Research Article Downregulation of Heparanase Expression ...

Research ArticleDownregulation of Heparanase Expression Results inSuppression of Invasion Migration and Adhesion Abilities ofHepatocellular Carcinoma Cells

Xiao-Peng Chen Jun-Sheng Luo Ye Tian Chen-Lin Nie Wei Cui and Wei-Dong Zhang

Department of Hepatobiliary Surgery Affiliated Yijishan Hospital of Wannan Medical College Wuhu 241001 China

Correspondence should be addressed to Xiao-Peng Chen drcxpqqcom

Received 11 August 2015 Revised 31 October 2015 Accepted 3 November 2015

Academic Editor Shinichi Aishima

Copyright copy 2015 Xiao-Peng Chen et alThis is an open access article distributed under theCreativeCommonsAttributionLicensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective Heparanase (HPSE) is high-expressed in most malignant tumors including hepatocellular carcinoma (HCC) andpromotes cancer cell invasion and migration The aim of the study is to explore whether HPSE enhances adhesion in metastasis ofHCC cellsMethods HPSE expressions in humanHCC cells weremeasured with real-time RT-PCR andWestern blot analysis Fourrecombinant miRNA vectors pcDNATM62-GWEmGFP-miR-HPSE (pmiR-HPSE) were transfected into HCCLM3 cell HPSEexpression in transfected cell was measuredThe cell invasion migration and adhesion abilities were detected respectively ResultsBothHPSEmRNA and protein relative expression levels were higher inHepG2 BEL-7402 andHCCLM3 cells than those in normalhepatocyte (119875 lt 005) HPSE showed highest expression level in HCCLM3 cell (119875 lt 005) Transfection efficiencies of four miRNAvectors were 75ndash85The recombinant vectors significantly decreased HPSE expression in transfected HCCLM3 cells (119875 lt 001)and pmiR-HPSE-1 showed best interference effect (119875 lt 005) pmiR-HPSE-1 significantly decreased the penetrated and migratingcells numbers and adherence rate of HCCLM3 cells (119875 lt 005) Conclusion HPSE is a potentiator of cell adhesion in metastasis ofHCC

1 Introduction

Globally hepatocellular carcinoma is the sixth most com-mon cancer and the third most common cause of cancer-related deaths [1] The high mortality of HCC is mainly dueto the occurrence of intrahepatic metastases Multicentricoccurrence of HCC is essentially the result of intrahepaticmetastasis [2ndash4]

Intrahepatic metastasis of HCC is a complex and mul-tistep biological process that includes cancer cell adhesioninvasion migration and proliferation Formation of tumormicroembolus in portal vein is one of the crucial linksand HCC cell adhesion is the prerequisites Cancer cells inblood vessel can activate blood coagulation to induce tumormicroembolus through multiple mechanisms [5ndash7] But it isstill unclear what molecules contribute to cell adhesion andtumor microembolus of HCC and the precise pathogenesisof intrahepatic metastasis remains to be determined

Heparanase (HPSE) is an endo-beta-glucuronidase thatis capable of cleaving heparan sulfate (HS) side chains ofheparan sulfate proteoglycans (HSPGs) on cell surfaces andextracellular matrices (ECM) of basement membrane (BM)and plays critical roles in tumor cell invasion migrationand angiogenesis by remodeling ECM and delivering somecytokines such as basic fibroblast growth factor (bFGF)and vascular endothelial cell growth factor (VEGF) [8ndash10]Increased HPSE expression was found in numerous tumortypes and correlates with poor prognosis [11 12] and down-regulation of heparanase expression results in suppressionof tumor invasion and migration especially in HCC cells[13ndash15] In recent 15 years HPSE has become a researchhotspot [16 17] On the other hand HPSE may neutralizethe anticoagulation properties of heparin and low-molecular-weight heparin and shows procoagulation activity resultingin cancer progression [18ndash20] Therefore HPSE might bea promising target for potential antiadhesive agents [21]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 241983 10 pageshttpdxdoiorg1011552015241983

2 BioMed Research International

Table 1 Primer sequences for PCR amplification

Primers Primer sequences Length of products (bp)HPSE-F 51015840-GCACAAACACTGACAATCCAAG-31015840 101HPSE-R 51015840-AAAAGGATAGGGTAACCGCAA-31015840

GAPDH-F 51015840-GTGGTCTCCTCTGACTTCAACA-31015840 136GAPDH-R 51015840-CCACCACCCTGTTGCTGTAG-31015840

Against this background we hypothesize that HPSE mightplay a proadhesive role in adhesion and tumormicroembolusof HCC cells

The aim of this study is to explore whether HPSE enhancecancer cell adhesion in metastasis of HCC

