Research Article PELP1 Suppression Inhibits Colorectal Cancer through …downloads.hindawi.com/journals/omcl/2014/193523.pdf · Research Article PELP1 Suppression Inhibits Colorectal

Research ArticlePELP1 Suppression Inhibits Colorectal Cancer throughc-Src Downregulation

Zhifeng Ning12 Youzhi Zhang3 Hanwei Chen4 Jiliang Wu1 Tieshan Song2

Qian Wu2 and Fuxing Liu2

1 Hubei Province Key Laboratory on Cardiovascular Cerebrovascular and Metabolic DisordersHubei University of Science and Technology Xianning 437100 China

2The Basic Medical School Hubei University of Science and Technology Xianning 437100 China3 School of Pharmacy Hubei University of Science and Technology Xianning 437100 China4Wuhan Blood Center Wuhan 430030 China

Correspondence should be addressed to Fuxing Liu liufx6505126com

Received 13 February 2014 Accepted 20 April 2014 Published 22 May 2014

Academic Editor Xiaoqian Chen

Copyright copy 2014 Zhifeng Ning et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Proline- glutamic acid- and leucine-rich protein 1 (PELP1) a coregulator of estrogen receptors alpha and beta is a potentialprotooncogene implicated in several human cancers including sexual hormone-responsive or sexual hormone-nonresponsivecancersHowever the functions of PELP1 in colorectal cancer remain unclear In this study western blot and bioinformatics revealedthat PELP1 expression was higher in several colorectal cancer cell lines than in immortalized normal colorectal epithelium PELP1silencing by short hairpin RNA promoted the senescence and inhibited the proliferation colony formation migration invasionand xenograft tumor formation of the CRC cell line HT-29 Moreover PELP1 silencing was accompanied by c-Src downregulationc-Src upregulation partly alleviated the damage in HT-29 malignant behavior induced by PELP1 RNA interference In conclusionPELP1 exhibits an oncogenic function in colorectal cancer through c-Src upregulation

1 Introduction

Colorectal cancer (CRC) is the second leading cause ofcancer-related death in the developed countries [1 2] InChina themorbidity andmortality rates of CRC have rapidlyincreased over the past two decades these rates are affectedby many factors including age population westernizationlifestyle and industrial pollution Early-stage CRC lacksspecific symptoms thus this disease is usually diagnosed ata relatively late stage The five-year survival rate of early-stage CRC is 85 after surgical resection whereas that ofstage III CRC with lymph node metastasis is lt50 aftersurgery [3] Cancer results from the activation of numerousprotooncogenes and the inhibition of tumor suppressorsCRC is characterized by the mutation of tumor suppressorgenes protooncogenes DNA repair genes growth factorsand their receptor genes apoptosis-related genes and cellcycle checkpoint genes [4] These diverse genes constitute a

complicated regulated network implicated in CRC oncogen-esis However interference with a single gene is not enough tocure cancer Therefore the master regulators of downstreameffector genes involved in carcinogenesis must be urgentlyidentified [5]

The specific functions of estrogen signaling in CRCremain unclear Many studies implicated the dysregulationof estrogen receptors (ERs) in CRC [6ndash9] However thespecific functions of ER120572 and ER120573 in the cancerogenesis ofcolorectal epithelium remain controversial Some researches[6 8] implied that ER120573 is the predominant form expressedin the normal colorectal epithelium and reduced in CRCThus ER120573 exerts protective effects against CRC this find-ing is contradictory to the report that the ER120573 proteinis significantly upregulated in colorectal epithelial cells ofcarcinomas [10] ER120572 is reportedly rarely expressed in normalcolorectal epithelium and lacks functions in CRC carcino-genesis However several studies reported that the ER120572 gene

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 193523 9 pageshttpdxdoiorg1011552014193523

2 Oxidative Medicine and Cellular Longevity

Table 1 Interference sequence for shRNA

shRNA number Sequence (31015840-51015840)Negative control GCAAGCTGACCCTGAAGTTGAGAACTTTGAAGTCCCAGTCGAACG1 GGAGAAACAAAGGUUAUUUGAGAACTUUUAUUGGAAACAAAGAGG2 CACAGUCUCACAUCGUUUAGAGAACTAUUUGCUACACUCUGACAC3 CCAGGAGCTTGTTGAAGAAGAGAACTAAGAAGTTGTTCGAGGACC

is frequently hypermethylated in CRC [11 12] suggestingthat ER120572 hypermethylation can predict CRC progressionER120572 upregulation in ER120572-negative CRC cell lines can sup-press cell growth [13] The reversal of a hypermethylatedinactive ER120572 can inhibit CRC in vitro and in vivo [14]The estrogen-ER signaling pathway is evidently involvedin CRC development Therefore comprehensive researchmust be conducted to elucidate the functions of ER inCRC

Proline- glutamic acid- and leucine-rich protein 1(PELP1) is a modulator of nongenomic actions of estrogenreceptors (NMAR) and a coregulator of ER PELP1 has beenimplicated in many physiological [15ndash17] and pathologicalprocesses [18] Research has demonstrated that PELP1 isa protooncogene in hormone-responsive cancers such asbreast [19 20] ovarian [21] endometrial [22 23] and prostatecancers [24] Moreover some researchers determined thefunctions of PELP1 in hormone-nonresponsive cancers suchas brain tumor [25] lung cancer [26] and colorectal cancer[27 28]

PELP1 is upregulated in CRC tissues [27 28] Howeverthe exact function of PELP1 in CRC remains unknown Inthe present study we investigated the functions of PELP1in several CRC cell lines Bioinformatics and western blotshowed that PELP1 expression was higher in CRC celllines than in normal colorectal epithelium PELP1 silencingby short hairpin RNA (shRNA) promoted the senescenceand inhibited the proliferation colony formation migrationinvasion and xenograft tumor formation of the CRC cell lineHT-29 Moreover PELP1 silencing was accompanied by c-Src downregulation c-Src upregulation partly recovered theoncogenic function of PELP1 These results demonstratedthat PELP1 suppression can inhibit CRC in vitro and in vivothrough c-Src downregulation

