Research Article Long Noncoding RNA-LET Suppresses Tumor...

Research ArticleLong Noncoding RNA-LET Suppresses Tumor Growth and EMTin Lung Adenocarcinoma

Bin Liu Chun-Feng Pan Zhi-Cheng He Jun Wang Peng-Li Wang Teng MaYang Xia and Yi-Jiang Chen

Department of Thoracic and Cardiovascular Surgery The First Affiliated Hospital of Nanjing Medical UniversityNanjing Jiangsu 210029 China

Correspondence should be addressed to Yi-Jiang Chen yijiangchenkxy126com

Received 22 August 2016 Revised 12 October 2016 Accepted 19 October 2016

Academic Editor Noriyoshi Sawabata

Copyright copy 2016 Bin Liu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Recently many studies showed that long noncoding RNAs (lncRNAs) are involved in tumor progression It is reported thatlncRNA-LET is downregulated and has antitumor effect on several types of cancer This study focuses on the role of lncRNA-LET on lung adenocarcinoma (LAC) progression RT-PCR results indicated that frequent downregulation of lncRNA-LET in LACtissues was related to clinicopathologic factors lncRNA-LET knockdown significantly promoted LAC cell proliferation invasionand migration while lncRNA-LET overexpression obviously inhibited LAC cell proliferation invasion and migration indicatinga tumor inhibition of lncRNA-LET in LAC progression Besides lncRNA-LET inhibited EMT and negatively regulated Wnt120573-catenin pathway in part Our study suggests that lncRNA-LET exhibits an important tumor-suppressive effect on LAC progressionby inhibiting EMT and Wnt120573-catenin pathway which provides potential therapeutic targets for LAC

1 Introduction

As the leading cause of cancer death there were about1800000 new lung cancer casesrsquo occurrence in 2012 about13 of total cancer diagnoses [1] Lung cancer mainly con-tains two types small cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) accounting for 15 and 85 oftotal lung cancer diagnoses respectively [2] As the mostwidespread histological type of NSCLC lung adenocarci-noma (LAC) leads to more than 500000 deaths every yeararound the world [3] Therefore it is of great clinical value tofurther reveal themolecularmechanisms of LAC progressionand development and develop new therapeutic targets forLAC patients

As a type of long noncoding RNA transcripts longnoncoding RNAs (lncRNAs) are longer than 200 nucleotidesin length [4] Increasing evidences have shown that lncRNAsare involved in tumor progression [5ndash7]

LncRNA-Low Expression in Tumor (lncRNA-LET) arecently identified lncRNA located at chromosome 15q241has been demonstrated to be downregulated and plays atumor-suppressive role in several types of cancer such as

hepatocellular carcinomas nasopharyngeal carcinoma cer-vical cancer and gallbladder cancer [8ndash11] However to ourknowledge the effect of lncRNA-LET in progression anddevelopment of LAC is still unknown

In this study we detected the expression level of lncRNA-LET in LAC tumor tissues and paracarcinoma tissues andinvestigated its relationship with clinicopathologic factors inLAC Then tumor inhibition of lncRNA-LET was exploredin vitro and in vivo Moreover we also assayed the molecularmarker levels of EMT and 120573-catenin to reveal inhibition oflncRNA-LET on EMT and Wnt120573-catenin pathway There-fore this study showed that lncRNA-LET has a significantrole in LAC progression and development and may act as apotential therapeutic target for LAC

2 Materials and Methods

21 Patients and Tissue Samples In all 60 LAC tissuesand matched paracarcinoma tissues used in this study wereobtained from patients undergoing thoracoscopic and opencardiac surgery in Jiangsu Province People Hospital from

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4693471 9 pageshttpdxdoiorg10115520164693471

2 BioMed Research International

2013 to 2014 In this study LAC patients had not receivedchemotherapy or radiotherapy treatment All surgical spec-imens were immediately frozen in liquid nitrogen until useThis research was approved by Jiangsu Province People Hos-pital and had received informed consents from all patientsLAC patients were staged in accordance with the tumor nodemetastasis (TNM) staging system (the 7th edition) of theAJCC staging system

22 Cell Culture 16HBE cells PC-9 cells H358 cells SPCA1cells H1975 cells H1299 cells and A549 cells were culturedin RPMI1640 medium containing 10 fetal calf serum100UmL penicillin and 100 120583gmL streptomycin within ahumidified atmosphere at 37∘C containing 5 CO

2

23 Plasmid andTransfection For overexpression of lncRNA-LET in H1299 cells the lncRNA-LET gene was clonedinto the lentiviral-vector pCDH-CMV-MCS-EF1-copGFP(System Biosciences Mountain View CA) and the primersequences are as follows lncRNA-LET sense 51015840-GTTGTT-GTTGCATTGGGGT-31015840 antisense 51015840-AAGATGGAGAGT-GGAGCCT-31015840 H1299 cells were transfected with LV-Vectoror LV-LncRNA-LET For knockdown of lncRNA-LET inA549 cells the sequences of shRNA targeting for lncRNA-LET were designed as 51015840-GGAGGGUGCUUGACAAUA-AUU-31015840 and the NC-shRNA was purchased from Genechem(Shanghai China) A549 cells were transfected with NC-shRNA or LncRNA-shRNA

24 RNA Extraction and qRT-PCR lncRNA-LET and nu-clear 120573-catenin expression level were measured by qRT-PCR In brief trizol reagent (Invitrogen Carlsbad CA)was used to extract the RNA according to the manufac-turerrsquos instruction Reverse TranscriptionKit (Takara DalianChina) was used to synthesize cDNAs The real-time reversetranscription PCR was carried out by using Power SYBRGreen (Takara Dalian China) GAPDH was used as aninternal control qRT-PCR and data collection were carriedout by the ABI7500 system (Applied Biosystems Foster CA)The relative levels of mRNAs were calculated using the 2minusΔΔCtmethod lncRNA-LET 51015840-CCTTCCTGACAGCCAGTGTG-31015840 (forward) 51015840-CAGAATGGAAATACTGGAGCAAG-31015840 (reverse) 120573-catenin 51015840-GCTTTCAGTTGAGCTGAC-CA-31015840 (forward) 51015840-CAAGCTCAAGATCAGCAGTCTC-31015840 (reverse) GADPH 51015840-GTCAACGGATTTGGTCTG-TATT-31015840 (forward) 51015840-AGTCTTCTGGGTGGCAGTGAT-31015840 (reverse)

