 4 Day 5

Transgene sod3Endogenous SOD3

Rela

tive e

xpre

ssio

n

01

1

10

(e)

Control

SOD3

lowastlowast0

10

20

30

40

Rela

tive e

xpre

ssio

n

lowastlowastlowast

lowastlowastlowast

lowast

1120583M 100120583M 1mM 10mMH2O2 H2O2 H2O2 H2O2

(f)

Figure 5 Continued


0

1

2

3

4

5

6

SOD3low

SOD3high

SOS1

Relat

ive e

xpre

ssio

n

lowast

(g)

SOS2

lowastlowast

0

1

2

3

4

5

6

SOD3low

SOD3high

Relat

ive e

xpre

ssio

n

(h)

Figure 5 Self-regulation of SOD3 expression is mediated by GEF small GTPase regulatory proteins (a-b) The analysis of the effects of lowand high SOD3 transgene transfection on endogenous sod3 mRNA synthesis in rat PC E1A cells Transfection of low amounts of humanSOD3 into PC E1A cells significantly increased the endogenous rat sod3 expression whereas high amounts of human SOD3 levels in PC E1Acells significantly reduced the endogenous rat sod3mRNA synthesis (c-d) Western blot analysis of ERK12 phosphorylation in PC E1A cellssuggested greater MAPK ERK12 activation at low SOD3 transgene expression levels compared to high transgene levels (e) Comparison ofrabbit transgene sod3 expression to human endogenous SOD3 mRNA synthesis in HEK 293T cells The expression analysis performed onfive consecutive days demonstrated a gradual decrease in the transgene expression that correlated with a gradual increase in the endogenousSOD3 mRNA synthesis (f) The effect of the H

2O2concentration on SOD3 mRNA expression HEK 293T cells were treated with different

concentrations of H2O2 Low H

2O2concentrations caused increased SOD3mRNA synthesis and high H

2O2levels caused decreased SOD3

expression (g)The effects of low and high SOD3 transgene expression on SOS1 and SOS2GEFmRNA expression in 8505c cells A low level oftransgene expression caused an increase in SOS1 and SOS2mRNA production whereas high transgene SOD3 levels decreased the expressionof both GEFs The data are expressed as the mean plusmn SD The 119901 values are represented (lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001)

causing this downregulation we investigated the influenceof the known regulatory factors in well-described thyroidcancer models systems in the current study

Notably the expression of SOD3 correlated with theactivation level of RAS (Figure 1) suggesting that SOD3 isa ras oncogene-downregulated gene Based on the data theregulation of SOD3 mRNA expression in the thyroid cellmodels can be divided into immediate regulatory eventswhich are reversible and depend on the RAS activity andlate regulatory events which cause long-term permanentsilencing of the gene The self-regulation mediated throughRAS small GTPase regulatory genes may represent the fastestmeans of regulating SOD3 expression which is required tomaintain stable expression of the mRNA production and theredox balance in tissues The guanine nucleotide exchangefactors (GEF) small GTPase regulatory genes contributeto RAS activation by active removal of GDP that is thenreplaced by GTP with consequent immediate response in thedownstream signal transduction The current data suggestthat even minor alterations in SOD3 expression significantlyaffect SOS1 and SOS2 GEF expression via a feedback loophence enabling response to sudden changes in signaling andfine adjustments of SOD3 production

Recent papers have suggested that induction of mir21transcription occurs through RAS-AP1 signaling [39] in adose-dependent manner that correlates with RAS activityWhile low RAS activity showed a minor impact on mir21production a striking increase in expression in cells with high

RAS activity [19] was observed these results are in accordwith the current data Thus the microRNA mir21 representsanother immediate reversible regulatory step however thismechanism of regulation might require greater changes inRAS activation than GEFndashmediated SOD3 regulation Theexpression of mir21 which binds to the 31015840UTR region ofSOD3 mRNA [31] showed an inverse correlation with sod3mRNA expression (Figures 1(c) and 1(d)) suggesting asudden increase in mir21 and decrease in sod3 expressionat 10-fold RAS activity Therefore the data suggest theimportance of 10-fold RAS activity for cellular redox balanceand mir21 target gene silencing The microRNA mir21 isa well-characterized oncomir that is upregulated by RASin the early stages of tumorigenesis A recent paper hassuggested that knockdown ofmir21 in cells transplanted intomice significantly reduces tumorigenesis [40] underliningthe importance of this microRNA in cancer development

The third reversible RAS-dependent regulation stepoccurs through p38 MAPK activation The ability of p38MAPK to regulate SOD3 mRNA expression is noteworthybecause the mitogen signaling cascade BRAF MEK12 andERK12 are primarily responsible for the stimulation ofSOD3 synthesis Although MAPK kinases follow specificsignal transduction pathways to phosphorylate their cognateMAPKs (eg MEK12 activates ERK12 but not JNK) p38MAPK displays selectivity between cognate and noncognatedocking sites that might explain the interference betweentwo signal transduction pathways [41] However we cannot


exclude the possibility that the p38MAPK inhibitor SB202190is not specific for p38 or that negative feedback could activatealternative signal transduction pathways [42]

DNAmethylation and histone acetylation representmorepermanent DNA regulatory mechanisms compared to regu-lation controlled by GEFs mir21 and p38 MAPK activationDNA methylation a stable modification that is often pro-voked by RAS controls the expression of a number of genesthrough epigenetic mechanisms that recruit different tran-scriptional repressors in an orderly fashion A recent publica-tion has described step-by-step methylation silencing of thefas gene suggesting coordinated action of 28RAS-related epi-genetic silencing effectors [17] that employ a well-organizedsignal transduction pathway The silencing of RAS down-stream genes is initiated by recruitment of PDK1 MAPK1and S100Z as the first step followed by 14 other steps thateventually culminate in DNMT1 activation and consequentDNA methylation [43] Although methylation changes areobserved even in precancerous stages with chronic inflam-mation the current data suggest the most prominent rolefor methylation-mediated SOD3 silencing in the presence ofhigh RAS activity in advanced well-differentiated and poorlydifferentiated aggressive cancers The first the second andthe third exon of human SOD3 locate inside or at close prox-imity of CpG islands and repetitive elements such as Alu andLINE 2B retrotransposons [33] Although SOD3 promoterregion is incompletely analyzed it has been demonstratedthat CpG methylation downregulates Sp1Sp3 transcriptionfactor binding to putative promoter region inhibiting SOD3promoter activity and mRNA synthesis in vitro in humanA549 adenocarcinoma lung alveolar basal epithelial cells [37]thus being in line with the current data