2 Materials and Methods

21 Cell Line The CRC cell lines COLO205 HT-29 SW-620 HCC-2998 and HCT-15 and normal colon epitheliumcell line FHC were purchased from American Type CultureCollection (Manassas VA) The five CRC cell lines werecultured in RPMI1640 medium supplemented with 10 fetalbovine serum 100UmL penicillin and 100 120583gmL strepto-mycin FHC was cultured in DMEMF12 medium with thesame supplement as the CRC cell lines All cell lines weremaintained at 37∘C in a humidified atmosphere of 5 CO

2

in an open-air incubator

22 Bioinformatics We performed a bioinformatics analy-sis on Oncomine (httpswwwoncomineorgresourceloginhtml) to analyze PELP1 expression in the CRC cell lines Weused a personal account to enter the Oncomine website Weinput PELP1 on the search box to begin the search Afterentering the page for PELP1 expression survey in almost allhuman cancer cell lines or tissues we focused on CRC andobtained PELP1 expression data from the Lee et al [29]Shankavaram et al [30] and Garnett [31] cell line datasetsAfter data standardization we presented PELP1 expressiondata in the five CRC cell lines as mean plusmn standard deviation(SD)

23 shRNA Interference of PELP1 HT-29 cells were seededin 24-well plates (Corning) until they reached 50 to 60confluence prior to transfection Then stable transfectionwas performedThree shRNA sequences targeting the PELP1gene were designed and synthesized by Shanghai Jima Phar-maceutical Technology China The three shRNA sequencesof PELP1 and the negative control sequence are listed inTable 1 Lipofectamine LTX and Plus Reagent (Invitrogen)were used to transfect shRNA into HT-29 cells accordingto the manufacturerrsquos protocol The medium containingtransfection reagents was replaced with RPMI1640 mediumsupplemented with 10 FBS at 18 h after the transfectionThecells were collected at 48 h after the transfection processedin the following experiments and then prepared for proteinextraction The silence efficiency of PELP1 was tested bywestern blot

24 Western Blot Total protein was extracted from HT-29-shRNA or HT-29-control cells using RIPA lysate buffer asdescribed in our previous research [32] After quantifica-tion by a bicinchoninic acid protein assay kit (BeyotimeBiotechnology China) equal amounts of proteins (20120583gto 25 120583g) were separated by 12 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and processedfor immunoblotting with a rabbit multiclonal antibody forPELP1 (diluted at 1 100 Santa Cruz) and c-Src (dilutedat 1 500 Santa Cruz) A mouse polyclonal anti-120573-actinantibody (diluted at 1 2000 Boster Biological EngineeringChina)was used as an internal control All protein bandswerescanned using ChemiImager 5500 V203 software and theintegrated density values were calculated by a computerizedimage analysis system (Fluor Chen 20) and normalized withthat of 120573-actin

25 MTT Cell viability and proliferation activity were deter-mined with the MTT assay HT-29-shRNA or HT-29-control

Oxidative Medicine and Cellular Longevity 3

cells were seeded in 96-well plates (Costar) at a densityof 5000 cells per well in complete medium (RPMI1640supplemented with 10 FBS 1 ps) and then incubatedfor 12 h under standard conditions (37∘C and 5 CO

2) The

total volume in each well was 200 120583L From the next dayto the seventh day 20120583L of MTT (5mgmL) was addedinto each well After additional incubation for 4 h thesolution in each well was replaced with dimethyl sulfoxide(Sigma USA) to solubilize formazan the metabolic productof MTT The plates were kept on a shaking mixer for 10minto guarantee complete solubilization of formazan and theoptical density was recorded at 490 nm using a microplateluminometer Results were expressed as means plusmn SD and agrowth curve was constructed Data were analyzed by one-way ANOVA with the post hoc Tukey test applied for pairedcomparisons

26 Plate Colony Forming Assay Colony forming ability wasexamined by the plate colony formation assay HT-29-shRNAor HT-29-control cells were seeded into six-centimeter plates(Costar) at a density of 3000 cells per plate in completemedium and then incubated for approximately 2weeks understandard conditions (37∘C and 5 CO

2) The cells were

washed twice with phosphate-buffered saline (PBS) and thenfixed with methanol for 15min After staining with 01crystal violet for 20min the number of positive colonieswith diameters exceeding 50 120583m was counted under a lightmicroscopewith 100timesmagnificationThe colony forming ratewas calculated by dividing the number of positive colonies bythe total number of cells seeded

27 Transwell Small Chamber Invasion and Migration AssayFor the invasion assay Transwell small chambers with 8 120583mpore filters were coated with 12 120583L of ice-cold Matrigel(75mgmL protein) In total 50000 HT-29-shRNA or HT-29-control cells were added to the upper chamber of theseMatrigel chambers in 200120583L of serum-free RPMI 1640medium Then the cells were placed in 24-well plates in500120583L of RPMI 1640 medium containing 10 FBS After22 h of incubation the cells were fixed with methanol andthen stained with 01 crystal violet Cotton tips were usedto remove the cells that remained in the Matrigel or attachedto the upper side of the filterThe number of cells on the lowerside of the filter was counted under a light microscope

Themethods used for themigration assaywere almost thesame as for the invasion assay described above except that noMatrigel was used to coat the well and the incubation timewas 16 h

28 120573-Galactosidase (120573-Gal) Staining The cells were washedin PBS fixed for 3min to 5min (room temperature) in 2formaldehyde02 glutaraldehyde (or 3 formaldehyde)washed again and then incubated at 37∘C (no CO

2) with a

fresh senescence-associated 120573-Gal stain solution containing1mg of 5-bromo-4-chloro-3-indolyl 120573-D-galactoside per mL(stock = 20mg of dimethylformamide per mL)40mM cit-ric acidsodium phosphate pH 605mM potassium ferro-cyanide5mM potassium ferricyanide150mM NaCl2mM

MgCl2 Staining was evident within 2 h to 4 h and peaked

within 12 h to 16 h To detect lysosomal 120573-Gal the citricacidsodium phosphate was pH 40

29 Nude Mice Subcutaneous Xenograft Assay Male BALBc(nunu) mice aged 6 to 8 weeks were obtained from theExperimental Animal Center of Guangdong Province Themice were housed and maintained in laminar flow cabinetsunder specific pathogen-free conditions according to theregulations and standards approved by the Animal Care andEthics Committee of Shantou University Medical College

To establish sc tumors 15 times 106 HT-29-shRNA or HT-29-control cells were resuspended in 200120583L of RPMI1640serum-free medium and injected via an 18-gauge needle intothe sc space of both flanks of the mice Tumor progres-sion was documented once weekly by measurements usingcalipers and tumor volumes were calculated by the followingformula length times width times height times 052 (in mm) The micewere given ethane anesthesia and then euthanized by cervicaldislocation