2 Materials and Methods

21 Materials Human normal liver cell line LO-2 and HCCcell lines (HepG2 and BEL-7402) were from Cell BankNational Academy of Science of China (Shanghai China)Human highly metastatic liver cancer cell line HCCLM3was from Liver Cancer Institute (Zhongshan Hospital FudanUniversity Shanghai China) Markers dNTP primers werefrom Shanghai Shenggong Company (Shanghai China)DMEM medium RPMI-1640 medium FBS Trizol solutionand Lipofectamine 2000 were from Invitrogen Co (CarlsbadCA USA) Reverse transcription kits restrictive endonucle-ase BglII SalI and T4 DNA ligase were fromMBI FermentasChina Co Ltd (Shenzhen China) BCA protein quantitativekit and real-time qPCR kit (SYBR Green) were from TiangenBiotech Co Ltd (Beijing china) Plasmid pcDNATM62-GWEmGFP-miR (pmiR) and pcDNATM62-GWEmGFP-miR-Negative Control (pmiR-NC) were fromBeinuo BiotechCo Ltd (Shanghai China) PCRpurification kits andplasmidextraction kit were from Axygen Scientific Inc (Carls-bad CA USA) Rabbit-anti-HPSE polyclonal antibody andhorseradish peroxidase- (HRP-) conjugated anti-rabbit IgGwere from Jinqiao Biotechnology (Beijing China) Rabbit-anti-phosphoglyceraldehyde dehydrogenase (GAPDH) poly-clonal antiserum was from Santa Cruz Biotechnology Inc(Santa Cruz CA USA) Matrigel was from BD Bioscience(San Jose CA USA)

22 Determination of HPSE Expression in HCC Cells

221 Quantitative Real-Time Reverse Transcription Poly-merase Chain Reaction (qRT-PCR) Human HCC cells andnormal liver cell line LO-2 were cultured in RPMI-1640medium with 10 FBS According to Gene ID of HPSEmRNA sequence (NM 006665) PCR primers were designedand synthesized (Table 1) The qRT-PCR was performedaccording to the methods as described in our paper [12] andGAPDH was used as loading control The experiments wereperformed for three times

222 Western Blotting Cells were lysed in lysis bufferincubated on ice for 30min and centrifuged for 20minto remove cell debris Total cell lysate was subjected to

SDS-polyacrylamide gel electrophoresis The proteins weremeasured withWestern blotting according to the methods asdescribed in our paper [12] and GAPDHwas used as loadingcontrol The experiments were performed for three times

23 Plasmid Construction and Identification In the exper-iment four HPSE RNAi vectors were constructed usingmiRNA technique which is the same as the short hairpinRNA (shRNA) in essence but an artificial flanking pri-miRNA sequence is extended from the ends of shRNAtarget sequences Based on above principle four pairs ofmiRNA single-stranded oligonucleotide were designed andsynthesized according to Gene ID of HPSE mRNA sequence(Table 2) and then converted into double-stranded formby denaturation and subsequent annealing The 4 specificdouble-stranded miRNA sequences were cloned into thepmiR vector respectively The products were then trans-formed into Escherichia coli competent cells and cultivatedon a plate containing spectinomycin overnight at 37∘CPlasmid DNAs were extracted and sequenced The sampleswith correct sequence were named pcDNA-miR-HPSE-1pcDNA-miR-HPSE-2 pcDNA-miR-HPSE-3 and pcDNA-miR-HPSE-4 respectively

24 Transfection and Assessment Recombinant plasmidspmiR-HPSE-1 pmiR-HPSE-2 pmiR-HPSE-3 and pmiR-HPSE-4 were respectively transfected into HCCLM3 cellusing Lipofectamine 2000 following the manufacturerrsquos pro-tocol No plasmid was used in blank control group and pmiR-NC was used as negative control Transfection efficiencywas observed with invert fluorescence microscope 24 h aftertransfection Five hundred cells were randomly countedand the percentage of EGFP-positive cells was calculatedHPSE expressions in transfected cells were measured byreal-time RT-PCR and Western blot analysis 48 h later Theexperiments were performed for three times According tothe expression levels of HPSE one miRNA plasmid with bestinhibitory effect was chosen for following experiment

25 Determination of Cell Invasion Migrationand Adhesion Abilities

251 Transwell Invasion and Migration Assay The exper-iments were performed as previously described [22] Forinvasion assay 72 hours after transfection 5 times 104 transfectedHCC cells in serum-free RPMI-1640 were seeded into theupper chambers of each well of 24-well plate with insert(8mm pore size Millipore Billerica MA USA) coated withMatrigel For migration assay the upper chambers were not