The transcriptional silencing of genes caused by DNAmethylation is further reinforced by histone deacetylase(HDAC) activity which inhibits histone acetylation andcauses the formation of closed chromatin structure andreduced DNA transcriptional activity CpG dimer methyl-ation-associated proteins such as the transcriptional repres-sor MeCP2 attract multiprotein complexes that containcorepressor histone deacetylases [44] which then inhibit theexpression of genes controlling the cell cycle such as p21and promote uncontrolled growth and carcinogenesis [45]The inhibition of HDAC function by TSA treatment had onlya minor effect on sod3 expression in cancer cells or FRLT5cells harboring high RAS activity (Figure 4) Instead HDACinhibition significantly decreased sod3mRNA production incells that modeled normal rat thyroid In normal thyroidthyroid stimulating hormone (TSH) production is linked tohistone acetylation TSA treatment inhibits TSH promoteractivity and therefore TSH production [46] We have previ-ously demonstrated that hormonal starvation reduces sod3expression in thyroid cells while TSH treatment stimulatessod3 production that is mediated by the cAMP-PKA andPLC-Ca2+ signal transduction pathways and affects normalthyroid cell proliferation [30] The TSA-mediated reductionin SOD3 expression in humanNthy cells which growwithouthormone supplementation suggests other TSH-independentmechanisms for gene regulation However the differences

between human and rat cells could be caused by species-specific signal transduction [47] although indirect generegulatory systems might also be involved

In conclusion the regulation of SOD3 expression in car-cinogenesis involves ras oncogene-driven sequential eventsconsisting of both reversible mechanisms that correlate withthe RAS activation levels and more stable epigenetic eventsImportantly the use of multiple cell lines harboring differentoncogenes and representing varying degrees of transforma-tion in different species suggests that the regulation of SOD3gene expression is not cell line-specific Our current resultsthus suggest that SOD3 is a ras oncogene-downregulatedgene

Conflict of Interests

The authors have no conflict of interests

References

[1] P-O Sjoquist and S L Marklund ldquoEndothelium bound extra-cellular superoxide dismutase type C reduces damage in reper-fused ischaemic rat heartsrdquoCardiovascular Research vol 26 no4 pp 347ndash350 1992

[2] M O Laukkanen P Leppanen P Turunen T Tuomisto JNaarala and S Yla-Herttuala ldquoEC-SOD gene therapy reducesparacetamol-induced liver damage in micerdquo Journal of GeneMedicine vol 3 no 4 pp 321ndash325 2001

[3] M O Laukkanen A Kivela T Rissanen et al ldquoAdenovirus-mediated extracellular superoxide dismutase gene therapyreduces neointima formation in balloon-denuded rabbit aortardquoCirculation vol 106 no 15 pp 1999ndash2003 2002

[4] KOzumi H Tasaki H Takatsu et al ldquoExtracellular superoxidedismutase overexpression reduces cuff-induced arterial neoin-timal formationrdquo Atherosclerosis vol 181 no 1 pp 55ndash62 2005

[5] E D van Deel Z Lu X Xu et al ldquoExtracellular superoxidedismutase protects the heart against oxidative stress and hyper-trophy after myocardial infarctionrdquo Free Radical Biology andMedicine vol 44 no 7 pp 1305ndash1313 2008

[6] J P Laurila L E Laatikainen M D Castellone and M OLaukkanen ldquoSOD3 reduces inflammatory cell migration byregulating adhesion molecule and cytokine expressionrdquo PLoSONE vol 4 no 6 Article ID e5786 2009

[7] M D Castellone A Langella S Cantara et al ldquoExtracellularsuperoxide dismutase inducesmouse embryonic fibroblast pro-liferative burst growth arrest immortalization and consequentin vivo tumorigenesisrdquo Antioxidants and Redox Signaling vol21 no 10 pp 1460ndash1474 2014

[8] M O Laukkanen F Cammarota T Esposito M Salvatore andM D Castellone ldquoExtracellular superoxide dismutase regulatesthe expression of small gtpase regulatory proteins GEFs GAPsand GDIrdquo PLoS ONE vol 10 no 3 Article ID e0121441 2015

[9] J P Laurila M D Castellone A Curcio et al ldquoExtracellularsuperoxide dismutase is a growth regulatory mediator of tissueinjury recoveryrdquoMolecular Therapy vol 17 no 3 pp 448ndash4542009

[10] H L Maecker Z Yun H T Maecker and A J GiaccialdquoEpigenetic changes in tumor Fas levels determine immuneescape and response to therapyrdquo Cancer Cell vol 2 no 2 pp139ndash148 2002


[11] J Sung J Turner S McCarthy et al ldquoOncogene regulation oftumor suppressor genes in tumorigenesisrdquo Carcinogenesis vol26 no 2 pp 487ndash494 2005

[12] S Cairns-Smith andPKarran ldquoEpigenetic silencing of theDNArepair enzyme O6-methylguanine-DNA methyltransferase inMex- human cellsrdquo Cancer Research vol 52 no 19 pp 5257ndash5263 1992

[13] H Takada I Imoto H Tsuda et al ldquoADAM23 a possible tumorsuppressor gene is frequently silenced in gastric cancers byhomozygous deletion or aberrant promoter hypermethylationrdquoOncogene vol 24 no 54 pp 8051ndash8060 2005