210 Stable Transfection of c-Src The human c-Src codingregion gene with a 376 bp sequence was amplified fromhomogenized HT-29 by RT-PCR The sequences of thePCR primers used for c-Src in this study were as followssense 51015840-TGTTCGGAGGCTTCAACTCC-31015840 and antisense51015840-CAGTAGGCACCTTTCGTGGT-31015840 The PCR productswere cloned into a TA expression vector (Invitrogen Carls-bad CA USA) and the sequence of the c-Src coding regionwas confirmed by sequencing The resulting plasmids (pc-Src) were propagated in Escherichia coli and then purifiedthrough cesium chloride gradient For gene transfectionHT-29-shRNA or HT-29-control cells were seeded in six-well plates at a concentration of 5 times 105 cells per well HT-29-shRNA or HT-29-control cells approaching 80 to 90confluence were transfected with 4mg pc-Src with 10mLof Lipofectamine 2000 (Invitrogen) following the manufac-turerrsquos protocol The cells transfected with empty plasmidpcDNA31 (mock) were used as negative controls

211 Quantitative RT-PCR The cells or tissues were har-vested with Trizol Reagent (Invitrogen Carlsbad CA USA)and total RNA was isolated according to the manufac-turerrsquos instructions cDNA synthesis was performed usingthe Superscript III RT-PCR kit (Invitrogen) Real-time PCRwas carried out using a Cepheid SmartCycler II (Sunny-vale CA USA) with gene-specific real-time PCR primersResults were normalized to GAPDH transcript levels andthe difference in fold expression was calculated using theΔΔ CT method The primers used for c-Src were as followssense 51015840-CTCTTCAGAGCCCTTGCTCA-31015840 and antisense51015840-ATTCACCCTCCCCCAAGGAA-31015840 The length of thePCR products was 193 bp

212 Statistics Analysis Data from all quantitative assayswere expressed as mean plusmn SD andwere analyzed using one-way ANOVA and independent-samples 119905-test All statistical


HT-29 HCC-2998 SW-620 HCT-15 COLO205 FHCPELP1

120573-Actin

(a)

HT-29

HCC

-2998

SW-620

HCT

-15

COLO

205

00

05

10

15

20

Log2

med

ian-

cent

ered

inte

nsity

(b)

Figure 1 PELP1 expression was upregulated inCRC (a) Western blot revealed that PELP1 protein expression was higher in the CRC celllines HT-29 HCC-2998 SW-620 HCT-15 and COLO205 than in the normal colorectal epithelium FHC (b) Informatics data suggested thatPELP1 mRNA expression was increased in these five CRC cell lines

analyses were performed and visualized by GraphPad Prism50 119875 lt 005 was considered statistically significant

3 Results

31 PELP1 Was Upregulated in CRC Cell Lines as Revealedby Western Blot and Bioinformatics We performed westernblot to test the protein expression of PELP1 in the CRC linesCOLO205 HT-29 SW-620 HCC-2998 and HCT-15 and inthe normal cell line FHC As shown in Figure 1(a) PELP1protein expression was higher in the CRC cell lines than inthe immortalized colorectal epithelium The bioinformaticsdata (Figure 1(b)) obtained from the Oncomine database alsoshowed that PELP1 was upregulated in the CRC cell linesThese results implied that PELP1 served oncogenic functionsin CRC

32 PELP1 Downregulation by shRNA Inhibited CRC In VitroTo identify the oncogenic function of PELP1 in CRC weutilized shRNA to silence PELP1 expression in HT-29 Aftertransfection shRNA 3 of PELP1 into HT-29 PELP1 expres-sion decreased by 90 (Figure 2(a)) PELP1 silencing reducedthe proliferation colony formation (by 575) migration (by693) and invasion (by 58) abilities of HT-29 (Figures2(b)ndash2(e)) 120573-Gal senescence assay demonstrated that PELP1silencing promoted the senescence of HT-29 (Figure 2(f))These results suggested that PELP1 downregulation inhibitedthe malignant behavior of CRC in vitro

33 PELP1 Downregulation by shRNA Inhibited XenograftFormation Ability of CRC in NudeMice Xenograft formationability reflects themalignant characteristic of cancer cellsWeperformed a subcutaneous xenograft formation experimenton nude mice to assess whether or not PELP1 can influence

the xenograft formation ability of HT-29 After stable trans-fection with PELP1-shRNA in HT-29 HT-29-control andHT-29-shRNA cells were inoculated subcutaneously in bothflanks of the nude mice Results showed that PELP1-shRNAreduced the xenograft formation ability of HT-29 in nudemice (Figure 3)

34 Oncogenic Function of PELP1 in CRCWas Mediated by c-Src Upregulation c-Src is a known protooncogene in coloncancer Thus we focused on the relationship between PELP1and c-Src in CRC carcinogenesis We tested c-Src expres-sion by quantitative RT-PCR and western blot after PELP1silencing by shRNA in HT-29 to explore the mechanism ofPELP1 downregulation in suppressing CRC carcinogenesisc-Src was reduced after PELP1 silencing at the mRNA(Figure 4(a)) and protein (Figure 4(b)) expression levels Toanalyze whether or not c-Src participates in promoting thefunction of PELP1 in CRC we upregulated the expressionof c-Src after PELP1 silencing As stated previously PELP1downregulation can inhibit the malignant behavior of CRCSurprisingly Src elevation can counteract the inhibitionof CRC induced by PELP1 silencing (Figures 4(c)ndash4(g))Therefore the suppressing function of PELP1 downregulationin CRC carcinogenesis was mediated by c-Src inhibition

4 Discussion

In the present study the oncogenic function of the ER coreg-ulator PELP1 or NMAR was identified in CRC a commonand highly invasive type of cancer We primarily clarifiedthe oncogenic mechanism of PELP1 in relation to c-Src animportant oncogene Although CRC cannot be completelyregarded as a hormone-responsive cancer emerging evidenceimplied that steroid hormones such as estrogen and androgenparticipate in CRC pathogenesis However the functions of