BioMed Research International 3

Table2miRNAoligo-DNAsequ

ence

ofhu

man

heparanase

gene

Oligon

ucleotide

Sequ

enceso

fmiRNAoligo

Correspon

ding

sequ

ence

ofmRN

Apm

iR-H

PSE-1-F

51015840-TGCT

GTA

TCCT

GGTT

GAC

TTGAG

ATTG

TTTT

GGCC

ACTG

ACTG

ACAAT

CTCA

ACAAC

CAGGAT

A-31015840

356ndash

376b

ppm

iR-H

PSE-1-R

51015840-C

CTGTA

TCCT

GGTT

GTT

GAG

ATTG

TCAG

TCAG

TGGCC

AAAAC

AAT

CTCA

AGTC

AAC

CAGGAT

AC-31015840

pmiR-H

PSE-2-F

51015840-TGCT

GTA

CAGAG

CTTC

TTGAG

TAGGTG

TTTT

GGCC

ACTG

ACTG

ACAC

CTAC

TCGAAG

CTCT

GTA

-31015840

490ndash

570b

ppm

iR-H

PSE-2-R

51015840-C

CTGTA

CAGAG

CTTC

GAG

TAGGTG

TCAG

TCAG

TGGCC

AAAAC

ACCT

ACTC

AAG

AAG

CTCT

GTA

C-31015840

pmiR-H

PSE-3-F

51015840-TGCT

GTA

TACT

CGAAG

CTTC

CTTC

TCGTT

TTGGCC

ACTG

ACTG

ACGAG

AAG

GAC

TTCG

AGTA

TA-31015840

1280ndash1301b

ppm

iR-H

PSE-3-R

51015840-C

CTGTA

TACT

CGAAG

TCCT

TCTC

GTC

AGTC

AGTG

GCC

AAAAC

GAG

AAG

GAAG

CTTC

GAG

TATA

C-31015840

pmiR-H

PSE-4-F

51015840-TGCT

GAAAG

CTGGCA

AGCC

CAGTG

AAG

TTTT

GGCC

ACTG

ACTG

ACTT

CACT

GGTT

GCC

AGCT

TT-31015840

1560ndash1580b

ppm

iR-H

PSE-4-R

51015840-C

CTGAAAG

CTGGCA

ACCA

GTG

AAG

TCAG

TCAG

TGGCC

AAAAC

TTCA

CTGGGCT

TGCC

AGCT

TTC-

31015840


LO-2

cell

Hep

G2

cell

BEL-7402

cell

HCC

LM3

cell

Hep

G2

cell

BEL-7402

cell

HCC

LM3

cell

HPSE

GAPDH

mRNA Protein

LO-2

cell

(a)

LO-2 HepG2 HCCLM3BEL-7402

HPS

E re

lativ

e exp

ress

ion

leve

ls

8

7

6

5

4

3

2

1

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast^^

lowastlowast^^

mRNAProtein

(b)

Figure 1HPSEmRNAandprotein expressions inHCCcells (a) Expressions ofHPSE inHCCcells were determined via RT-PCR andWesternblot analysis (b) HPSE mRNA and protein relative expression levels in HCC cells Data presented means plusmn SD lowastlowast119875 lt 001 compared withthat in LO-2 cell andand119875 lt 001 compared with that in HepG2 cell

coated with Matrigel and cells were seeded after 48-hourtransfection RPMI-1640 containing 10 FBS was placed inthe lower chambers as a chemoattractant After 24 hours ofincubation cells on the upper membrane surface were wipedoff and the cells that invaded across the Matrigel membranewere fixed with paraformaldehyde and stained with crystalviolet The number of invasive cells was then counted (fiverandomly chosen fields for each membrane) under an invertmicroscope (200x) Each condition was done in triplicate

252 Adhesion Experiment Matrigel glue (20mgL) wasadded to a 96-well plate at 100120583L per well The plate wasincubated in a Clean Bench overnight The redundant gluewas washed away with appropriate RPMI-1640 mediumHCC cells were transfected for 48 h Cells were trypsinizedsuspended in PBS counted and then seeded on the 96-wellplate at 5 times 104 per well The plate was then incubated in the5 CO

2incubator at 37∘C for 2 h After adding 10 120583L of MTT

solution (5mgmL) per well the cells were continuouslycultured for 4 h Following adding 200120583LDMSO to eachwell

the plate was gently oscillated at 110 strokesmin for 10minThe absorbance at 490 nm of the colored solution (119860

570 nm)was measured by a microplate reader Negative control andblank control were both used Cell adherence rate () =(119860570 nm of experimental group119860

570 nm of negative controlgroup) times 100 Each assay was performed in triplicate wells

26 Statistical Analysis All the data are expressed as meanvalues plusmn standard deviation (SD) Comparisons among mul-tiple groups were made with a one-way analysis of variance(ANOVA) followed by 119902-test119875 lt 005was used for statisticalsignificance

3 Results

31 HPSE Expression in HCC Cells HPSE mRNA relativeexpression levels were higher in HepG2 BEL-7402 andHCCLM3 cells than that in normal hepatocyte (119875 lt 001)Of all 3 kinds of HCC cells HPSE showed highest expressionlevel in HCCLM3 cell (119875 lt 001) (Figure 1) HPSE protein


110 120 130 140 150 160 170

TGCTGTATCCTGGTTGACTTGAGATTGTTTTGGCCACTGACTGACAATCTCAACAACCAGGATA

(a)

110 120 130 140 150 160

TGCTGTACAGAGCTTCTTGAGTAGGTGTTTTGGCCACTGACTGACACCTACTCGAAGCTCTGTATGCTGTACAGAGCTTCTTGAGTAGGTGTTTTGGCCACTGACTGGGGGGGGGGGGGACACCTACTCGAAGCTCTGTA

(b)

170110 120 130 140 150 160

TGCTGTATACTCGAAGCTTCCTTCTCGTTTTGGCCACTGACTGACGAGAAGGACTTCGAGTAT

(c)