[14] XQian G Li H K Asmussen et al ldquoOncogenic inhibition by adeleted in liver cancer gene requires cooperation between tensinbinding and Rho-specific GTPase-activating protein activitiesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 21 pp 9012ndash9017 2007

[15] R Holliday ldquoA new theory of carcinogenesisrdquo British Journal ofCancer vol 40 no 4 pp 513ndash522 1979

[16] J Covault M Perry and R Chalkley ldquoEffects of histonehyperacetylation and hypoacetylation on RNA synthesis inHTC cellsrdquoThe Journal of Biological Chemistry vol 257 no 22pp 13433ndash13440 1982

[17] C Gazin N Wajapeyee S Gobeil C-M Virbasius and MR Green ldquoAn elaborate pathway required for Ras-mediatedepigenetic silencingrdquo Nature vol 449 no 7165 pp 1073ndash10772007

[18] O A Kent and J TMendell ldquoA small piece in the cancer puzzlemicroRNAs as tumor suppressors and oncogenesrdquo Oncogenevol 25 no 46 pp 6188ndash6196 2006

[19] G De Vita L Bauer V M Correa Da Costa et al ldquoDose-dependent inhibition of thyroid differentiation by RAS onco-genesrdquoMolecular Endocrinology vol 19 no 1 pp 76ndash89 2005

[20] M O Laukkanen P Lehtolainen P Turunen et al ldquoRabbitextracellular superoxide dismutase expression and effect onLDL oxidationrdquo Gene vol 254 no 1-2 pp 173ndash179 2000

[21] F S Ambesi-Impiombato L A M Parks and H G CoonldquoCulture of hormone-dependent functional epithelial cells fromrat thyroidsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 77 no 6 I pp 3455ndash3459 1980

[22] A Fusco M T Berlingieri P P Di Fiore G Portella M Griecoand G Vecchio ldquoOne- and two-step transformations of ratthyroid epithelial cells by retroviral oncogenesrdquo Molecular andCellular Biology vol 7 no 9 pp 3365ndash3370 1987

[23] M Grieco M Santoro M T Berlingieri et al ldquoMolecularcloning of PTC a new oncogene found activated in humanthyroid papillary carcinomas and their lymph nodemetastasesrdquoAnnals of the New York Academy of Sciences vol 551 pp 380ndash381 1988

[24] M Grieco M Santoro M T Berlingieri et al ldquoPTC is a novelrearranged form of the ret proto-oncogene and is frequentlydetected in vivo in human thyroid papillary carcinomasrdquo Cellvol 60 no 4 pp 557ndash563 1990

[25] R Ralston and J M Bishop ldquoThe protein products of the mycandmyb oncogenes and adenovirus E1a are structurally relatedrdquoNature vol 306 no 5945 pp 803ndash806 1983

[26] M Tiainen D Spitkovsky P Jansen-Durr A Sacchi andM Crescenzi ldquoExpression of E1A in terminally differentiatedmuscle cells reactivates the cell cycle and suppresses tissue-specific genes by separablemechanismsrdquoMolecular andCellularBiology vol 16 no 10 pp 5302ndash5312 1996

[27] N R Lemoine E S Mayall T Jones et al ldquoCharacterisation ofhuman thyroid epithelial cells immortalised in vitro by simianvirus 40 DNA transfectionrdquo British Journal of Cancer vol 60no 6 pp 897ndash903 1989

[28] T Ito T Seyama T Mizuno et al ldquoUnique association ofp53 mutations with undifferentiated but not with differentiatedcarcinomas of the thyroid glandrdquo Cancer Research vol 52 no5 pp 1369ndash1371 1992

[29] M Xing ldquoBRAFmutation in thyroid cancerrdquo Endocrine-RelatedCancer vol 12 no 2 pp 245ndash262 2005

[30] L E Laatikainen M D Castellone A Hebrant et al ldquoExtracel-lular superoxide dismutase is a thyroid differentiation markerdown-regulated in cancerrdquo Endocrine-Related Cancer vol 17no 3 pp 785ndash796 2010

[31] X Zhang W-L Ng P Wang et al ldquoMicroRNA-21 modulatesthe levels of reactive oxygen species by targeting SOD3 andTNF120572rdquo Cancer Research vol 72 no 18 pp 4707ndash4713 2012

[32] T Kamiya H Hara H Yamada H Imai N Inagaki andT Adachi ldquoCobalt chloride decreases EC-SOD expressionthrough intracellular ROS generation and p38-MAPKpathwaysin COS7 cellsrdquo Free Radical Research vol 42 no 11-12 pp 949ndash956 2008

[33] M O Laukkanen S Mannermaa M O Hiltunen et al ldquoLocalhypomethylation in atherosclerosis found in rabbit ec-sodgenerdquoArteriosclerosisThrombosis and Vascular Biology vol 19no 9 pp 2171ndash2178 1999

[34] L E Laatikainen M Incoronato M D Castellone J P LaurilaM Santoro and M O Laukkanen ldquoSOD3 decreases ischemicinjury derived apoptosis through phosphorylation of Erk12Akt and Foxo3ardquo PLoS ONE vol 6 no 8 Article ID e244562011

[35] P Stralin and S L Marklund ldquoMultiple cytokines regulatethe expression of extracellular superoxide dismutase in humanvascular smoothmuscle cellsrdquoAtherosclerosis vol 151 no 2 pp433ndash441 2000

[36] T Kamiya H Hara N Inagaki and T Adachi ldquoThe effect ofhypoxia mimetic cobalt chloride on the expression of EC-SODin 3T3-L1 adipocytesrdquo Redox Report vol 15 no 3 pp 131ndash1372010

[37] I N Zelko M R Mueller and R J Folz ldquoCpG methylationattenuates Sp1 and Sp3 binding to the human extracellularsuperoxide dismutase promoter and regulates its cell-specificexpressionrdquo Free Radical Biology and Medicine vol 48 no 7pp 895ndash904 2010