PELP1

120573-Actin

Con

trol

siRN

A1

siRN

A2

siRN

A3

0 1 2 3 4 5 6 700

05

10

15

20

HT-29-controlHT-29-shRNA

Days

Cel

l via

bilit

y

lowast

lowast

PELP1-control PELP1-shRNA

PELP1-control PELP1-shRNA PELP1-control PELP1-shRNA

0

20

40

60

80

100

Col

ony

num

ber (

)

lowast

0

200

400

600

Mig

ratio

n ce

ll nu

mbe

r

lowast

0

50

100

150

200

Inva

sive c

ell n

umbe

r


(a)

(b) (c)

(d) (e)

(f)

Figure 2 PELP1 downregulation inhibited the CRC cell line HT-29 in vitro (a) After transfection with shRNA PELP1 protein expressionwas decreased by 90 shRNA 3 was selected for further investigations (b) After PELP1 silencing the cell viability of HT-29 was inhibited(c) PELP1 silencing inhibited the colony formation ability of HT-29 by 575 (lower panel) A representative colony formation assay is shown(upper panel) (d) PELP1 silencing inhibited the migration ability of HT-29 by 693 (lower panel) A representative migration assay is shown(upper panel) (e) PELP1 silencing inhibited the invasion ability of HT-29 by 58 (lower panel) A representative invasive assay is shown(upper panel) (f) Senescence was induced by PELP1 silencing in HT-29 A representative 120573-Gal assay is shown

ER and AR in CRC are complex and controversial becausemany complicated signaling pathways are involved in estro-gen and androgen signaling ER120573 is usually regarded as aprotective factor in CRC and ER120572 is believed to have noparticipation in CRC [33] Different from ERs androgenreceptors (ARs) serve no [34] suppressive [35] or promotingfunctions [36] in CRC As a nuclear receptor coregulator

PELP1 can interact with ERs ARs glucocorticoid receptorsand progesterone receptors [37] Therefore we analyzed thefunctions of PELP1 in CRC to elucidate the complex signalingnetwork involving ER and AR

Two recent studies have evaluated the expression ofPELP1 in CRC tissues by using immunohistochemistry [2728] However their findings were inconsistent Tzelepi et al


0 1 2 3 4 5

0

500

1000

1500

PELP1-controlPELP1-shRNA

Time after inoculation of cells (weeks)

Tum

or v

olum

e (m

m3)

lowast

(a) (b)

Figure 3 PELP1 downregulation inhibited the CRC cell line HT-29 in nude mice xenograft assay PELP1 silencing inhibited CRC growth innude mice (a) A representative nude mice xenograft assay is shown in (b)

[27] claimed that PELP1 expression decreases from normalcolorectal epithelium to cancerous colorectal epithelium bycontrast Grivas et al [28] found that PELP1 protein expres-sion in epithelial cells increases during colorectal tumorigen-esis despite the fact that PELP1 overexpression in epithelialcells correlates with prolonged overall survival MoreoverGrivas et al [28] found that ER120573 expression in epithelialcells is upregulated during colorectal tumorigenesis in malepatients They also found that ER120573 expression correlateswith increased risk of relapse implying that ER120573 servesan oncogenic function in male CRC patients The presentexperimental results seemed to be partly consistent withthose of Grivas et al That is bioinformatics and western blotassay demonstrated that the mRNA and protein expressionlevels of PELP1 increased in the CRC cell lines COLO205HT-29 SW-620 HCC-2998 and HCT-15 Moreover wefound that the malignant behavior of CRC was inhibited invitro and in vivo after PELP1 silencing by shRNA Theseresults suggested that PELP1 served an oncogenic function inCRC

A decade ago Peyton Rous described a filterable agent(ie virus) that can induce solid tumor formation in birdsAfter approximately 50 years the Rous sarcoma virus wasidentified from Rousrsquo filterable agent Extensive researchinto the molecular biology and genetics of Rous sarcomavirus identified v-Src as a viral oncogene responsible formalignant transformation Then Bishop and Varmus [38]demonstrated that v-Src has a cellular counterpart thefirst identified protooncogene c-Src c-Src is a nonreceptortyrosine kinase that is abnormally expressed in many humancancers and is linked with malignant biological behavior

related to proliferation adhesion migration invasion andmetastasis [39] The oncogenic function of c-Src in CRC hasbeen explored in different studies The dysregulation of c-Src contributed to the initiation and development of CRCIn the present study we linked PELP1 with c-Src in CRCSeveral scholars have identified the fact that an interactionexists between PELP1 and c-Src For example Chakravartyet al [40] found that estrogen-mediated extranuclear sig-naling promotes cytoskeleton reorganization through theER-Src-PELP1-phosphoinositide 3-kinase-ILK1 pathway inbreast cancer PELP1 silencing can significantly inhibit c-Src activation Dimple et al [41] suggested that PELP1downregulation reduces the proliferation and tumorigenicpotential of ovarian cancer cells and affects the magnitudeof c-Src and protein kinase B (AKT) signaling in a nudemouse model Rajhans et al [42] found that the PELP1-mediated induction of aromatase requires functional Srcand phosphatidylinositol-3-kinase pathways Conversely c-Src phosphorylates PELP1 at the C terminal (tyrosine 920)domain [43] However PELP1 can function independent ofc-Src Kayahara et al [44] found that PELP1 functionallyinteracts with both NH2- and COOH-terminal glucocorti-coid receptor domains to modulate transactivation they alsofound through inhibitor and c-Src knockdown studies thatthe function of PELP1 is independent of c-Src activity In thepresent study we found that the oncogenic function of PELP1was partially mediated by c-Src in CRC PELP1 silencingby shRNA downregulated c-Src expression In addition c-Src upregulation partly recovered the oncogenic function ofPELP1 Our research suggested that the oncogenic effect ofPELP1 in CRC was partly mediated by c-Src


HT-29-controlHT-29-c-Src

PELP1

c-Src

120573-Actin

c-Src00

05

10

15


Rela

tive q

uant

ifica

tion

lowast

0 1 2 3 4 5 6 7

00

05

10

15

20

Days

Cel

l via

bilit

y

HT-29-control

HT-29-control

HT-29-c-Src

HT-29-c-Src

0

20

40

60

80

Col

ony

num

ber (

)

HT-29-control HT-29-c-Src0

100

200

300

400

500

Mig

ratio

n ce

ll nu

mbe

r

HT-29-control HT-29-c-Src

0

50

100

150

200

Inva

sive c

ell n

umbe

r

(a)