110 170120 130 140 150 160

TGCTGAAAGCTGGCAAGCCCAGTGAAGTTTTGGCCACTGACTGACTTCACTGGTTGCCAGCTTT

(d)

Figure 2 Sequencing graphs of recombinant vectors ((a)ndash(d)) Sequencing graphs of 4 target sequences of recombinant vectors pmiR-HPSE-1 pmiR-HPSE-2 pmiR-HPSE-3 and pmiR-HPSE-4 respectively

expression was the same as the mRNA expression (Figure 1)According to above results the HCCLM3 cell was used forsubsequent research

32 Identification of Recombinant Vectors The sequencingresults showed that all 4 kinds of miRNA vectors weretotally consistent with the designing sequence No deletioninsertion or mutation was detected (Figure 2) The resultssuggested HPSE RNAi vector pmiR-HPSE was successfullyconstructed with miRNA technique

33 Transfection Efficiency After cell transfection no fluores-cence was found in blank control group Bright fluorescencein negative control or 4 kinds of recombinant plasmid trans-fected cells could be observed using fluorescence analysis 48 hlater The average transfection efficiencies of negative controland recombinant plasmids ranged from 75 to 85 withoutsignificant difference among them (119875 gt 005) but were all sig-nificantly higher than that of blank control group (119875 lt 001)(Figure 3)These results suggested that recombinant plasmidswere successfully transfected into the specific HCC cells

34 Effect of Recombinant Plasmids on HPSE Expression inHCC Cells Both HPSE mRNA and protein expressions inpmiR-HPSE transfected HCCLM3 cells were significantlylower than those in control groups (119875 lt 001) There wasno obvious difference between blank control and pmiR-NCgroups (119875 gt 005) The maximal decrease was shown inpmiR-HPSE-1 group (119875 lt 005) and the inhibition ratioapproached to 70 (Figure 4) Therefore plasmid pmiR-HPSE-1 was selected for following invasion and adhesionexperiments

35 Effect of Plasmid Transfection on Invasion Migration andAdhesion Abilities of HCC Cells The number of penetratedHCCLM3 cells in pmiR-HPSE-1 group was significantly lessthan those in blank control and pmiR-NC groups (119875 lt005) There was no obvious difference between two groups(119875 gt 005) (Figure 5) Cell migration showed similarresults (Figure 5)The adherence rate in pmiR-HPSE-1 groupshowed significant decrease compared with those in blankcontrol and pmiR-NC groups (119875 lt 005) and there was noobvious difference between two control groups (119875 gt 005)(Figure 6)

4 Discussion

High levels of HPSE mRNA and protein are expressedin most malignant tumors including HCC and are closelyassociated with tumor metastasis angiogenesis and otherdiverse pathological processes [8ndash10] In this study we foundthat both HPSE mRNA and protein expressions in 3 kindsof HCC cells were higher than those in normal hepatocytewhich were similar to previous results [11 12] In additiontheHPSE expression levels are different amongdifferentHCCcells Of all 3 kinds of HCC cells highly metastatic HCCLM3cells showed highest HPSE expression level These findingssuggested that invasion and metastasis potentials of HCCwere positively correlated with HPSE expression level

HPSE is believed to play an important role in the processof tumor invasion and metastasis [8ndash15] In order to verify itsprometastasis function we constructed RNAi vector pmiR-HPSE using miRNA technique The RNAi based on miRNAcontext may provide an efficient and safe therapeutic knock-down effect on target gene HPSE [23] The results proved all


(a) (b)

(c) (d)

(e) (f)

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

Tran

sfect

ion

effici

enci

es o

f pla

smid

s 10

08

06

04

02

0

(g)

Figure 3 Photofluorograms and transfection efficiencies (a)Nofluorescence could be found in blank control group 48 h later (200x 48 h) (b)Bright fluorescence could be observed in pmiR-NC group (200x 48 h) ((c)ndash(f)) Bright fluorescence in pmiR-HPSE-1pmiR-HPSE-2pmiR-HPSE-3pmiR-HPSE-4 groups (200x 48 h) respectively (g) Transfection efficiencies of every group Data presented means plusmn SD lowastlowast119875 lt 001compared with that in blank control group


mRN

APr

otei

n

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4

HPSE

HPSE

GAPDH

GAPDH

(a)H

PSE

expr

essio

ns aft

er tr

ansfe

ctio

n

8

7

6

5

4

3

2

1

0

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4


lowastlowast lowastlowast


lowastlowast^lowastlowast^

mRNAProtein

(b)

Figure 4 HPSE expressions in pmiR-HPSE transfected HCCLM3 cells (a) Expressions of HPSE in transfected HCCLM3 cells weredetermined via RT-PCR and Western blot analysis (b) Relative expression levels of HPSE mRNA and protein Data presented means plusmnSD HPSE expressions in all pmiR-HPSE groups were significantly lower than those in control groups lowastlowast119875 lt 001 compared with those incontrol groups HPSE expression in pmiR-HPSE-1 group was significantly lower than those in pmiR-HPSE-2pmiR-HPSE-3pmiR-HPSE-4groups and119875 lt 005 compared with those in pmiR-HPSE-2pmiR-HPSE-3pmiR-HPSE-4 groups