25 Cell Proliferation Cells were seeded onto 96-well plateand incubated overnight at 37∘C containing 5CO

2 20120583L of

MTT solution per well was added into the 96-well plate andcultured for 4 h at 37∘C Then 150 120583L DMSO was added intothe 96-well plate and incubated for 10min The absorbancewas read by an enzyme-linked immunosorbent assay (ELISA)reader at 490 nm

26 Wound Healing Assay Cells were spread at the bottomof 12-well plates marked by a horizontal line on the back for

targeting the same field of vision a line wound was madeby scraping 100 120583L tips across the confluent cell layer Aftersucking cell culture and washing the cells for three times toremove detached cells and debris the serum-free mediumwas added into the 12-well plates and incubated under theusual culture conditions for 48 hwound closurewas capturedusing a lightmicroscope (DFC500 LeicaWetzlar Germany)AxioVision version 47 software (Carl Zeiss Meditec DublinCA USA) was used to measure the wound closure

27 Transwell Assay Cell invasion assay was operated bypolycarbonatemembrane Boyden chamber insert (EMDMil-lipore Billerica MA USA) The transfected cells were addedinto the upper invasion chamber the 500120583L culture mediumwas added into the lower invasion chamber 45120583g Matrigel(BD Biosciences San Jose CA USA) was used to precoateach insert The Matrigel invasion chambers were incubatedunder the usual culture conditions for 48 h After removingthe filter inserts and the cells on the upper side of the filterthe invaded cells on the lower chamber were suspendedin 100 precooling methanol for 10min Then cells werestained with 005 crystal violet for 30min washed with PBSbuffer and captured using microscopic inspection (DFC500Leica) according to the manufacturerrsquos instructions Finallythe values for invasion were carried out under three fields permembrane

For cell migration assay by transwell assay each insertneededwas not precoated withMatrigelThe other steps weresimilar to cell invasion assay

28 Western Blot In order to investigate expression level of120573-catenin E-cadherin N-cadherin c-Myc and COX-2 cellswere collected and cell lysis was performed by using RIPAlysis buffer (Cell Signal Technology Danvers MA) includingprotease inhibitors on ice according to the manufacturerrsquosinstruction The extracted protein was quantified by bicin-choninic acid quantification assay (Pierce BiotechnologyInc Rockford IL USA) Then the total cellular proteinswere subjected to SDS-PAGE (10) for western analysisAfter transferring to polyvinylidene difluoride (PVDF)mem-branes blots were incubated with 5 BSA in Trisbufferedsaline containing 05 Tween 20 for 60min and incubatedovernight at 4∘C on a rocker with the following primaryantibodies polyclonal rabbit anti-human 120573-catenin antibody(1 1000) monoclonal mouse anti-human E-cadherin anti-body (1 1000) polyclonal rabbit anti-human N-cadherinantibody (1 1000) monoclonal rabbit anti-human c-Mycantibody (1 1000) and polyclonal rabbit anti-human COX-2 antibody (1 1000) Following washing three times withTBS-T for 5min the blots were incubated with horseradishperoxidase- (HRP-) conjugated goat anti-rabbit IgG HampLpolyclonal secondary antibody (1 1000) at room temper-ature for 1 h All antibodies were purchased from Abcam(Cambridge MA USA) The bands were captured under anenhanced chemiluminescence detection system (GE Health-care Life Sciences)

BioMed Research International 3

0

1

2

3

4

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET

lowastlowastlowast

P (n = 60) T (n = 60)

(a)

00

05

10

15

16H

BE

A54

9

PC-9

H35

8

SPCA

1

H12

99

H19

75

20

25

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET

(b)

0

1

220

30

40

A549H1299

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET

lowast

lowast

NC-

shRN

A

lncR

NA

-shR

NA

LV-V

ecto

r

LV-ln

cRN

A

(c)

Figure 1 Expression of lncRNA-LET in LAC patients and cell lines (a) Analysis of lncRNA-LET expression level in paracarcinoma tissue(P) and tumor tissue (T) Total RNA was detected by quantitative real-time PCR (qRT-PCR) and GAPDH was used as an internal controllncRNA-LET was significantly downregulated in LAC tumor tissues compared with the paracarcinoma tissue (b) Analysis of lncRNA-LETexpression level in seven cell lines was detected by using RT-PCR GAPDH was used as an internal control (c) Analysis of transfectionefficiency in A549 cells and H1299 cells lncRNA-LET expression in A549 cell line after being transfected with lncRNA-LET-shRNA or NC-shRNA was determined by qRT-PCR lncRNA-LET expression in H1299 cell line after being transfected with LV-LncRNA-LET or LV-Vectorwas determined by qRT-PCR Data are presented as the mean plusmn SD of three independent experiments lowastlowastlowast119875 lt 0001 lowast119875 lt 005

29 Xenograft Model All mice experiments were operatedwith the approval of the Animal Studies Ethics Committeeof the First Affiliated Hospital of Nanjing Medical Universityaccording to the Guide for the Care and Use of LaboratoryAnimals published by the US National Institutes of Health(NIH publication number 85-23 revised 1996) H1299 cells(6 times 104mL) transfected with LV-LncRNA-LET or LV-Vectorwere implanted into the flanks ofNODSCIDmice (4-5weeksold) subcutaneously The tumor volumes were evaluated byusing calipers The mice were sacrificed and the grafts wereremoved after 6 weeks

210 HE Staining The grafts were fixed by formalin andembedded by paraffin Tumor tissue sections were cut to4 120583m thickness and mounted in Poly-Lysine (Sigma USA)coated glass slides Sections were deparaffinized in xylenedehydrated in a decreasing ethanol series and performed byHE staining

211 Immunohistochemistry After sections were deparaf-finized and dehydrated sections were immersed in methanolwith 03 (volvol) H