(b)

(c) (d)

(e) (f)

(g)

Figure 4 PELP1 silencing suppressed CRC through c-Src downregulation (a) PELP1 silencing was accompanied by the downregulation ofc-Src mRNA as determined by quantitative RT-PCR (b) PELP1 silencing was accompanied by c-Src protein downregulation as determinedby western blot (c) Decreased cell viability induced by PELP1 silencing was recovered by c-Src upregulation in HT-29 (d) Decreased colonyformation ability was recovered by c-Src upregulation in HT-29 (e) Decreased migration ability was recovered by c-Src upregulation in HT-29 (f) Decreased invasion ability was recovered by c-Src upregulation in HT-29 (g) Induced senescence by PELP1 silencing was inhibitedafter c-Src upregulation


5 Conclusions

PELP1 a nuclear receptor coregulator exerts oncogenicaction in CRC PELP1 silencing by shRNA promoted thesenescence and inhibited the proliferation colony forma-tion migration invasion and xenograft tumor formationof CRC Moreover PELP1 silencing was accompanied byc-Src downregulation c-Src upregulation partly recoveredthe oncogenic function of PELP1 This study is the first toidentify the oncogenic function of PELP1 inCRC a hormone-nonresponsive cancer Therefore PELP1 can be regarded as atherapeutic target in hormone-nonresponsive cancers

Abbreviations

PELP1 Proline- glutamic acid- and leucine-rich protein 1c-Src Cellular counterpart of Rous sarcoma virusER Estrogen receptorCRC Colorectal cancer

Conflict of Interests

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

Authorsrsquo Contribution

Zhifeng Ning Youzhi Zhang and Hanwei Chen contributedequally to this work

Acknowledgment

This work was supported by the Key Project of HubeiProvince Education Department (D20122802)

References

[1] TGansler P AGanzMGrant et al ldquoSixty years of ca A cancerjournal for cliniciansrdquo CA Cancer Journal for Clinicians vol 60no 6 pp 345ndash350 2010

[2] A Jemal R Siegel J Xu and E Ward ldquoCancer statistics 2010rdquoCA Cancer Journal for Clinicians vol 60 no 5 pp 277ndash3002010

[3] J B OrsquoConnell M A Maggard and C Y Ko ldquoColon cancersurvival rates with the new American Joint Committee onCancer sixth edition stagingrdquo Journal of the National CancerInstitute vol 96 no 19 pp 1420ndash1425 2004

[4] S Narayan andD Roy ldquoRole of APC andDNAmismatch repairgenes in the development of colorectal cancersrdquo MolecularCancer vol 2 article 41 2003

[5] D M Lonard and B W OrsquoMalley ldquoNuclear receptor coregu-lators judges juries and executioners of cellular regulationrdquoMolecular Cell vol 27 no 5 pp 691ndash700 2007

[6] A Rudolph C Toth M Hoffmeister et al ldquoExpression ofoestrogen receptor beta and prognosis of colorectal cancerrdquoBritish Journal of Cancer vol 107 pp 831ndash839 2012

[7] Y Q He J Q Sheng X L Ling et al ldquoEstradiol regulatesmiR-135b and mismatch repair gene expressions via estro-gen receptor-120573 in colorectal cellsrdquo Experimental amp MolecularMedicine vol 44 pp 723ndash732 2012

[8] D Saleiro G Murillo R V Benya M Bissonnette J Hart andR G Mehta ldquoEstrogen receptor-beta protects against colitis-associated neoplasia in micerdquo International Journal of Cancervol 131 pp 2553ndash2561 2012

[9] R M Hasson A Briggs A M Carothers et al ldquoEstrogenreceptor 120572 or 120573 loss in the colon of Min+ mice promotescrypt expansion and impairs TGF120573 and HNF3120573 signalingrdquoCarcinogenesis vol 35 no 1 pp 96ndash102 2014

[10] P D Grivas V Tzelepi G Sotiropoulou-Bonikou Z Kefa-lopoulou A G Papavassiliou and H Kalofonos ldquoEstrogenreceptor 120572120573 AIB1 and TIF2 in colorectal carcinogenesisdo coregulators have prognostic significancerdquo InternationalJournal of Colorectal Disease vol 24 no 6 pp 613ndash622 2009

[11] A Jubb S Bell and P Quirke ldquoMethylation and colorectalcancerrdquoThe Journal of Pathology vol 195 pp 111ndash134 2001

[12] Y Kondo and J P J Issa ldquoEpigenetic changes in colorectalcancerrdquo Cancer and Metastasis Reviews vol 23 no 1-2 pp 29ndash39 2004

[13] J-P J Issa Y L Ottaviano P Celano S R Hamilton NE Davidson and S B Baylin ldquoMethylation of the oestrogenreceptor CpG island links ageing and neoplasia in humancolonrdquo Nature Genetics vol 7 no 4 pp 536ndash540 1994

[14] F Al-Azzawi and M Wahab ldquoEstrogen and colon cancercurrent issuesrdquo Climacteric vol 5 no 1 pp 3ndash14 2002

[15] J Wang S Song L Shi et al ldquoTemporal expression of pelp1during proliferation and osteogenic differentiation of rat bonemarrow mesenchymal stem cellsrdquo PLoS ONE vol 8 Article ID0075477 2013

[16] Y Yan W Zeng S Song et al ldquoVitamin C induces periodontalligament progenitor cell differentiation via activation of ERKpathway mediated by PELP1rdquo Protein Cell vol 4 pp 620ndash6272013

[17] J Wang Q Zhu S Song et al ldquoIncreased PELP1 expressionin rat periodontal ligament tissue in response to estrogenstreatmentrdquo Journal of Molecular Histology vol 44 pp 347ndash3562013

[18] B J Girard A R Daniel C A Lange and J H OstranderldquoPELP1 a review of PELP1 interactions signaling and biologyrdquoMolecular and Cellular Endocrinology vol 8 pp 00326ndash003222013

[19] I M Bennani-Baiti ldquoIntegration of ERalpha-PELP1-HER2signaling by LSD1 (KDM1AAOF2) offers combinatorial ther-apeutic opportunities to circumventing hormone resistance inbreast cancerrdquo Breast Cancer Research vol 14 article 112 2012

[20] V Cortez M Mann S Tekmal et al ldquoTargeting the PELP1-KDM1 axis as a potential therapeutic strategy for breast cancerrdquoBreast Cancer Research vol 14 article R108 2012