4 recombinant plasmids could significantly decrease HPSEexpression in HCCLM3 cells and pmiR-HPSE-1 showedstrongest inhibitory effect In the following experiment wedemonstrated that pmiR-HPSE-1 can lead to the obviousdecrease in the invasion and metastasis capabilities of HCCcells Therefore downregulation of HPSE expression couldresult in suppression of invasion and metastasis abilities ofHCC cells which were similar to other studies [13ndash15]

On the other hand HPSE can exert proadhesion orprocoagulation activity in hematogenous metastasis andinflammation HPSE can augment the adhesion of humanneutrophils and mononuclear cells to human umbilicalvein endothelial cells in a concentration-dependent manner[24] HPSE-miRNA transfection significantly decreases theadhesion ability of melanoma cells besides the invasionand migration abilities [23] and mollusk heparan sulfateinhibits LS180 colon carcinoma cell adhesion [25] Ouradhesion experiment also found pmiR-HPSE-1 significantlyattenuated the adherence rate of HCC cells while it obviouslyinhibited the invasion and migration abilities Tumor cellshows different adhesion capability in different environmentcondition In our adhesion experiment the plate was gentlyoscillated at 110 strokesmin after it was incubated We thinkit could imitate shaken adhesion or rolling adhesion in bloodvessel [26] Adhered cells in blood vessel possess a protectivebarrier and escape from immune surveillance resulting inhigher capability of invasion and metastasis [27] Based onabove findings and analysis we conclude that HPSE not onlyplays an important role in tumor invasion and migrationbut also contributes to the adhesion of HCC cells To our

knowledge this is the first report that HPSE plays a proadhe-sive role in cell adhesion and tumor microembolus of HCCThere are three possible reasons The first is HPSE-mediateddegradation ofHSPGs in ECMof BM Integrity of BMbarrieris destroyed Second various vascular growth factor andadhesion molecule were delivered P-selectin is known toparticipate in interactions involving tumor cells plateletsleukocytes and endothelium and heparin has been shownto inhibit P-selectin and as a consequence it blunts metastasisand inflammation [25] Third HPSE and membrane HSPGsactivate signaling molecules such as Akt Src epidermalgrowth factor receptor and Rac [28] Of course further studyis needed to explore the concrete mechanism

5 Conclusions

In conclusion HPSE is a potentiator of cell adhesion inmetastasis of HCC

Abbreviations

HCC Hepatocellular carcinomaHPSE HeparanaseHSPG Heparan sulfate proteoglycanECM Extracellular matricesBM Basement membranebFGF Basic fibroblast growth factorVEGF Vascular endothelial cell growth factorRT-PCR Reverse transcriptase-polymerase chain

reaction


Blank control

Mig

ratio

n

pmiR-NC pmiR-HPSE-1

Inva

sion

(a)

Blank control pmiR-NC pmiR-HPSE-1

120

100

80

60

40

20

0

HCC

LM3

cell

num

bers

lowastlowast

Penetrated cellsMigrated cells

(b)

Figure 5 Effect of silencing HPSE gene on invasion and migration ability of HCC cells (a) Invasion and migration experiments Penetratedand migrating cells were both decreased after HCCLM3 cells were transfected with pmiR-HPSE-1 (b) Penetrated and migrating HCCLM3cell numbers Data presented means plusmn SD lowast119875 lt 005 compared with those in control groups


20

40

60

80

100

120

lowast

Adhe

renc

e rat

e (

)

Figure 6 Effect of silencing HPSE gene on adhesion ability of HCC cells HCCLM3 cell adherence rate significantly decreased after pmiR-HPSE-1 transfection Data presented means plusmn SD lowast119875 lt 005 compared with those in control groups


pmiR pcDNATM62-GWEmGFP-miRpmiR-NC pcDNATM62-GWEmGFP-miR-Negative

ControlGAPDH Phosphoglyceraldehyde dehydrogenaseshRNA Short hairpin RNA

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Conflict of Interests

The authors declare that they have no competing interests

Authorsrsquo Contribution

Xiao-Peng Chen conceived the study carried out the designand drafted the paper and participated in the RT-PCR studyand Western blot analysis Jun-Sheng Luo and Wei-DongZhang constructed the recombinant vectors and carried outtransfection the RT-PCR study andWestern blot analysis YeTian and Chen-Lin Nie carried out the invasion migrationand adhesion test Wei Cui performed the statistical analysisand helped to draft the paper All authors read and approvedthe final paper

Acknowledgments

The authors thank Dr Kong Lv Dr Min Zhong and DrMengYing Zhangwho provided excellent technical assistancein experiments on behalf of Central Laboratory AffiliatedYijishanHospital ofWannanMedical CollegeThis study wassupported by the National Natural Science Foundation ofChina (Grant no 81272412) and Natural Science Foundationof Anhui province China (Grant no 11040606M181)