2O2for 30min Sections were heated

in citrate buffer (10mM pH 60) at 120∘C for 5min

Then the sections were incubated with primary antibody(polyclonal rabbit anti-human PCNA antibody) overnightat 4∘C After washing with PBS sections were incubatedwith biotinylated secondary antibody at room temperaturefor 1 h Diaminobenzidine (005 for 10min at temperature)was employed to be as chromogen Slides were stainedwith Mayerrsquos hematoxylin solution and mounted in Entellan(Merck KGaA Darmstadt Germany)

212 Statistical Analysis All statistical analyses were carriedout by SPSS 170 and the data were expressed as the means plusmnSD Comparison of lncRNA-LET level between tumors andparacarcinoma was performed using the Wilcoxon test Thecorrelation between lncRNA-LET level and clinical charac-teristics was analyzed using the chi-squared test119875 lt 005wasconsidered to indicate a statistically significant difference

3 Results

31 Expression of lncRNA-LET in LAC Patients and CellLines The expression of lncRNA-LET in LAC tumor tissuesand paracarcinoma tissues from 60 patients was detected byquantitative real-time PCR (qRT-PCR) Figure 1(a) showed


Table 1 lncRNA-LET expression and clinicopathologic factors in lung adenocarcinoma (LAC)

lncRNA-LET expressionCharacteristics Case number Low High 119875 valueGender

Male 23 11 12 0791Female 37 19 18

Age (year)lt60 38 20 18 0592ge60 22 10 12

Site of tumorLeft lung 25 14 11 0757Right lung 35 21 14

Histological gradeModerately 36 15 21 0027lowastPoorly 24 17 7

Tumor stageIII 36 14 22 0015lowastIIIIV 24 17 7

Lymph node metastasisNegative 20 6 14 0028lowastPositive 40 24 16

Tumor sizeT1T2 38 17 21 0284T3T4 22 13 9lowast indicates 119875 lt 005

that expression of lncRNA-LET was significantly down-regulated in lung adenocarcinoma tissues compared withparacarcinoma tissues (119875 lt 0001) suggesting that frequentdownregulation of lncRNA-LET in LAC may be related toLAC pathogenesis To identify the correlation of lncRNAexpression with clinicopathologic factors we divided the 60LACpatients into a high level and a low level group accordingto the mean level of lncRNA-LET The clinicopathologicfactors were analyzed in Table 1 Compared with high levelgroup of lncRNA-LET low level group of lncRNA-LET wassignificantly associated with a less differentiated histologyhigher tumor stage and more lymph node metastasis (119875 lt005) but not correlated with gender age site of tumor andtumor size (119875 gt 005)

In addition we detected the expression level of lncRNA-LET in several normal and LAC cell lines via using RT-PCR and showed that comparing with 16HBE cell line A549and H358 cells expressed the higher level of lncRNA-LETwhile other cell lines expressed lower level of lncRNA-LET(Figure 1(b)) To explore the role of lncRNA-LET in LACprogression and development we chose A549 cell line forlncRNA-LET knockdown and H1299 cell line for lncRNA-LET overexpression and the transfection efficiency wassubsequently detected via RT-PCR as shown in Figure 1(c)The lncRNA-LET expression was effectively suppressed inA549 cells by lncRNA-shRNA and elevated in H1299 cells byLV-lncRNA

32 Effects of lncRNA-LET on LAC Cell Proliferation Migra-tion and Invasion Since lncRNA-LET is downregulated inLAC and associated with the progression of LAC we nextexplored the role of lncRNA-LET in LAC cell lines Asshowed in Figure 2(a)MTT assays showed that lncRNA-LETknockdown significantly promoted cell proliferation of A549cells while lncRNA-LET overexpression obviously inhibitedcell proliferation of H1299 cells Then wound healing assaywas employed to determine cell migration Compared withthe control group A549 cells with lncRNA-LET knock-down exhibited stronger migration while overexpressionof lncRNA-LET significantly impaired migration ability inH1299 cells (Figure 2(b)) On the other hand as showedin Figure 2(c) effects of lncRNA-LET on cell migration byusing transwell assay were the same as that performed bywound healing assay Besides Transwell assay also revealedeffects of lncRNA-LET on cell invasion (Figure 2(c)) showingthat lncRNA-LET knockdown markedly accelerated A549cell invasion and lncRNA-LET overexpression obviouslyinhibited H1299 cell invasion These results demonstratedthat lncRNA-LET might be involved in progression anddevelopment of LAC

33 lncRNA-LET Inhibited EMT and the Canonical Wnt120573-Catenin Pathway To demonstrate whether the lncRNA-LETaffected EMT we employed western blot to assay themolecu-lar marker levels of EMT Figure 3(a) displayed that lncRNA-LET knockdown inhibited E-cadherin level and elevated


Day 1 Day 2 Day 300

05

10

15

20

25

30

NC-shRNAlncRNA-shRNA

Day 1 Day 2 Day 300

05

10

15

20

LV-VectorLV-lncRNA

A549 H1299

lowast

lowastlowast

lowast

Cel

l num

ber (

1lowast10

5)

Cel

l num

ber (

1lowast10

5)

(a)

A549 H1299NC-shRNA lncRNA-shRNA LV-Vector LV-lncRNA

0h

48

h

(b)Migration Invasion

NC-shRNA lncRNA-shRNA NC-shRNA lncRNA-shRNA

LV-Vector LV-lncRNA LV-Vector LV-lncRNA

A54

9H

1299 0

50

100

150

200600

700

800

A549H1299

Mig

ratio

n ce

ll nu

mbe

r

0

50

100

150

200700

800

900

A549H1299

Inva

sion

cell

num

ber

lowast lowast

lowastlowast

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

(c)

Figure 2 Effects of lncRNA-LET on LAC cell proliferation migration and invasion (a) MTT assays were performed to detect theproliferation of A549 cells and H1299 cells (b) Wound healing assays were performed to determine the migration of A549 cells and H1299cells (c) Transwell assay was employed to assay the migration and invasion of A549 cells and H1299 cells A549 cells were transfected withlncRNA-LET-shRNA or NC-shRNA and H1299 cells were transfected with lncRNA-LET-shRNA or NC-shRNA Data are presented as themean plusmn SD of three independent experiments lowast119875 lt 005

levels of N-cadherin c-Myc and COX-2 in A549 cells whilelncRNA-LET overexpression elevated levels of E-cadherinand inhibited levels of N-cadherin c-Myc and COX-2 inH1299 cells indicating that lncRNA-LET could inhibit EMT