[21] S Aust P Horak D Pils et al ldquoThe prognostic value of estrogenreceptor beta and proline- glutamic acid- and leucine-richprotein 1 (PELP1) expression in ovarian cancerrdquo BMC Cancervol 13 pp 1471ndash2407 2013

[22] J Wan and X Li ldquoPELP1MNAR suppression inhibits prolifer-ation and metastasis of endometrial carcinoma cellsrdquo OncologyReports vol 28 pp 2035ndash2042 2012

[23] R K Vadlamudi S Balasenthil R R Broaddus J-A Gustafs-son and R Kumar ldquoDeregulation of estrogen receptor coac-tivator proline- glutamic acid- and leucine-rich protein-1modulator of nongenomic activity of estrogen receptor inhuman endometrial tumorsrdquo Journal of Clinical Endocrinologyand Metabolism vol 89 no 12 pp 6130ndash6138 2004


[24] P Ravindranathan T K Lee L Yang et al ldquoPeptidomimetictargeting of critical androgen receptor-coregulator interactionsin prostate cancerrdquo Nature Communications vol 4 article 19232013

[25] Z Kefalopoulou V Tzelepi V Zolota et al ldquoPrognostic value ofnovel biomarkers in astrocytic brain tumors nuclear receptorco-regulators AIB1 TIF2 and PELP1 are associated with hightumor grade and worse patient prognosisrdquo Journal of Neuro-Oncology vol 106 no 1 pp 23ndash31 2012

[26] D C Marquez-Garban H W Chen M C Fishbein LGoodglick and R J Pietras ldquoEstrogen receptor signalingpathways in human non-small cell lung cancerrdquo Steroids vol72 no 2 pp 135ndash143 2007

[27] V Tzelepi P Grivas Z Kefalopoulou H Kalofonos J NVarakis and G Sotiropoulou-Bonikou ldquoExpression of estrogenreceptor co-regulators NCoR and PELP1 in epithelial cells andmyofibroblasts of colorectal carcinomas cytoplasmic transloca-tion ofNCoR in epithelial cells correlates withworse prognosisrdquoVirchows Archiv vol 454 no 1 pp 41ndash53 2009

[28] P D Grivas V Tzelepi G Sotiropoulou-Bonikou Z Kefalo-poulou A G Papavassiliou and H Kalofonos ldquoExpression ofERaERszlig and co-regulator PELP1MNAR in colorectal cancerprognostic significance and clinicopathologic correlationsrdquoCel-lular Oncology vol 31 no 3 pp 235ndash247 2009

[29] J K LeeDMHavaleshkoH Cho et al ldquoA strategy for predict-ing the chemosensitivity of human cancers and its application todrug discoveryrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 104 no 32 pp 13086ndash130912007

[30] U T Shankavaram W C Reinhold S Nishizuka et al ldquoTran-script and protein expression profiles of the NCI-60 cancercell panel an integromic microarray studyrdquo Molecular CancerTherapeutics vol 6 no 3 pp 820ndash832 2007

[31] M J Garnett E J Edelman S J Heidorn et al ldquoSystematicidentification of genomic markers of drug sensitivity in cancercellsrdquo Nature vol 483 no 7391 pp 570ndash575 2012

[32] H Dong H Guo L Xie et al ldquoThe metastasis-associatedgene MTA3 a component of the Mi-2NuRD transcriptionalrepression complex predicts prognosis of gastroesophagealjunction adenocarcinomardquoPLoSONE vol 8 Article ID e629862013

[33] A Barzi A M Lenz M J Labonte and H J Lenz ldquoMolecularpathways estrogen pathway in colorectal cancerrdquo Clinical Can-cer Research vol 19 pp 5842ndash5848 2013

[34] M N Passarelli A I Phipps J D Potter et al ldquoCommonsingle-nucleotide polymorphisms in the estrogen receptor betapromoter are associated with colorectal cancer survival inpostmenopausal womenrdquo Cancer Research vol 73 pp 767ndash7752013

[35] M L Slattery C Sweeney M Murtaugh et al ldquoAssociationsbetween vitamin D vitamin D receptor gene and the androgenreceptor gene with colon and rectal cancerrdquo InternationalJournal of Cancer vol 118 no 12 pp 3140ndash3146 2006

[36] M Mariani G F Zannoni S Sioletic et al ldquoGender influencesthe class III and V beta-tubulin ability to predict poor outcomein colorectal cancerrdquoClinical Cancer Research vol 18 pp 2964ndash2975 2012

[37] R K Vadlamudi and R Kumar ldquoFunctional and biologicalproperties of the nuclear receptor coregulator PELP1MNARrdquoNuclear receptor signaling vol 5 article e004 2007

[38] M GS ldquoThe hunting of the Srcrdquo Nature Reviews Molecular CellBiology vol 2 pp 467ndash475 2001

[39] S Zhang and D Yu ldquoTargeting Src family kinases in anti-cancer therapies turning promise into triumphrdquo Trends inPharmacological Sciences vol 33 no 3 pp 122ndash128 2012

[40] D Chakravarty S S Nair B Santhamma et al ldquoExtranuclearfunctions of ER impact invasive migration and metastasis bybreast cancer cellsrdquo Cancer Research vol 70 no 10 pp 4092ndash4101 2010

[41] C Dimple S S Nair R Rajhans et al ldquoRole of PELP1MNARsignaling in ovarian tumorigenesisrdquo Cancer Research vol 68no 12 pp 4902ndash4909 2008

[42] R Rajhans H B Nair S S Nair et al ldquoModulation of in situestrogen synthesis by proline- glutamic acid- and leucine-richprotein-1 potential estrogen receptor autocrine signaling loopin breast cancer cellsrdquo Molecular Endocrinology vol 22 no 3pp 649ndash664 2008

[43] B J Cheskis J Greger N Cooch et al ldquoMNAR plays animportant role in ERa activation of SrcMAPK and PI3KAktsignaling pathwaysrdquo Steroids vol 73 no 9-10 pp 901ndash9052008

[44] M Kayahara J Ohanian V Ohanian A Berry R Vadlamudiand D W Ray ldquoMNAR functionally interacts with both NH2-and COOH-terminal GR domains to modulate transactiva-tionrdquo The American Journal of PhysiologymdashEndocrinology andMetabolism vol 295 no 5 pp E1047ndashE1055 2008