[38] I N Zelko M W Stepp A L Vorst and R J Folz ldquoHistoneacetylation regulates the cell-specific and interferon-gamma-inducible expression of extracellular superoxide dismutase inhuman pulmonary arteriesrdquo American Journal of RespiratoryCell and Molecular Biology vol 45 no 5 pp 953ndash961 2011

[39] F Talotta A Cimmino M R Matarazzo et al ldquoAn autoregu-latory loop mediated by miR-21 and PDCD4 controls the AP-1activity in RAS transformationrdquoOncogene vol 28 no 1 pp 73ndash84 2009

[40] D Frezzetti M De Menna P Zoppoli et al ldquoUpregulation ofmiR-21 by Ras in vivo and its role in tumor growthrdquo Oncogenevol 30 no 3 pp 275ndash286 2011

[41] A J Bardwell E Frankson and L Bardwell ldquoSelectivity ofdocking sites in MAPK kinasesrdquo The Journal of BiologicalChemistry vol 284 no 19 pp 13165ndash13173 2009

[42] P Salerno V De Falco A Tamburrino et al ldquoCytostatic activityof adenosine triphosphate-competitive kinase inhibitors in


BRAF mutant thyroid carcinoma cellsrdquo Journal of ClinicalEndocrinology andMetabolism vol 95 no 1 pp 450ndash455 2010

[43] NWajapeyee S KMalonia R K Palakurthy andM R GreenldquoOncogenic RAS directs silencing of tumor suppressor genesthrough ordered recruitment of transcriptional repressorsrdquoGenes and Development vol 27 no 20 pp 2221ndash2226 2013

[44] P L Jones G J C Veenstra P AWade et al ldquoMethylated DNAandMeCP2 recruit histone deacetylase to repress transcriptionrdquoNature Genetics vol 19 no 2 pp 187ndash191 1998

[45] V M Richon T W Sandhoff R A Rifkind and P A MarksldquoHistone deacetylase inhibitor selectively induces p21WAF1expressjon and gene-associated histone acetylationrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 97 no 18 pp 10014ndash10019 2000

[46] D Wang X Xia Y Liu et al ldquoNegative regulation of tsh120572target gene by thyroid hormone involves histone acetylation andcorepressor complex dissociationrdquo Molecular Endocrinologyvol 23 no 5 pp 600ndash609 2009

[47] Y Song C Massart V Chico-Galdo et al ldquoSpecies specificthyroid signal transduction conserved physiology divergentmechanismsrdquo Molecular and Cellular Endocrinology vol 319no 1-2 pp 56ndash62 2010

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


2 Methods

21 Cells Rat thyroid PC Cl3 PC PTC1 PC E1A andFRLT5 and the related cell clones V13 V21 and V27 stablytransfected with an H-RasV12 expression plasmid [19] werecultured in Hamrsquos F12 medium Coonrsquos modified (Sigma StLouis MO USA) supplemented with 5 calf serum (LifeTechnologies Inc Carlsbad CA) penicillin (100UmL)(Sigma) and streptomycin (100mgL) (Sigma) PC Cl3 andFRLT5 cells (modeling normal thyroid cells) were addition-ally supplemented with 10 nM TSH 10 nM hydrocortisone100 nM insulin 5mgmL transferrin 5 nM somatostatin and20mgmL glycyl-histidyl-lysine Human thyroid NTHY-ori31 (Nthy) and anaplastic thyroid cancer 8505c cells weregrown in RPMI medium (Life Technologies Inc) supple-mented with 10 fetal bovine serum (Sigma) penicillin(100UmL) and streptomycin (100mgL) Human papillarythyroid cancer TPC1 cells were grown in DMEM supple-mented with 10 fetal bovine serum penicillin (100UmL)and streptomycin (100mgL) HEK 293T cells were grownin DMEM supplemented with 10 fetal bovine serumpenicillin (100UmL) and streptomycin (100mgL) To studythe regulation of sod3 gene expression the cells were sup-plemented with 10 120583M 5-azacytidine (Sigma) 100 nM TSA(Sigma) and 10120583M SB202190 p38 MAPK inhibitor (Sigma)for 24 hours or with H

2O2(Sigma) for 6 hours

The human SOD3 expression vector (a gift fromProfessorStefan L Marklund University of Umea Sweden) rabbitsod3 expression vector previously cloned in our group [20]or pcDNA3 control (Invitrogen Paisley UK) or H-RasV12plasmid was transfected with Fugene 6 (Promega WI USA)according to standard protocols

22 Real-TimeReverse Transcription PCR Cells were pelletedfor RNA isolation using an RNeasyMini Kit (Qiagen HildenGermany) and cDNA synthesis using a QuantiTect ReverseTranscription Kit (Qiagen)The PCR reaction was performedusing an iCycler (BioRad Hercules CA USA) and SYBRGreen PCR Master Mix (Applied Biosystems Foster CityCA) The primers were designed with (httpwwwncbinlmnihgovtoolsprimer-blast) Human 120573-actin and rat 120573-actinwere used to normalize human and rat templates respec-tively Relative fluorescence expression values were calculatedas follows ΔCt = Ct (gene of interest)-Ct (120573-actin) ΔΔCt =ΔCt (treated)-ΔCt (control) Relative expression values =2(minusΔΔCt) The primers are listed in Table 1

23 MicroRNA Isolation MicroRNA was isolated using anmiRNeasy Kit (Qiagen) for reverse transcription with amiScript RT II Kit (Qiagen) The miR21 and RNU5 miScriptPrimer Assays (Qiagen) were used for amplification and tonormalize themiR21 expression

24 Western Blot The cells were harvested in lysis buffer(50mM HEPES pH 75 150mM NaCl 10 glycerol 1 Tri-ton X-100 1mM EGTA 15mM MgCl