TheWnt120573-catenin signaling pathway plays an importantrole in the development of EMT and cancer metastasis [12]Thus we further determine the 120573-catenin protein level incell nucleus by using western blot We found that A549 cellswith lncRNA-LET knockdown exhibited more accumulationof 120573-catenin than control group and H1299 cells withlncRNA-LET overexpression showed a contrary tendency(Figure 3(b)) Besides the mRNA expression of nuclear120573-catenin in LAC tissues was also detected by using RT-PCR and we demonstrated that accumulation of nuclear 120573-catenin in LAC tissues compared with paracarcinoma tissues(Figure 3(c)) Taken together these results suggested that

lncRNA-LET expression inhibited EMT and the canonicalWnt120573-catenin pathway in part

34 Effects of lncRNA-LET on LAC Tumor Growth In VivoH1299 cells transfected with LV-lncRNA-LET or LV-Vectorwere injected intomice subcutaneously lncRNA-LET expres-sion in tumor tissues was detected by qRT-PCR and showedthe lncRNA-LET overexpression in LV-lncRNA-LET groupcompared with that in LV-Vector group (Figure 4(a)) Tumorvolume in lncRNA-LET overexpression group was signifi-cantly lower than tumor in the control group (Figure 4(b))Immunohistochemical staining of tumor tissues showed thatthere was a decrease in proliferation marker (PCNA) in LV-lncRNA-LET group versus LV-Vector group (Figure 4(c))These data verified the tumor inhibition of lncRNA-LET invivo


A549NC-shRNA

lncRNA-shRNAE-cadherin

N-cadherin

C-Myc

COX-2

H1299LV-Vector

LV-lncRNAminus

minus+

+minus

minus+

+

E-cadherin

N-cadherin

C-Myc

COX-2

E-cadherin N-cadherin C-Myc COX-20

1

2

3


LV-VectorLV-lncRNA

Relat

ive p

rote

in ex

pres

sion

lowast

lowast lowast

lowast

lowast

lowastlowastlowast

120573-Actin 120573-Actin

(a)

H1299

LV-Vector LV-lncRNA

A549


NC-shRNA lncRNA-shRNA0

1

2

3

A549LV-Vector LV-lncRNA

00

05

10

15

H1299

lowastlowast

120573-Catenin

120573-Actin

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

(b)

0

1

2

3

4lowastlowast

Relat

ive e

xpre

ssio

n of

nuc

lear

120573-c

aten

in

P (n = 60) T (n = 60)

(c)

Figure 3 lncRNA-LET expression inhibited EMT and canonical Wnt120573-catenin pathway in part (a) Western blot was performed to assaythe molecular marker levels of EMT (b) Western blot was performed to assay the protein level of nuclear 120573-catenin in LAC cell lines Therelative protein levels were normalized to 120573-actin A549 cells were transfected with lncRNA-LET-shRNA or NC-shRNA andH1299 cells weretransfected with lncRNA-LET-shRNA or NC-shRNA Relative protein expression was quantified by the Image J software (c) RT-PCR wasperformed to assay the mRNA level of nuclear 120573-catenin in paracarcinoma tissue (P) and tumor tissue (T) Data are presented as the mean plusmnSD of three independent experiments lowastlowast119875 lt 001 lowast119875 lt 005

4 Discussion

In the present study we demonstrated that the expressionlevel of lncRNA-LETwas significantly downregulated in lungadenocarcinoma tissues compared with adjacent nontumortissues and low level group of lncRNA-LET was significantlyassociated with a less differentiated histology higher tumorstage and more lymph node metastasis compared with

high level group of lncRNA-LET suggesting a crucial roleof lncRNA-LET in progression and development of LACFurthermore we also found that lncRNA-LET knockdownsignificantly promoted A549 cell proliferation invasionand migration while lncRNA-LET overexpression obviouslyinhibited H1299 cell proliferation invasion and migrationIn addition we further tested the effect of lncRNA-LETin xenograft model and found that lncRNA-LET could


LV-Vector LV-lncRNA0

5

10

15

20

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET lowast

(a)


100

200

300

400

3 6 9 12 150

100

200

300

400

LV-VectorLV-lncRNA

(Days)

lowast

Tum

or v

olum

e (m

m3)

Tum

or v

olum

e (m

m3)

(b)

LV-Vector LV-lncRNA

HampE

PCNA

(c)

Figure 4 lncRNA-LET inhibits LAC tumor growth and cell proliferation in vivo (a) Analysis of lncRNA-LET expression level in tumortissues was detected by using RT-PCR GAPDHwas used as an internal control (b) Tumor volume in lncRNA-LET overexpression group wasdetermined by using calipers (c) HE and immunohistochemical staining showed that lncRNA-LET overexpression inhibited the aggressivephenotype of LAC cells in vivo as indicated by the expression of PCNA-positive cells Data are presented as the mean plusmn SD of threeindependent experiments lowast119875 lt 005

inhibit tumor growth and proliferation Thus our resultsdemonstrated that lncRNA-LET acts as a tumor suppressorin progression and development of LAC

Previous studies have showed that EMT was involvedin cell motility and invasiveness in several types of cancerprogression including LAC and control or suppression oftumor cell epithelial-mesenchymal transition (EMT) couldsignificantly inhibit metastasis and growth of LAC [13ndash16]EMT is a conserved cellular process in which epithelial tumorcell lacks its polarity and transforms into a mesenchymalphenotype [16 17] The feature of EMT occurrence is thatthe epithelial marker E-cadherin is downregulated and mes-enchymal marker like N-cadherin is upregulated [18 19]To demonstrate whether the lncRNA-LET affected EMT wedetected the molecular marker levels of EMT and showed theinhibition of lncRNA-LET on EMT