References

[1] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010

[2] K Shirabe S Wakiyama T Gion et al ldquoClinicopathologicalrisk factors linked to recurrence pattern after curative hepaticresection for hepatocellular carcinomamdashresults of 152 resectedcasesrdquo Hepato-Gastroenterology vol 54 no 79 pp 2084ndash20872007

[3] O Nakashima and M Kojiro ldquoRecurrence of hepatocellularcarcinoma multicentric occurrence or intrahepatic metastasisA viewpoint in terms of pathologyrdquo Journal of Hepato-Biliary-Pancreatic Surgery vol 8 no 5 pp 404ndash409 2001

[4] A C Y Chan S C Chan K S H Chok et al ldquoTreatmentstrategy for recurrent hepatocellular carcinoma salvage trans-plantation repeated resection or radiofrequency ablationrdquoLiver Transplantation vol 19 no 4 pp 411ndash419 2013

[5] S Noble and J Pasi ldquoEpidemiology and pathophysiology ofcancer-associated thrombosisrdquo British Journal of Cancer vol102 no 1 pp S2ndashS9 2010

[6] A Falanga M Panova-Noeva and L Russo ldquoProcoagulantmechanisms in tumour cellsrdquo Best Practice and ResearchClinical Haematology vol 22 no 1 pp 49ndash60 2009

[7] T Tsuruo and N Fujita ldquoPlatelet aggregation in the formationof tumormetastasisrdquo Proceedings of the Japan Academy Series BPhysical and Biological Sciences vol 84 no 6 pp 189ndash198 2008

[8] I Vlodavsky Y Friedmann M Elkin et al ldquoMammalianheparanase gene cloning expression and function in tumorprogression and metastasisrdquo Nature Medicine vol 5 no 7 pp793ndash802 1999

[9] P H Kussie J D Hulmes D L Ludwig et al ldquoCloning andfunctional expression of a human heparanase generdquo Biochem-ical and Biophysical Research Communications vol 261 no 1pp 183ndash187 1999

[10] M Toyoshima and M Nakajima ldquoHuman heparanase Purifi-cation characterization cloning and expressionrdquo Journal ofBiological Chemistry vol 274 no 34 pp 24153ndash24160 1999

[11] G Chen Y-W Dang D-Z Luo Z-B Feng and X-L TangldquoExpression of heparanase in hepatocellular carcinoma hasprognostic significance a tissue microarray studyrdquo OncologyResearch vol 17 no 4 pp 183ndash189 2008

[12] B Chen X-P Chen M-S Wu W Cui and M ZhongldquoExpressions of heparanase and upstream stimulatory factorin hepatocellular carcinomardquo European Journal of MedicalResearch vol 19 no 1 pp 45ndash52 2014

[13] S-J Yu X-H Kang J-N Zhang et al ldquoEffects of smallinterfering RNA targeting heparanase-1 combined with heparinon invasiveness of mouse hepatocellular carcinoma cell linesrdquoChinese Journal of Cancer vol 29 no 9 pp 816ndash823 2010

[14] Z Xiong M-H Lu Y-H Fan et al ldquoDownregulation ofheparanase by RNA interference inhibits invasion and tumori-genesis of hepatocellular cancer cells in vitro and in vivordquoInternational Journal of Oncology vol 40 no 5 pp 1601ndash16092012

[15] L-D Zheng G-S Jiang J-R Pu et al ldquoStable knockdown ofheparanase expression in gastric cancer cells in vitrordquo WorldJournal of Gastroenterology vol 15 no 43 pp 5442ndash5448 2009

[16] C Pisano I Vlodavsky N Ilan and F Zunino ldquoThe potentialof heparanase as a therapeutic target in cancerrdquo BiochemicalPharmacology vol 89 no 1 pp 12ndash19 2014

[17] E A McKenzie ldquoHeparanase a target for drug discovery incancer and inflammationrdquo British Journal of Pharmacology vol151 no 1 pp 1ndash14 2007

[18] Y Nadir and B Brenner ldquoHeparanase procoagulant effectsand inhibition by heparinsrdquo Thrombosis Research vol 125supplement 2 pp S72ndashS76 2010

[19] Y Nadir B Brenner L Fux I Shafat J Attias and I VlodavskyldquoHeparanase enhances the generation of activated factor Xin the presence of tissue factor and activated factor VIIrdquoHaematologica vol 95 no 11 pp 1927ndash1934 2010

[20] YNadir andB Brenner ldquoHeparanasemultiple effects in cancerrdquoThrombosis Research vol 133 supplement 2 pp S90ndashS94 2014

[21] O Goldshmidt E Zcharia M Cohen et al ldquoHeparanasemediates cell adhesion independent of its enzymatic activityrdquoThe FASEB Journal vol 17 no 9 pp 1015ndash1025 2003

[22] C Peng Z Zhang J Wu et al ldquoA critical role for ZDHHC2 inmetastasis and recurrence in human hepatocellular carcinomardquoBioMed Research International vol 2014 Article ID 832712 9pages 2014