2 10mM NaF 10mM

sodium pyrophosphate 1mM Na3VO4 10 120583g aprotininmL

and 10 120583g leupeptinmL) separated by sodium dodecyl

Table 1 PCR primer sequences used in the study

Human SOD3 forward cttcgcctctgctgaagtctHuman SOD3 reverse gggtgtttcggtacaaatggRat sod3 forward gacctggagatctggatggaRat sod3 reverse gtggttggaggtgttctgctlowastRabbit sod3 forward gttgcgtgagcggaaagaRabbit sod3 reverse gtgagcgcctgccagatctcHuman SOS1 forward cacctcctcctcaaacacctHuman SOS1 reverse gtgtgtgtgctcccttttgtHuman SOS2 forward ttttgaagaacgggtggcagHuman SOS2 reverse ttttcctttcctgcagtgccHuman 120573-actin forward tgcgtgacattaaggagaagHuman 120573-actin reverse gctcgtagctcttctccaRat 120573-actin forward tcgtgcgtgacattaaggagRat 120573-actin reverse gtcaggcagctcgtagctctlowastPrimer designed against EF1alfa promoter

sulfatendashpolyacrylamide gel electrophoresis and transferredto Hybond C Extra nitrocellulose membranes (GE Health-careWI USA)The following antibodies were used pErk12Erk12 p-p38 MAPK and p38 MAPK (Cell Signaling Tech-nology MA USA)

25 Statistical Analysis The experiments were repeated atleast three times All of the results were expressed as themeanvalues plusmn SDThe 119901 values (lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt0001) were determined using two-tailed independent sample119905-tests

3 Results

31Thyroid Cell Models Representing Different Degrees ofThy-roid Cancer Thyroid stimulating hormone hydrocortisoneinsulin transferrin somatostatin and glycyl-histidyl-lysinesupplementation-dependent FRLT5 cells are a clonal cell linederived from rat normal thyroid FRLT cells by purificationthrough successive isolation of colonies [21] FRLT5 clonesV13 K20 K2 V21 V39 and V27 were created by stabletransfection with H-RasV12 oncogene followed by clonalexpansion of the cell lines derived from a single cellThe cloneV13 harbors 14-fold K20 31-fold K2 63-fold V21 10-foldV39 35-fold and V27 46-fold RAS activity relative to parentalcells This increased RAS activity drives the growth ofthe cells without hormone supplementation thus modelingthe hormone-independent growth characteristics of thyroidcancers [19] Similar to FRLT5 cells PC Cl3 cells representrat normal thyroid cells and are dependent on hormonesupplementation [22] PC PTC1 and PC E1A clones are PCCl3 derivatives that were transformed with PTC1 and E1Aoncogenes [23] PTC1 is a papillary thyroid cancer- (PTC-)associated oncogene derived from a genomic rearrangementof the RET oncogene with the CDC61H4 gene [24] The E1Aoncogene shows structural and functional similarities to theMYC and MYB oncogenes [25] that mechanistically bypass


Nthy TPC1 8505c

SOD3

0

05

1

15Re

lativ

e exp

ress

ion

lowastlowast

lowastlowast


sod3

lowastlowastlowast

lowastlowastlowast

0

05

1

15

Rela

tive e

xpre

ssio

n

(b)

FRTL5 V13 K20 K2 V21 V39 V27

sod3

lowastlowast

lowastlowast

lowast


0

2

4

6

8

Rela

tive e

xpre

ssio

n


lowastlowastlowast

(c)

FRLT5 V13 K20 K2 V21 V39 V27


mir21

0

2

4

6

8

10

Rela

tive e

xpre

ssio

n

lowast

lowast

lowastlowast

(d)

















Nthycntr

NthySB

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(a)

TPC1cntr

TPC1 SB0

1

2

3

4

5

Rela

tive e

xpre

ssio

n lowast

(b)

8505ccntr

8505cSB

lowastlowastlowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(c)

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

PC Cl3cntr

PC Cl3SB

(d)

PC PTC1cntr

PC PTC1SB

lowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(e)

PC E1Acntr

PC E1ASB

lowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(f)

FRLT5cntr

FRLT5SB

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(g)

FRLT5V13 cntr

FRLT5V13 SB

lowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(h)

FRLT5V21 cntr

FRLT5V21 SB

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

lowastlowastlowast

(i)

FRLT5V39 cntr

FRLT5V39 SB

lowast

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(j)

p-p-38

FRLT5cntr

FRLT5V13

FRLT5V21

FRLT5V39

p-38

(k)

p-p-38

p-38

Ras 01 120583g

Ras 05 120583g

Ras 1120583g

Ras 5120583g

(l)

Figure 2 Continued


FRLT5 V13 V21 V39

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

05

1

15

2Re

lativ

e int

ensit

y

(m)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

05

1

15

2

Rela

tive i

nten

sity


(n)



2O2increased the






4 Discussion



Nthy cntr Nthy aza0

2

4

6

8

Rela

tive e

xpre

ssio

n


0

2

4

6

8

Rela

tive e

xpre

ssio

n

lowastlowast


0

1

2

3

4

5

6

7

Rela

tive e

xpre

ssio

n

lowastlowastlowast

(c)

0

05

1

15

2


Rela

tive e

xpre

ssio

n

(d)

0

05

1

15

2


Rela

tive e

xpre

ssio

nlowast

(e)

0

05

1

15

2


Rela

tive e

xpre

ssio

n lowastlowast

(f)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(g)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(h)

0

1

2

3

4

5

6


Relat

ive e

xpre

ssio

n

lowastlowast

(i)


lowastlowastlowast

0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n

(j)



Nthy cntr Nthy TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n

(c)

PC Cl3cntr

PC Cl3TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n

(d)

PC PTC1cntr

PC PTC1TSA

lowast

0

05

1

15

2Re

lativ

e exp

ress

ion

(e)

PC E1Acntr

PC E1ATSA

0

05

1

15

2

Rela

tive e

xpre

ssio

n(f)

FRLT5cntr

FRLT5TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(g)

FRLT5V13 cntr

FRLT5V13 TSA

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(h)

FRLT5V21 cntr

FRLT5V21 TSA

0

05

1

15

2Re

lativ

e exp

ress

ion

(i)