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


Table 1 Interference sequence for shRNA

shRNA number Sequence (31015840-51015840)Negative control GCAAGCTGACCCTGAAGTTGAGAACTTTGAAGTCCCAGTCGAACG1 GGAGAAACAAAGGUUAUUUGAGAACTUUUAUUGGAAACAAAGAGG2 CACAGUCUCACAUCGUUUAGAGAACTAUUUGCUACACUCUGACAC3 CCAGGAGCTTGTTGAAGAAGAGAACTAAGAAGTTGTTCGAGGACC

is frequently hypermethylated in CRC [11 12] suggestingthat ER120572 hypermethylation can predict CRC progressionER120572 upregulation in ER120572-negative CRC cell lines can sup-press cell growth [13] The reversal of a hypermethylatedinactive ER120572 can inhibit CRC in vitro and in vivo [14]The estrogen-ER signaling pathway is evidently involvedin CRC development Therefore comprehensive researchmust be conducted to elucidate the functions of ER inCRC

Proline- glutamic acid- and leucine-rich protein 1(PELP1) is a modulator of nongenomic actions of estrogenreceptors (NMAR) and a coregulator of ER PELP1 has beenimplicated in many physiological [15ndash17] and pathologicalprocesses [18] Research has demonstrated that PELP1 isa protooncogene in hormone-responsive cancers such asbreast [19 20] ovarian [21] endometrial [22 23] and prostatecancers [24] Moreover some researchers determined thefunctions of PELP1 in hormone-nonresponsive cancers suchas brain tumor [25] lung cancer [26] and colorectal cancer[27 28]

PELP1 is upregulated in CRC tissues [27 28] Howeverthe exact function of PELP1 in CRC remains unknown Inthe present study we investigated the functions of PELP1in several CRC cell lines Bioinformatics and western blotshowed that PELP1 expression was higher in CRC celllines than in normal colorectal epithelium PELP1 silencingby short hairpin RNA (shRNA) promoted the senescenceand inhibited the proliferation colony formation migrationinvasion and xenograft tumor formation of the CRC cell lineHT-29 Moreover PELP1 silencing was accompanied by c-Src downregulation c-Src upregulation partly recovered theoncogenic function of PELP1 These results demonstratedthat PELP1 suppression can inhibit CRC in vitro and in vivothrough c-Src downregulation

2 Materials and Methods

21 Cell Line The CRC cell lines COLO205 HT-29 SW-620 HCC-2998 and HCT-15 and normal colon epitheliumcell line FHC were purchased from American Type CultureCollection (Manassas VA) The five CRC cell lines werecultured in RPMI1640 medium supplemented with 10 fetalbovine serum 100UmL penicillin and 100 120583gmL strepto-mycin FHC was cultured in DMEMF12 medium with thesame supplement as the CRC cell lines All cell lines weremaintained at 37∘C in a humidified atmosphere of 5 CO

2

in an open-air incubator

22 Bioinformatics We performed a bioinformatics analy-sis on Oncomine (httpswwwoncomineorgresourceloginhtml) to analyze PELP1 expression in the CRC cell lines Weused a personal account to enter the Oncomine website Weinput PELP1 on the search box to begin the search Afterentering the page for PELP1 expression survey in almost allhuman cancer cell lines or tissues we focused on CRC andobtained PELP1 expression data from the Lee et al [29]Shankavaram et al [30] and Garnett [31] cell line datasetsAfter data standardization we presented PELP1 expressiondata in the five CRC cell lines as mean plusmn standard deviation(SD)

23 shRNA Interference of PELP1 HT-29 cells were seededin 24-well plates (Corning) until they reached 50 to 60confluence prior to transfection Then stable transfectionwas performedThree shRNA sequences targeting the PELP1gene were designed and synthesized by Shanghai Jima Phar-maceutical Technology China The three shRNA sequencesof PELP1 and the negative control sequence are listed inTable 1 Lipofectamine LTX and Plus Reagent (Invitrogen)were used to transfect shRNA into HT-29 cells accordingto the manufacturerrsquos protocol The medium containingtransfection reagents was replaced with RPMI1640 mediumsupplemented with 10 FBS at 18 h after the transfectionThecells were collected at 48 h after the transfection processedin the following experiments and then prepared for proteinextraction The silence efficiency of PELP1 was tested bywestern blot

24 Western Blot Total protein was extracted from HT-29-shRNA or HT-29-control cells using RIPA lysate buffer asdescribed in our previous research [32] After quantifica-tion by a bicinchoninic acid protein assay kit (BeyotimeBiotechnology China) equal amounts of proteins (20120583gto 25 120583g) were separated by 12 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and processedfor immunoblotting with a rabbit multiclonal antibody forPELP1 (diluted at 1 100 Santa Cruz) and c-Src (dilutedat 1 500 Santa Cruz) A mouse polyclonal anti-120573-actinantibody (diluted at 1 2000 Boster Biological EngineeringChina)was used as an internal control All protein bandswerescanned using ChemiImager 5500 V203 software and theintegrated density values were calculated by a computerizedimage analysis system (Fluor Chen 20) and normalized withthat of 120573-actin

25 MTT Cell viability and proliferation activity were deter-mined with the MTT assay HT-29-shRNA or HT-29-control



2) The



2) The cells were





2) with a











120573-Actin

(a)

HT-29

HCC

-2998

SW-620

HCT

-15

COLO

205

00

05

10

15

20

Log2

med

ian-

cent

ered

inte

nsity

(b)



3 Results






4 Discussion



PELP1

120573-Actin

Con

trol

siRN

A1

siRN

A2

siRN

A3

0 1 2 3 4 5 6 700

05

10

15

20


Days

Cel

l via

bilit

y

lowast

lowast



0

20

40

60

80

100

Col

ony

num

ber (

)

lowast

0

200

400

600

Mig

ratio

n ce

ll nu

mbe

r

lowast

0

50

100

150

200

Inva

sive c

ell n

umbe

r


(a)

(b) (c)

(d) (e)

(f)






0 1 2 3 4 5

0

500

1000

1500



Tum

or v

olum

e (m

m3)

lowast

(a) (b)







PELP1

c-Src

120573-Actin

c-Src00

05

10

15


Rela

tive q

uant

ifica

tion

lowast

0 1 2 3 4 5 6 7

00

05

10

15

20

Days

Cel

l via

bilit

y

HT-29-control

HT-29-control

HT-29-c-Src

HT-29-c-Src

0

20

40

60

80

Col

ony

num

ber (

)