Recently it has been reported that overexpression ofWnt signaling pathway is involved in metastasis of lungcancer [12 20] Wnt120573-catenin pathway can control cellproliferation by regulating the expression of cell cycle-relatedgene including c-Myc andmodulate EMT by downregulatingE-cadherin and upregulating N-cadherin [21 22] Besidesaccumulation of 120573-catenin in cell nucleus indicates thatcanonical Wnt120573-catenin pathway is activated and relatedto tumor progression and metastasis [23 24] Thus wefurther determined the 120573-catenin level in cell nucleus andLAC tissues and found that accumulation of nuclear 120573-catenin in LAC tissues compared with paracarcinoma tissuesMoreover lncRNA-LET expression could block activation ofcanonical Wnt120573-catenin pathway In our previous study wehave demonstrated that lncRNA-LET functions as a tumorsuppressor gene by activating p53 in esophageal squamous


cell carcinoma cells [25] In addition p53 acts upstreamof Wnt to suppress canonical Wnt signaling pathway [26]Thus we speculate that lncRNA-LET may suppress Wnt120573-catenin pathway by regulating p53 in LAC Although betterevidence is necessary the present study also supports therationale of an important role for lncRNA-LET that playstumor-suppressive roles in progression and development ofLAC by inhibiting Wnt120573-catenin pathway

In conclusion our study demonstrated that lncRNA-LET had tumor-suppressive effect on LAC progression anddevelopment in vitro and vivo and was correlated withclinicopathologic factors Besides lncRNA-LET could inhibitEMT and negatively regulated Wnt120573-catenin pathway inpart Our findings suggest that lncRNA-LET can act as apotential therapeutic target for LAC

Competing Interests

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

Acknowledgments

The study was funded by the National Natural ScienceFoundation of China (Grant no 81572263)

References

[1] L A Torre F Bray R L Siegel J Ferlay J Lortet-Tieulent andA Jemal ldquoGlobal cancer statistics 2012rdquo CA Cancer Journal forClinicians vol 65 no 2 pp 87ndash108 2015

[2] R S Herbst J V Heymach and S M Lippman ldquoLung cancerrdquoThe New England Journal of Medicine vol 359 no 13 pp 1367ndash1380 2008

[3] M Imielinski A H Berger P S Hammerman et al ldquoMappingthe hallmarks of lung adenocarcinoma with massively parallelsequencingrdquo Cell vol 150 no 6 pp 1107ndash1120 2012

[4] M Esteller ldquoNon-coding RNAs in human diseaserdquo NatureReviews Genetics vol 12 no 12 pp 861ndash874 2011

[5] X-S Ge H-J Ma X-H Zheng et al ldquoHOTAIR a prognosticfactor in esophageal squamous cell carcinoma inhibits WIF-1expression and activatesWnt pathwayrdquo Cancer Science vol 104no 12 pp 1675ndash1682 2013

[6] J-F Huang Y-J Guo C-X Zhao et al ldquoHepatitis B virus Xprotein (HBx)-related long noncoding RNA (lncRNA) down-regulated expression by HBx (Dreh) inhibits hepatocellularcarcinoma metastasis by targeting the intermediate filamentprotein vimentinrdquoHepatology vol 57 no 5 pp 1882ndash1892 2013

[7] F Yang L Zhang X-S Huo et al ldquoLong noncoding RNA highexpression in hepatocellular carcinoma facilitates tumor growththrough enhancer of zeste homolog 2 in humansrdquo Hepatologyvol 54 no 5 pp 1679ndash1689 2011

[8] F Yang X S Huo S X Yuan et al ldquoRepression of the longnoncoding RNA-LET by histone deacetylase 3 contributes tohypoxia-mediated metastasisrdquoMolecular Cell vol 49 no 6 pp1083ndash1096 2013

[9] Q Sun H Liu L Li et al ldquoLong noncoding RNA-LET whichis repressed by EZH2 inhibits cell proliferation and inducesapoptosis of nasopharyngeal carcinoma cellrdquoMedical Oncologyvol 32 no 9 article 226 2015

[10] S Jiang H-L Wang and J Yang ldquoLow expression of long non-coding RNA LET inhibits carcinogenesis of cervical cancerrdquoInternational Journal of Clinical and Experimental Pathologyvol 8 no 1 pp 806ndash811 2015

[11] M-Z Ma X Kong M-Z Weng et al ldquoLong non-codingRNA-LET is a positive prognostic factor and exhibits tumor-suppressive activity in gallbladder cancerrdquo Molecular Carcino-genesis vol 54 no 11 pp 1397ndash1406 2015

[12] J Qu M Li J An et al ldquoMicroRNA-33b inhibits lung adeno-carcinoma cell growth invasion and epithelial-mesenchymaltransition by suppressing Wntbeta-cateninZEB1 signalingrdquoInternational Journal of Oncology vol 47 pp 2141ndash2152 2015

[13] Y Wang H Dong M Xu et al ldquo37-kDa laminin receptorprecursor promotes lung adenocarcinoma cell invasion andmetastasis by epithelial-to-mesenchymal transitionrdquo CancerGene Therapy vol 21 no 4 pp 150ndash157 2014

[14] J Hu D Yang H Zhang et al ldquoUSP22 promotes tumorprogression and induces epithelial-mesenchymal transition inlung adenocarcinomardquo Lung Cancer vol 88 no 3 pp 239ndash2452015

[15] L Gao J Liu B Zhang et al ldquoFunctional MUC4 suppressepithelial-mesenchymal transition in lung adenocarcinomametastasisrdquo Tumor Biology vol 35 no 2 pp 1335ndash1341 2014

[16] H Long Z Wang J Chen et al ldquomicroRNA-214 promotesepithelial-mesenchymal transition and metastasis in lung ade-nocarcinoma by targeting the suppressor-of-fused protein(Sufu)rdquo Oncotarget vol 6 no 36 pp 38705ndash38718 2015

[17] C-H Heldin M Vanlandewijck and A Moustakas ldquoRegula-tion of EMT by TGF120573 in cancerrdquo FEBS Letters vol 586 no 14pp 1959ndash1970 2012

[18] E Giarnieri C De Vitis A Noto et al ldquoEMT markers inlung adenocarcinoma pleural effusion spheroid cellsrdquo Journalof Cellular Physiology vol 228 no 8 pp 1720ndash1726 2013