[23] X Liu H Fang H Chen et al ldquoAn artificial miRNA againstHPSE suppresses melanoma invasion properties correlatingwith a down-regulation of chemokines and MAPK phospho-rylationrdquo PLoS ONE vol 7 no 6 Article ID e38659 2012


[24] R Lever M J Rose E A McKenzie and C P Page ldquoHep-aranase induces inflammatory cell recruitment in vivo by pro-moting adhesion to vascular endotheliumrdquoAmerican Journal ofPhysiologymdashCell Physiology vol 306 no 12 pp C1184ndashC11902014

[25] A M Gomes E O Kozlowski L Borsig F C Teixeira IVlodavsky and M S Pavao ldquoAntitumor properties of a newnon-anticoagulant heparin analog from themolluskNodipectennodosus effect on P-selectin heparanase metastasis and cellu-lar recruitmentrdquo Glycobiology vol 25 no 4 pp 386ndash393 2015

[26] L Zhang J-N Wang J-M Tang et al ldquoVEGF is essential forthe growth and migration of human hepatocellular carcinomacellsrdquo Molecular Biology Reports vol 39 no 5 pp 5085ndash50932012

[27] B Zbytek J A Carlson J Granese J Ross M Mihm Jr andA Slominski ldquoCurrent concepts of metastasis in melanomardquoExpert Review of Dermatology vol 3 no 5 pp 569ndash585 2008

[28] F Levy-Adam N Ilan and I Vlodavsky ldquoTumorigenic andadhesive properties of heparanaserdquo Seminars in Cancer Biologyvol 20 no 3 pp 153ndash160 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Primer sequences for PCR amplification

Primers Primer sequences Length of products (bp)HPSE-F 51015840-GCACAAACACTGACAATCCAAG-31015840 101HPSE-R 51015840-AAAAGGATAGGGTAACCGCAA-31015840

GAPDH-F 51015840-GTGGTCTCCTCTGACTTCAACA-31015840 136GAPDH-R 51015840-CCACCACCCTGTTGCTGTAG-31015840

Against this background we hypothesize that HPSE mightplay a proadhesive role in adhesion and tumormicroembolusof HCC cells

The aim of this study is to explore whether HPSE enhancecancer cell adhesion in metastasis of HCC

2 Materials and Methods

21 Materials Human normal liver cell line LO-2 and HCCcell lines (HepG2 and BEL-7402) were from Cell BankNational Academy of Science of China (Shanghai China)Human highly metastatic liver cancer cell line HCCLM3was from Liver Cancer Institute (Zhongshan Hospital FudanUniversity Shanghai China) Markers dNTP primers werefrom Shanghai Shenggong Company (Shanghai China)DMEM medium RPMI-1640 medium FBS Trizol solutionand Lipofectamine 2000 were from Invitrogen Co (CarlsbadCA USA) Reverse transcription kits restrictive endonucle-ase BglII SalI and T4 DNA ligase were fromMBI FermentasChina Co Ltd (Shenzhen China) BCA protein quantitativekit and real-time qPCR kit (SYBR Green) were from TiangenBiotech Co Ltd (Beijing china) Plasmid pcDNATM62-GWEmGFP-miR (pmiR) and pcDNATM62-GWEmGFP-miR-Negative Control (pmiR-NC) were fromBeinuo BiotechCo Ltd (Shanghai China) PCRpurification kits andplasmidextraction kit were from Axygen Scientific Inc (Carls-bad CA USA) Rabbit-anti-HPSE polyclonal antibody andhorseradish peroxidase- (HRP-) conjugated anti-rabbit IgGwere from Jinqiao Biotechnology (Beijing China) Rabbit-anti-phosphoglyceraldehyde dehydrogenase (GAPDH) poly-clonal antiserum was from Santa Cruz Biotechnology Inc(Santa Cruz CA USA) Matrigel was from BD Bioscience(San Jose CA USA)

22 Determination of HPSE Expression in HCC Cells

221 Quantitative Real-Time Reverse Transcription Poly-merase Chain Reaction (qRT-PCR) Human HCC cells andnormal liver cell line LO-2 were cultured in RPMI-1640medium with 10 FBS According to Gene ID of HPSEmRNA sequence (NM 006665) PCR primers were designedand synthesized (Table 1) The qRT-PCR was performedaccording to the methods as described in our paper [12] andGAPDH was used as loading control The experiments wereperformed for three times

222 Western Blotting Cells were lysed in lysis bufferincubated on ice for 30min and centrifuged for 20minto remove cell debris Total cell lysate was subjected to

SDS-polyacrylamide gel electrophoresis The proteins weremeasured withWestern blotting according to the methods asdescribed in our paper [12] and GAPDHwas used as loadingcontrol The experiments were performed for three times