FRLT5V39 cntr

FRLT5V39 TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(j)





0

3

6

9

12

15Re

lativ

e exp

ress

ion

lowastlowastlowast

(a)


lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n


(b)

Erk12

Control

p-Erk12

SOD3 low SOD3 high

(c)

Control SOD3low

SOD3high

lowastlowast

lowastlowast

0

2

4

6

8

10

12

Rela

tive i

nten

sity

(d)



Rela

tive e

xpre

ssio

n

01

1

10

(e)

Control

SOD3

lowastlowast0

10

20

30

40

Rela

tive e

xpre

ssio

n

lowastlowastlowast

lowastlowastlowast

lowast

1120583M 100120583M 1mM 10mMH2O2 H2O2 H2O2 H2O2

(f)

Figure 5 Continued


0

1

2

3

4

5

6

SOD3low

SOD3high

SOS1

Relat

ive e

xpre

ssio

n

lowast

(g)

SOS2

lowastlowast

0

1

2

3

4

5

6

SOD3low

SOD3high

Relat

ive e

xpre

ssio

n

(h)




















References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nthy TPC1 8505c

SOD3

0

05

1

15Re

lativ

e exp

ress

ion

lowastlowast

lowastlowast


sod3

lowastlowastlowast

lowastlowastlowast

0

05

1

15

Rela

tive e

xpre

ssio

n

(b)

FRTL5 V13 K20 K2 V21 V39 V27

sod3

lowastlowast

lowastlowast

lowast


0

2

4

6

8

Rela

tive e

xpre

ssio

n


lowastlowastlowast

(c)

FRLT5 V13 K20 K2 V21 V39 V27


mir21

0

2

4

6

8

10

Rela

tive e

xpre

ssio

n

lowast

lowast

lowastlowast

(d)

















Nthycntr

NthySB

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(a)

TPC1cntr

TPC1 SB0

1

2

3

4

5

Rela

tive e

xpre

ssio

n lowast

(b)

8505ccntr

8505cSB

lowastlowastlowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(c)

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

PC Cl3cntr

PC Cl3SB

(d)

PC PTC1cntr

PC PTC1SB

lowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(e)

PC E1Acntr

PC E1ASB

lowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(f)

FRLT5cntr

FRLT5SB

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(g)

FRLT5V13 cntr

FRLT5V13 SB

lowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(h)

FRLT5V21 cntr

FRLT5V21 SB

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

lowastlowastlowast

(i)

FRLT5V39 cntr

FRLT5V39 SB

lowast

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(j)

p-p-38

FRLT5cntr

FRLT5V13

FRLT5V21

FRLT5V39

p-38

(k)

p-p-38

p-38

Ras 01 120583g

Ras 05 120583g

Ras 1120583g

Ras 5120583g

(l)

Figure 2 Continued


FRLT5 V13 V21 V39

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

05

1

15

2Re

lativ

e int

ensit

y

(m)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

05

1

15

2

Rela

tive i

nten

sity


(n)



2O2increased the






4 Discussion



Nthy cntr Nthy aza0

2

4

6

8

Rela

tive e

xpre

ssio

n


0

2

4

6

8

Rela

tive e

xpre

ssio

n

lowastlowast


0

1

2

3

4

5

6

7

Rela

tive e

xpre

ssio

n

lowastlowastlowast

(c)

0

05

1

15

2


Rela

tive e

xpre

ssio

n

(d)

0

05

1

15

2


Rela

tive e

xpre

ssio

nlowast

(e)

0

05

1

15

2


Rela

tive e

xpre

ssio

n lowastlowast

(f)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(g)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(h)

0

1

2

3

4

5

6


Relat

ive e

xpre

ssio

n

lowastlowast

(i)


lowastlowastlowast

0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n

(j)



Nthy cntr Nthy TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n

(c)

PC Cl3cntr

PC Cl3TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n

(d)

PC PTC1cntr

PC PTC1TSA

lowast

0

05

1

15

2Re

lativ

e exp

ress

ion

(e)

PC E1Acntr

PC E1ATSA

0

05

1

15

2

Rela

tive e

xpre

ssio

n(f)

FRLT5cntr

FRLT5TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(g)

FRLT5V13 cntr

FRLT5V13 TSA

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(h)

FRLT5V21 cntr

FRLT5V21 TSA

0

05

1

15

2Re

lativ

e exp

ress

ion

(i)

FRLT5V39 cntr

FRLT5V39 TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(j)





0

3

6

9

12

15Re

lativ

e exp

ress

ion

lowastlowastlowast

(a)


lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n


(b)

Erk12

Control

p-Erk12

SOD3 low SOD3 high

(c)

Control SOD3low

SOD3high

lowastlowast

lowastlowast

0

2

4

6

8

10

12

Rela

tive i

nten

sity

(d)



Rela

tive e

xpre

ssio

n

01

1

10

(e)

Control

SOD3

lowastlowast0

10

20

30

40

Rela

tive e

xpre

ssio

n

lowastlowastlowast

lowastlowastlowast

lowast

1120583M 100120583M 1mM 10mMH2O2 H2O2 H2O2 H2O2

(f)

Figure 5 Continued


0

1

2

3

4

5

6

SOD3low

SOD3high

SOS1

Relat

ive e

xpre

ssio

n

lowast

(g)

SOS2

lowastlowast

0

1

2

3

4

5

6

SOD3low

SOD3high

Relat

ive e

xpre

ssio

n

(h)




















References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Nthycntr

NthySB

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(a)

TPC1cntr

TPC1 SB0

1

2

3

4

5

Rela

tive e

xpre

ssio

n lowast

(b)

8505ccntr

8505cSB

lowastlowastlowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(c)

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

PC Cl3cntr

PC Cl3SB

(d)

PC PTC1cntr

PC PTC1SB

lowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(e)

PC E1Acntr

PC E1ASB

lowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(f)

FRLT5cntr

FRLT5SB

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(g)

FRLT5V13 cntr

FRLT5V13 SB

lowast

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

(h)