[19] L-K Liu X-Y Jiang X-X Zhou D-MWang X-L Song andH-B Jiang ldquoUpregulation of vimentin and aberrant expressionof E-cadherinΒ- catenin complex in oral squamous cell car-cinomas correlation with the clinicopathological features andpatient outcomerdquoModern Pathology vol 23 no 2 pp 213ndash2242010

[20] D X Nguyen A C Chiang X H-F Zhang et al ldquoWNTTCFsignaling through LEF1 and HOXB9mediates lung adenocarci-noma metastasisrdquo Cell vol 138 no 1 pp 51ndash62 2009

[21] Y Geng Y Ju F Ren et al ldquoInsulin receptor substrate 12(IRS12) regulates Wnt120573-Catenin signaling through blockingautophagic degradation of dishevelledrdquo Journal of BiologicalChemistry vol 289 no 16 pp 11230ndash11241 2014

[22] X Bao H Song Z Chen and X Tang ldquoWnt3a promotes epi-thelial-mesenchymal transition migration and proliferation oflens epithelial cellsrdquo Molecular Vision vol 18 pp 1983ndash19902012

[23] T-T Huynh Y K Rao W-H Lee et al ldquoDestruxin Binhibits hepatocellular carcinoma cell growth through modu-lation of the Wnt120573-catenin signaling pathway and epithelial-mesenchymal transitionrdquo Toxicology in Vitro vol 28 no 4 pp552ndash561 2014

[24] T-H Hu Y Yao S Yu et al ldquoSDF-1CXCR4 promotesepithelial-mesenchymal transition and progression of colorec-tal cancer by activation of the Wnt120573-catenin signaling path-wayrdquo Cancer Letters vol 354 no 2 pp 417ndash426 2014

[25] P Wang B Liu Y Xia C Pan T Ma and Y Chen ldquoLong non-coding RNA-Low Expression in Tumor inhibits the invasion


and metastasis of esophageal squamous cell carcinoma byregulating p53 expressionrdquoMolecular Medicine Reports vol 13pp 3074ndash3082 2016

[26] N H Kim H S Kim N-G Kim et al ldquop53 and microRNA-34are suppressors of canonical Wnt signalingrdquo Science Signalingvol 4 no 197 article ra71 pp 3242ndash3246 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


2013 to 2014 In this study LAC patients had not receivedchemotherapy or radiotherapy treatment All surgical spec-imens were immediately frozen in liquid nitrogen until useThis research was approved by Jiangsu Province People Hos-pital and had received informed consents from all patientsLAC patients were staged in accordance with the tumor nodemetastasis (TNM) staging system (the 7th edition) of theAJCC staging system

22 Cell Culture 16HBE cells PC-9 cells H358 cells SPCA1cells H1975 cells H1299 cells and A549 cells were culturedin RPMI1640 medium containing 10 fetal calf serum100UmL penicillin and 100 120583gmL streptomycin within ahumidified atmosphere at 37∘C containing 5 CO

2

23 Plasmid andTransfection For overexpression of lncRNA-LET in H1299 cells the lncRNA-LET gene was clonedinto the lentiviral-vector pCDH-CMV-MCS-EF1-copGFP(System Biosciences Mountain View CA) and the primersequences are as follows lncRNA-LET sense 51015840-GTTGTT-GTTGCATTGGGGT-31015840 antisense 51015840-AAGATGGAGAGT-GGAGCCT-31015840 H1299 cells were transfected with LV-Vectoror LV-LncRNA-LET For knockdown of lncRNA-LET inA549 cells the sequences of shRNA targeting for lncRNA-LET were designed as 51015840-GGAGGGUGCUUGACAAUA-AUU-31015840 and the NC-shRNA was purchased from Genechem(Shanghai China) A549 cells were transfected with NC-shRNA or LncRNA-shRNA

24 RNA Extraction and qRT-PCR lncRNA-LET and nu-clear 120573-catenin expression level were measured by qRT-PCR In brief trizol reagent (Invitrogen Carlsbad CA)was used to extract the RNA according to the manufac-turerrsquos instruction Reverse TranscriptionKit (Takara DalianChina) was used to synthesize cDNAs The real-time reversetranscription PCR was carried out by using Power SYBRGreen (Takara Dalian China) GAPDH was used as aninternal control qRT-PCR and data collection were carriedout by the ABI7500 system (Applied Biosystems Foster CA)The relative levels of mRNAs were calculated using the 2minusΔΔCtmethod lncRNA-LET 51015840-CCTTCCTGACAGCCAGTGTG-31015840 (forward) 51015840-CAGAATGGAAATACTGGAGCAAG-31015840 (reverse) 120573-catenin 51015840-GCTTTCAGTTGAGCTGAC-CA-31015840 (forward) 51015840-CAAGCTCAAGATCAGCAGTCTC-31015840 (reverse) GADPH 51015840-GTCAACGGATTTGGTCTG-TATT-31015840 (forward) 51015840-AGTCTTCTGGGTGGCAGTGAT-31015840 (reverse)

25 Cell Proliferation Cells were seeded onto 96-well plateand incubated overnight at 37∘C containing 5CO

2 20120583L of

MTT solution per well was added into the 96-well plate andcultured for 4 h at 37∘C Then 150 120583L DMSO was added intothe 96-well plate and incubated for 10min The absorbancewas read by an enzyme-linked immunosorbent assay (ELISA)reader at 490 nm

26 Wound Healing Assay Cells were spread at the bottomof 12-well plates marked by a horizontal line on the back for

targeting the same field of vision a line wound was madeby scraping 100 120583L tips across the confluent cell layer Aftersucking cell culture and washing the cells for three times toremove detached cells and debris the serum-free mediumwas added into the 12-well plates and incubated under theusual culture conditions for 48 hwound closurewas capturedusing a lightmicroscope (DFC500 LeicaWetzlar Germany)AxioVision version 47 software (Carl Zeiss Meditec DublinCA USA) was used to measure the wound closure