100

200

300

400

500

Mig

ratio

n ce

ll nu

mbe

r


0

50

100

150

200

Inva

sive c

ell n

umbe

r

(a)

(b)

(c) (d)

(e) (f)

(g)



5 Conclusions


Abbreviations






Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2) The



2) The cells were





2) with a











120573-Actin

(a)

HT-29

HCC

-2998

SW-620

HCT

-15

COLO

205

00

05

10

15

20

Log2

med

ian-

cent

ered

inte

nsity

(b)



3 Results






4 Discussion



PELP1

120573-Actin

Con

trol

siRN

A1

siRN

A2

siRN

A3

0 1 2 3 4 5 6 700

05

10

15

20


Days

Cel

l via

bilit

y

lowast

lowast



0

20

40

60

80

100

Col

ony

num

ber (

)

lowast

0

200

400

600

Mig

ratio

n ce

ll nu

mbe

r

lowast

0

50

100

150

200

Inva

sive c

ell n

umbe

r


(a)

(b) (c)

(d) (e)

(f)






0 1 2 3 4 5

0

500

1000

1500



Tum

or v

olum

e (m

m3)

lowast

(a) (b)







PELP1

c-Src

120573-Actin

c-Src00

05

10

15


Rela

tive q

uant

ifica

tion

lowast

0 1 2 3 4 5 6 7

00

05

10

15

20

Days

Cel

l via

bilit

y

HT-29-control

HT-29-control

HT-29-c-Src

HT-29-c-Src

0

20

40

60

80

Col

ony

num

ber (

)


100

200

300

400

500

Mig

ratio

n ce

ll nu

mbe

r


0

50

100

150

200

Inva

sive c

ell n

umbe

r

(a)

(b)

(c) (d)

(e) (f)

(g)



5 Conclusions


Abbreviations






Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















120573-Actin

(a)

HT-29

HCC

-2998

SW-620

HCT

-15

COLO

205

00

05

10

15

20

Log2

med

ian-

cent

ered

inte

nsity

(b)



3 Results






4 Discussion



PELP1

120573-Actin

Con

trol

siRN

A1

siRN

A2

siRN

A3

0 1 2 3 4 5 6 700

05

10

15

20


Days

Cel

l via

bilit

y

lowast

lowast



0

20

40

60

80

100

Col

ony

num

ber (

)

lowast

0

200

400

600

Mig

ratio

n ce

ll nu

mbe

r

lowast

0

50

100

150

200

Inva

sive c

ell n

umbe

r


(a)

(b) (c)

(d) (e)

(f)






0 1 2 3 4 5

0

500

1000

1500



Tum

or v

olum

e (m

m3)

lowast

(a) (b)







PELP1

c-Src

120573-Actin

c-Src00

05

10

15


Rela

tive q

uant

ifica

tion

lowast

0 1 2 3 4 5 6 7

00

05

10

15

20

Days

Cel

l via

bilit

y

HT-29-control

HT-29-control

HT-29-c-Src

HT-29-c-Src

0

20

40

60

80

Col

ony

num

ber (

)


100

200

300

400

500

Mig

ratio

n ce

ll nu

mbe

r


0

50

100

150

200

Inva

sive c

ell n

umbe

r

(a)

(b)

(c) (d)

(e) (f)

(g)



5 Conclusions


Abbreviations






Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PELP1

120573-Actin

Con

trol

siRN

A1

siRN

A2

siRN

A3

0 1 2 3 4 5 6 700

05

10

15

20


Days

Cel

l via

bilit

y

lowast

lowast



0

20

40

60

80

100

Col

ony

num

ber (

)

lowast

0

200

400

600

Mig

ratio

n ce

ll nu

mbe

r

lowast

0

50

100

150

200

Inva

sive c

ell n

umbe

r


(a)

(b) (c)

(d) (e)

(f)






0 1 2 3 4 5

0

500

1000

1500



Tum

or v

olum

e (m

m3)

lowast

(a) (b)







PELP1

c-Src

120573-Actin

c-Src00

05

10

15


Rela

tive q

uant

ifica

tion

lowast

0 1 2 3 4 5 6 7

00

05

10

15

20

Days

Cel

l via

bilit

y

HT-29-control

HT-29-control

HT-29-c-Src

HT-29-c-Src

0

20

40

60

80

Col

ony

num

ber (

)


100

200

300

400

500

Mig

ratio

n ce

ll nu

mbe

r


0

50

100

150

200

Inva

sive c

ell n

umbe

r

(a)

(b)

(c) (d)

(e) (f)

(g)



5 Conclusions


Abbreviations






Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 1 2 3 4 5

0

500

1000

1500



Tum

or v

olum

e (m

m3)

lowast

(a) (b)







PELP1

c-Src

120573-Actin

c-Src00

05

10

15


Rela

tive q

uant

ifica

tion

lowast

0 1 2 3 4 5 6 7

00

05

10

15

20

Days

Cel

l via

bilit

y

HT-29-control

HT-29-control

HT-29-c-Src

HT-29-c-Src

0

20

40

60

80

Col

ony

num

ber (

)


100

200

300

400

500

Mig

ratio

n ce

ll nu

mbe

r


0

50

100

150

200

Inva

sive c

ell n

umbe

r

(a)

(b)

(c) (d)

(e) (f)

(g)



5 Conclusions


Abbreviations






Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















PELP1

c-Src

120573-Actin

c-Src00

05

10

15


Rela

tive q

uant

ifica

tion

lowast

0 1 2 3 4 5 6 7

00

05

10

15

20

Days

Cel

l via

bilit

y

HT-29-control

HT-29-control

HT-29-c-Src

HT-29-c-Src

0

20

40

60

80

Col

ony

num

ber (

)


100

200

300

400

500

Mig

ratio

n ce

ll nu

mbe

r


0

50

100

150

200

Inva

sive c

ell n

umbe

r

(a)

(b)

(c) (d)

(e) (f)

(g)



5 Conclusions


Abbreviations






Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5 Conclusions


Abbreviations






Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article PELP1 Suppression Inhibits Colorectal Cancer through …downloads.hindawi.com/journals/omcl/2014/193523.pdf · Research Article PELP1 Suppression Inhibits Colorectal