FRLT5V21 cntr

FRLT5V21 SB

0

1

2

3

4

5

Rela

tive e

xpre

ssio

n

lowastlowastlowast

(i)

FRLT5V39 cntr

FRLT5V39 SB

lowast

0

1

2

3

4

5

Relat

ive e

xpre

ssio

n

(j)

p-p-38

FRLT5cntr

FRLT5V13

FRLT5V21

FRLT5V39

p-38

(k)

p-p-38

p-38

Ras 01 120583g

Ras 05 120583g

Ras 1120583g

Ras 5120583g

(l)

Figure 2 Continued


FRLT5 V13 V21 V39

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

05

1

15

2Re

lativ

e int

ensit

y

(m)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

05

1

15

2

Rela

tive i

nten

sity


(n)



2O2increased the






4 Discussion



Nthy cntr Nthy aza0

2

4

6

8

Rela

tive e

xpre

ssio

n


0

2

4

6

8

Rela

tive e

xpre

ssio

n

lowastlowast


0

1

2

3

4

5

6

7

Rela

tive e

xpre

ssio

n

lowastlowastlowast

(c)

0

05

1

15

2


Rela

tive e

xpre

ssio

n

(d)

0

05

1

15

2


Rela

tive e

xpre

ssio

nlowast

(e)

0

05

1

15

2


Rela

tive e

xpre

ssio

n lowastlowast

(f)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(g)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(h)

0

1

2

3

4

5

6


Relat

ive e

xpre

ssio

n

lowastlowast

(i)


lowastlowastlowast

0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n

(j)



Nthy cntr Nthy TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n

(c)

PC Cl3cntr

PC Cl3TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n

(d)

PC PTC1cntr

PC PTC1TSA

lowast

0

05

1

15

2Re

lativ

e exp

ress

ion

(e)

PC E1Acntr

PC E1ATSA

0

05

1

15

2

Rela

tive e

xpre

ssio

n(f)

FRLT5cntr

FRLT5TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(g)

FRLT5V13 cntr

FRLT5V13 TSA

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(h)

FRLT5V21 cntr

FRLT5V21 TSA

0

05

1

15

2Re

lativ

e exp

ress

ion

(i)

FRLT5V39 cntr

FRLT5V39 TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(j)





0

3

6

9

12

15Re

lativ

e exp

ress

ion

lowastlowastlowast

(a)


lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n


(b)

Erk12

Control

p-Erk12

SOD3 low SOD3 high

(c)

Control SOD3low

SOD3high

lowastlowast

lowastlowast

0

2

4

6

8

10

12

Rela

tive i

nten

sity

(d)



Rela

tive e

xpre

ssio

n

01

1

10

(e)

Control

SOD3

lowastlowast0

10

20

30

40

Rela

tive e

xpre

ssio

n

lowastlowastlowast

lowastlowastlowast

lowast

1120583M 100120583M 1mM 10mMH2O2 H2O2 H2O2 H2O2

(f)

Figure 5 Continued


0

1

2

3

4

5

6

SOD3low

SOD3high

SOS1

Relat

ive e

xpre

ssio

n

lowast

(g)

SOS2

lowastlowast

0

1

2

3

4

5

6

SOD3low

SOD3high

Relat

ive e

xpre

ssio

n

(h)




















References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















FRLT5 V13 V21 V39

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

05

1

15

2Re

lativ

e int

ensit

y

(m)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

05

1

15

2

Rela

tive i

nten

sity


(n)



2O2increased the






4 Discussion



Nthy cntr Nthy aza0

2

4

6

8

Rela

tive e

xpre

ssio

n


0

2

4

6

8

Rela

tive e

xpre

ssio

n

lowastlowast


0

1

2

3

4

5

6

7

Rela

tive e

xpre

ssio

n

lowastlowastlowast

(c)

0

05

1

15

2


Rela

tive e

xpre

ssio

n

(d)

0

05

1

15

2


Rela

tive e

xpre

ssio

nlowast

(e)

0

05

1

15

2


Rela

tive e

xpre

ssio

n lowastlowast

(f)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(g)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(h)

0

1

2

3

4

5

6


Relat

ive e

xpre

ssio

n

lowastlowast

(i)


lowastlowastlowast

0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n

(j)



Nthy cntr Nthy TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n

(c)

PC Cl3cntr

PC Cl3TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n

(d)

PC PTC1cntr

PC PTC1TSA

lowast

0

05

1

15

2Re

lativ

e exp

ress

ion

(e)

PC E1Acntr

PC E1ATSA

0

05

1

15

2

Rela

tive e

xpre

ssio

n(f)

FRLT5cntr

FRLT5TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(g)

FRLT5V13 cntr

FRLT5V13 TSA

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(h)

FRLT5V21 cntr

FRLT5V21 TSA

0

05

1

15

2Re

lativ

e exp

ress

ion

(i)

FRLT5V39 cntr

FRLT5V39 TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(j)





0

3

6

9

12

15Re

lativ

e exp

ress

ion

lowastlowastlowast

(a)


lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n


(b)

Erk12

Control

p-Erk12

SOD3 low SOD3 high

(c)

Control SOD3low

SOD3high

lowastlowast

lowastlowast

0

2

4

6

8

10

12

Rela

tive i

nten

sity

(d)



Rela

tive e

xpre

ssio

n

01

1

10

(e)

Control

SOD3

lowastlowast0

10

20

30

40

Rela

tive e

xpre

ssio

n

lowastlowastlowast

lowastlowastlowast

lowast

1120583M 100120583M 1mM 10mMH2O2 H2O2 H2O2 H2O2

(f)

Figure 5 Continued


0

1

2

3

4

5

6

SOD3low

SOD3high

SOS1

Relat

ive e

xpre

ssio

n

lowast

(g)

SOS2

lowastlowast

0

1

2

3

4

5

6

SOD3low

SOD3high

Relat

ive e

xpre

ssio

n

(h)




















References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nthy cntr Nthy aza0