27 Transwell Assay Cell invasion assay was operated bypolycarbonatemembrane Boyden chamber insert (EMDMil-lipore Billerica MA USA) The transfected cells were addedinto the upper invasion chamber the 500120583L culture mediumwas added into the lower invasion chamber 45120583g Matrigel(BD Biosciences San Jose CA USA) was used to precoateach insert The Matrigel invasion chambers were incubatedunder the usual culture conditions for 48 h After removingthe filter inserts and the cells on the upper side of the filterthe invaded cells on the lower chamber were suspendedin 100 precooling methanol for 10min Then cells werestained with 005 crystal violet for 30min washed with PBSbuffer and captured using microscopic inspection (DFC500Leica) according to the manufacturerrsquos instructions Finallythe values for invasion were carried out under three fields permembrane

For cell migration assay by transwell assay each insertneededwas not precoated withMatrigelThe other steps weresimilar to cell invasion assay

28 Western Blot In order to investigate expression level of120573-catenin E-cadherin N-cadherin c-Myc and COX-2 cellswere collected and cell lysis was performed by using RIPAlysis buffer (Cell Signal Technology Danvers MA) includingprotease inhibitors on ice according to the manufacturerrsquosinstruction The extracted protein was quantified by bicin-choninic acid quantification assay (Pierce BiotechnologyInc Rockford IL USA) Then the total cellular proteinswere subjected to SDS-PAGE (10) for western analysisAfter transferring to polyvinylidene difluoride (PVDF)mem-branes blots were incubated with 5 BSA in Trisbufferedsaline containing 05 Tween 20 for 60min and incubatedovernight at 4∘C on a rocker with the following primaryantibodies polyclonal rabbit anti-human 120573-catenin antibody(1 1000) monoclonal mouse anti-human E-cadherin anti-body (1 1000) polyclonal rabbit anti-human N-cadherinantibody (1 1000) monoclonal rabbit anti-human c-Mycantibody (1 1000) and polyclonal rabbit anti-human COX-2 antibody (1 1000) Following washing three times withTBS-T for 5min the blots were incubated with horseradishperoxidase- (HRP-) conjugated goat anti-rabbit IgG HampLpolyclonal secondary antibody (1 1000) at room temper-ature for 1 h All antibodies were purchased from Abcam(Cambridge MA USA) The bands were captured under anenhanced chemiluminescence detection system (GE Health-care Life Sciences)


0

1

2

3

4

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET

lowastlowastlowast

P (n = 60) T (n = 60)

(a)

00

05

10

15

16H

BE

A54

9

PC-9

H35

8

SPCA

1

H12

99

H19

75

20

25

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET

(b)

0

1

220

30

40

A549H1299

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET

lowast

lowast

NC-

shRN

A

lncR

NA

-shR

NA

LV-V

ecto

r

LV-ln

cRN

A

(c)









3 Results





Male 23 11 12 0791Female 37 19 18

Age (year)lt60 38 20 18 0592ge60 22 10 12











Day 1 Day 2 Day 300

05

10

15

20

25

30


Day 1 Day 2 Day 300

05

10

15

20

LV-VectorLV-lncRNA

A549 H1299

lowast

lowastlowast

lowast

Cel

l num

ber (

1lowast10

5)

Cel

l num

ber (

1lowast10

5)

(a)


0h

48

h




A54

9H

1299 0

50

100

150

200600

700

800

A549H1299

Mig

ratio

n ce

ll nu

mbe

r

0

50

100

150

200700

800

900

A549H1299

Inva

sion

cell

num

ber

lowast lowast

lowastlowast

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

(c)







A549NC-shRNA


N-cadherin

C-Myc

COX-2

H1299LV-Vector

LV-lncRNAminus

minus+

+minus

minus+

+

E-cadherin

N-cadherin

C-Myc

COX-2


1

2

3


LV-VectorLV-lncRNA

Relat

ive p

rote

in ex

pres

sion

lowast

lowast lowast

lowast

lowast

lowastlowastlowast


(a)

H1299

LV-Vector LV-lncRNA

A549



1

2

3


00

05

10

15

H1299

lowastlowast

120573-Catenin

120573-Actin

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

(b)

0

1

2

3

4lowastlowast

Relat

ive e

xpre

ssio

n of

nuc

lear

120573-c

aten

in

P (n = 60) T (n = 60)

(c)


4 Discussion





5

10

15

20

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET lowast

(a)


100

200

300

400

3 6 9 12 150

100

200

300

400

LV-VectorLV-lncRNA

(Days)

lowast

Tum

or v

olum

e (m

m3)

Tum

or v

olum

e (m

m3)

(b)

LV-Vector LV-lncRNA

HampE

PCNA

(c)








Competing Interests


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET

lowastlowastlowast

P (n = 60) T (n = 60)

(a)

00

05

10

15

16H

BE

A54

9

PC-9

H35

8

SPCA

1

H12

99

H19

75

20

25

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET

(b)

0

1

220

30

40

A549H1299

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET

lowast

lowast

NC-

shRN

A

lncR

NA

-shR

NA

LV-V

ecto

r

LV-ln

cRN

A

(c)









3 Results





Male 23 11 12 0791Female 37 19 18

Age (year)lt60 38 20 18 0592ge60 22 10 12











Day 1 Day 2 Day 300

05

10

15

20

25

30


Day 1 Day 2 Day 300

05

10

15

20

LV-VectorLV-lncRNA

A549 H1299

lowast

lowastlowast

lowast

Cel

l num

ber (

1lowast10

5)

Cel

l num

ber (

1lowast10

5)

(a)


0h

48

h




A54

9H

1299 0

50

100

150

200600

700

800

A549H1299

Mig

ratio

n ce

ll nu

mbe

r

0

50

100

150

200700

800

900

A549H1299

Inva

sion

cell

num

ber

lowast lowast

lowastlowast

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

(c)







A549NC-shRNA


N-cadherin

C-Myc

COX-2

H1299LV-Vector

LV-lncRNAminus

minus+

+minus

minus+

+

E-cadherin

N-cadherin

C-Myc

COX-2


1

2

3


LV-VectorLV-lncRNA

Relat

ive p

rote

in ex

pres

sion

lowast

lowast lowast

lowast

lowast

lowastlowastlowast


(a)

H1299

LV-Vector LV-lncRNA

A549



1

2

3


00

05

10

15

H1299

lowastlowast

120573-Catenin

120573-Actin

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

(b)