2

4

6

8

Rela

tive e

xpre

ssio

n


0

2

4

6

8

Rela

tive e

xpre

ssio

n

lowastlowast


0

1

2

3

4

5

6

7

Rela

tive e

xpre

ssio

n

lowastlowastlowast

(c)

0

05

1

15

2


Rela

tive e

xpre

ssio

n

(d)

0

05

1

15

2


Rela

tive e

xpre

ssio

nlowast

(e)

0

05

1

15

2


Rela

tive e

xpre

ssio

n lowastlowast

(f)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(g)

0

05

1

15

2


Relat

ive e

xpre

ssio

n

(h)

0

1

2

3

4

5

6


Relat

ive e

xpre

ssio

n

lowastlowast

(i)


lowastlowastlowast

0

1

2

3

4

5

6

Relat

ive e

xpre

ssio

n

(j)



Nthy cntr Nthy TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n

(c)

PC Cl3cntr

PC Cl3TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n

(d)

PC PTC1cntr

PC PTC1TSA

lowast

0

05

1

15

2Re

lativ

e exp

ress

ion

(e)

PC E1Acntr

PC E1ATSA

0

05

1

15

2

Rela

tive e

xpre

ssio

n(f)

FRLT5cntr

FRLT5TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(g)

FRLT5V13 cntr

FRLT5V13 TSA

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(h)

FRLT5V21 cntr

FRLT5V21 TSA

0

05

1

15

2Re

lativ

e exp

ress

ion

(i)

FRLT5V39 cntr

FRLT5V39 TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(j)





0

3

6

9

12

15Re

lativ

e exp

ress

ion

lowastlowastlowast

(a)


lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n


(b)

Erk12

Control

p-Erk12

SOD3 low SOD3 high

(c)

Control SOD3low

SOD3high

lowastlowast

lowastlowast

0

2

4

6

8

10

12

Rela

tive i

nten

sity

(d)



Rela

tive e

xpre

ssio

n

01

1

10

(e)

Control

SOD3

lowastlowast0

10

20

30

40

Rela

tive e

xpre

ssio

n

lowastlowastlowast

lowastlowastlowast

lowast

1120583M 100120583M 1mM 10mMH2O2 H2O2 H2O2 H2O2

(f)

Figure 5 Continued


0

1

2

3

4

5

6

SOD3low

SOD3high

SOS1

Relat

ive e

xpre

ssio

n

lowast

(g)

SOS2

lowastlowast

0

1

2

3

4

5

6

SOD3low

SOD3high

Relat

ive e

xpre

ssio

n

(h)




















References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nthy cntr Nthy TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n


0

05

1

15

2

Rela

tive e

xpre

ssio

n

(c)

PC Cl3cntr

PC Cl3TSA

lowastlowastlowast

0

05

1

15

2

Rela

tive e

xpre

ssio

n

(d)

PC PTC1cntr

PC PTC1TSA

lowast

0

05

1

15

2Re

lativ

e exp

ress

ion

(e)

PC E1Acntr

PC E1ATSA

0

05

1

15

2

Rela

tive e

xpre

ssio

n(f)

FRLT5cntr

FRLT5TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(g)

FRLT5V13 cntr

FRLT5V13 TSA

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(h)

FRLT5V21 cntr

FRLT5V21 TSA

0

05

1

15

2Re

lativ

e exp

ress

ion

(i)

FRLT5V39 cntr

FRLT5V39 TSA

lowastlowastlowast

0

05

1

15

2

Relat

ive e

xpre

ssio

n

(j)





0

3

6

9

12

15Re

lativ

e exp

ress

ion

lowastlowastlowast

(a)


lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n


(b)

Erk12

Control

p-Erk12

SOD3 low SOD3 high

(c)

Control SOD3low

SOD3high

lowastlowast

lowastlowast

0

2

4

6

8

10

12

Rela

tive i

nten

sity

(d)



Rela

tive e

xpre

ssio

n

01

1

10

(e)

Control

SOD3

lowastlowast0

10

20

30

40

Rela

tive e

xpre

ssio

n

lowastlowastlowast

lowastlowastlowast

lowast

1120583M 100120583M 1mM 10mMH2O2 H2O2 H2O2 H2O2

(f)

Figure 5 Continued


0

1

2

3

4

5

6

SOD3low

SOD3high

SOS1

Relat

ive e

xpre

ssio

n

lowast

(g)

SOS2

lowastlowast

0

1

2

3

4

5

6

SOD3low

SOD3high

Relat

ive e

xpre

ssio

n

(h)




















References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















0

3

6

9

12

15Re

lativ

e exp

ress

ion

lowastlowastlowast

(a)


lowastlowastlowast

01

1

10

100

1000

10000

Rela

tive e

xpre

ssio

n


(b)

Erk12

Control

p-Erk12

SOD3 low SOD3 high

(c)

Control SOD3low

SOD3high

lowastlowast

lowastlowast

0

2

4

6

8

10

12

Rela

tive i

nten

sity

(d)



Rela

tive e

xpre

ssio

n

01

1

10

(e)

Control

SOD3

lowastlowast0

10

20

30

40

Rela

tive e

xpre

ssio

n

lowastlowastlowast

lowastlowastlowast

lowast

1120583M 100120583M 1mM 10mMH2O2 H2O2 H2O2 H2O2

(f)

Figure 5 Continued


0

1

2

3

4

5

6

SOD3low

SOD3high

SOS1

Relat

ive e

xpre

ssio

n

lowast

(g)

SOS2

lowastlowast

0

1

2

3

4

5

6

SOD3low

SOD3high

Relat

ive e

xpre

ssio

n

(h)




















References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

6

SOD3low

SOD3high

SOS1

Relat

ive e

xpre

ssio

n

lowast

(g)

SOS2

lowastlowast

0

1

2

3

4

5

6

SOD3low

SOD3high

Relat

ive e

xpre

ssio

n

(h)




















References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























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 oncogene-mediated progressive silencing...

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