0

1

2

3

4lowastlowast

Relat

ive e

xpre

ssio

n of

nuc

lear

120573-c

aten

in

P (n = 60) T (n = 60)

(c)


4 Discussion





5

10

15

20

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET lowast

(a)


100

200

300

400

3 6 9 12 150

100

200

300

400

LV-VectorLV-lncRNA

(Days)

lowast

Tum

or v

olum

e (m

m3)

Tum

or v

olum

e (m

m3)

(b)

LV-Vector LV-lncRNA

HampE

PCNA

(c)








Competing Interests


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Male 23 11 12 0791Female 37 19 18

Age (year)lt60 38 20 18 0592ge60 22 10 12











Day 1 Day 2 Day 300

05

10

15

20

25

30


Day 1 Day 2 Day 300

05

10

15

20

LV-VectorLV-lncRNA

A549 H1299

lowast

lowastlowast

lowast

Cel

l num

ber (

1lowast10

5)

Cel

l num

ber (

1lowast10

5)

(a)


0h

48

h




A54

9H

1299 0

50

100

150

200600

700

800

A549H1299

Mig

ratio

n ce

ll nu

mbe

r

0

50

100

150

200700

800

900

A549H1299

Inva

sion

cell

num

ber

lowast lowast

lowastlowast

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

(c)







A549NC-shRNA


N-cadherin

C-Myc

COX-2

H1299LV-Vector

LV-lncRNAminus

minus+

+minus

minus+

+

E-cadherin

N-cadherin

C-Myc

COX-2


1

2

3


LV-VectorLV-lncRNA

Relat

ive p

rote

in ex

pres

sion

lowast

lowast lowast

lowast

lowast

lowastlowastlowast


(a)

H1299

LV-Vector LV-lncRNA

A549



1

2

3


00

05

10

15

H1299

lowastlowast

120573-Catenin

120573-Actin

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

(b)

0

1

2

3

4lowastlowast

Relat

ive e

xpre

ssio

n of

nuc

lear

120573-c

aten

in

P (n = 60) T (n = 60)

(c)


4 Discussion





5

10

15

20

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET lowast

(a)


100

200

300

400

3 6 9 12 150

100

200

300

400

LV-VectorLV-lncRNA

(Days)

lowast

Tum

or v

olum

e (m

m3)

Tum

or v

olum

e (m

m3)

(b)

LV-Vector LV-lncRNA

HampE

PCNA

(c)








Competing Interests


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Day 1 Day 2 Day 300

05

10

15

20

25

30


Day 1 Day 2 Day 300

05

10

15

20

LV-VectorLV-lncRNA

A549 H1299

lowast

lowastlowast

lowast

Cel

l num

ber (

1lowast10

5)

Cel

l num

ber (

1lowast10

5)

(a)


0h

48

h




A54

9H

1299 0

50

100

150

200600

700

800

A549H1299

Mig

ratio

n ce

ll nu

mbe

r

0

50

100

150

200700

800

900

A549H1299

Inva

sion

cell

num

ber

lowast lowast

lowastlowast

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

NC-

shRN

Aln

cRN

A-s

hRN

A

LV-V

ecto

rLV

-lncR

NA

(c)







A549NC-shRNA


N-cadherin

C-Myc

COX-2

H1299LV-Vector

LV-lncRNAminus

minus+

+minus

minus+

+

E-cadherin

N-cadherin

C-Myc

COX-2


1

2

3


LV-VectorLV-lncRNA

Relat

ive p

rote

in ex

pres

sion

lowast

lowast lowast

lowast

lowast

lowastlowastlowast


(a)

H1299

LV-Vector LV-lncRNA

A549



1

2

3


00

05

10

15

H1299

lowastlowast

120573-Catenin

120573-Actin

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

(b)

0

1

2

3

4lowastlowast

Relat

ive e

xpre

ssio

n of

nuc

lear

120573-c

aten

in

P (n = 60) T (n = 60)

(c)


4 Discussion





5

10

15

20

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET lowast

(a)


100

200

300

400

3 6 9 12 150

100

200

300

400

LV-VectorLV-lncRNA

(Days)

lowast

Tum

or v

olum

e (m

m3)

Tum

or v

olum

e (m

m3)

(b)

LV-Vector LV-lncRNA

HampE

PCNA

(c)








Competing Interests


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















A549NC-shRNA


N-cadherin

C-Myc

COX-2

H1299LV-Vector

LV-lncRNAminus

minus+

+minus

minus+

+

E-cadherin

N-cadherin

C-Myc

COX-2


1

2

3


LV-VectorLV-lncRNA

Relat

ive p

rote

in ex

pres

sion

lowast

lowast lowast

lowast

lowast

lowastlowastlowast


(a)

H1299

LV-Vector LV-lncRNA

A549



1

2

3


00

05

10

15

H1299

lowastlowast

120573-Catenin

120573-Actin

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

Rela

tive e

xpre

ssio

n of

120573-c

aten

in

(b)

0

1

2

3

4lowastlowast

Relat

ive e

xpre

ssio

n of

nuc

lear

120573-c

aten

in

P (n = 60) T (n = 60)

(c)


4 Discussion





5

10

15

20

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET lowast

(a)


100

200

300

400

3 6 9 12 150

100

200

300

400

LV-VectorLV-lncRNA

(Days)

lowast

Tum

or v

olum

e (m

m3)

Tum

or v

olum

e (m

m3)

(b)

LV-Vector LV-lncRNA

HampE

PCNA

(c)








Competing Interests


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5

10

15

20

Relat

ive e

xpre

ssio

n of

lncR

NA-

LET lowast

(a)


100

200

300

400

3 6 9 12 150

100

200

300

400

LV-VectorLV-lncRNA

(Days)

lowast

Tum

or v

olum

e (m

m3)

Tum

or v

olum

e (m

m3)

(b)

LV-Vector LV-lncRNA

HampE

PCNA

(c)








Competing Interests


Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Competing Interests


Acknowledgments


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 Long Noncoding RNA-LET Suppresses Tumor...

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Transcript of Research Article Long Noncoding RNA-LET Suppresses Tumor...