23 Plasmid Construction and Identification In the exper-iment four HPSE RNAi vectors were constructed usingmiRNA technique which is the same as the short hairpinRNA (shRNA) in essence but an artificial flanking pri-miRNA sequence is extended from the ends of shRNAtarget sequences Based on above principle four pairs ofmiRNA single-stranded oligonucleotide were designed andsynthesized according to Gene ID of HPSE mRNA sequence(Table 2) and then converted into double-stranded formby denaturation and subsequent annealing The 4 specificdouble-stranded miRNA sequences were cloned into thepmiR vector respectively The products were then trans-formed into Escherichia coli competent cells and cultivatedon a plate containing spectinomycin overnight at 37∘CPlasmid DNAs were extracted and sequenced The sampleswith correct sequence were named pcDNA-miR-HPSE-1pcDNA-miR-HPSE-2 pcDNA-miR-HPSE-3 and pcDNA-miR-HPSE-4 respectively

24 Transfection and Assessment Recombinant plasmidspmiR-HPSE-1 pmiR-HPSE-2 pmiR-HPSE-3 and pmiR-HPSE-4 were respectively transfected into HCCLM3 cellusing Lipofectamine 2000 following the manufacturerrsquos pro-tocol No plasmid was used in blank control group and pmiR-NC was used as negative control Transfection efficiencywas observed with invert fluorescence microscope 24 h aftertransfection Five hundred cells were randomly countedand the percentage of EGFP-positive cells was calculatedHPSE expressions in transfected cells were measured byreal-time RT-PCR and Western blot analysis 48 h later Theexperiments were performed for three times According tothe expression levels of HPSE one miRNA plasmid with bestinhibitory effect was chosen for following experiment

25 Determination of Cell Invasion Migrationand Adhesion Abilities

251 Transwell Invasion and Migration Assay The exper-iments were performed as previously described [22] Forinvasion assay 72 hours after transfection 5 times 104 transfectedHCC cells in serum-free RPMI-1640 were seeded into theupper chambers of each well of 24-well plate with insert(8mm pore size Millipore Billerica MA USA) coated withMatrigel For migration assay the upper chambers were not



ence

ofhu

man

heparanase

gene

Oligon

ucleotide

Sequ

enceso

fmiRNAoligo

Correspon

ding

sequ

ence

ofmRN

Apm

iR-H

PSE-1-F

51015840-TGCT

GTA

TCCT

GGTT

GAC

TTGAG

ATTG

TTTT

GGCC

ACTG

ACTG

ACAAT

CTCA

ACAAC

CAGGAT

A-31015840

356ndash

376b

ppm

iR-H

PSE-1-R

51015840-C

CTGTA

TCCT

GGTT

GTT

GAG

ATTG

TCAG

TCAG

TGGCC

AAAAC

AAT

CTCA

AGTC

AAC

CAGGAT

AC-31015840

pmiR-H

PSE-2-F

51015840-TGCT

GTA

CAGAG

CTTC

TTGAG

TAGGTG

TTTT

GGCC

ACTG

ACTG

ACAC

CTAC

TCGAAG

CTCT

GTA

-31015840

490ndash

570b

ppm

iR-H

PSE-2-R

51015840-C

CTGTA

CAGAG

CTTC

GAG

TAGGTG

TCAG

TCAG

TGGCC

AAAAC

ACCT

ACTC

AAG

AAG

CTCT

GTA

C-31015840

pmiR-H

PSE-3-F

51015840-TGCT

GTA

TACT

CGAAG

CTTC

CTTC

TCGTT

TTGGCC

ACTG

ACTG

ACGAG

AAG

GAC

TTCG

AGTA

TA-31015840

1280ndash1301b

ppm

iR-H

PSE-3-R

51015840-C

CTGTA

TACT

CGAAG

TCCT

TCTC

GTC

AGTC

AGTG

GCC

AAAAC

GAG

AAG

GAAG

CTTC

GAG

TATA

C-31015840

pmiR-H

PSE-4-F

51015840-TGCT

GAAAG

CTGGCA

AGCC

CAGTG

AAG

TTTT

GGCC

ACTG

ACTG

ACTT

CACT

GGTT

GCC

AGCT

TT-31015840

1560ndash1580b

ppm

iR-H

PSE-4-R

51015840-C

CTGAAAG

CTGGCA

ACCA

GTG

AAG

TCAG

TCAG

TGGCC

AAAAC

TTCA

CTGGGCT

TGCC

AGCT

TTC-

31015840


LO-2

cell

Hep

G2

cell

BEL-7402

cell

HCC

LM3

cell

Hep

G2

cell

BEL-7402

cell

HCC

LM3

cell

HPSE

GAPDH

mRNA Protein

LO-2

cell

(a)


HPS

E re

lativ

e exp

ress

ion

leve

ls

8

7

6

5

4

3

2

1

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast^^

lowastlowast^^

mRNAProtein

(b)










3 Results



110 120 130 140 150 160 170


(a)

110 120 130 140 150 160


(b)

170110 120 130 140 150 160


(c)

110 170120 130 140 150 160


(d)







4 Discussion




(a) (b)

(c) (d)

(e) (f)

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4



Tran

sfect

ion

effici

enci

es o

f pla

smid

s 10

08

06

04

02

0

(g)



mRN

APr

otei

n

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4

HPSE

HPSE

GAPDH

GAPDH

(a)H

PSE

expr

essio

ns aft

er tr

ansfe

ctio

n

8

7

6

5

4

3

2

1

0

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4





mRNAProtein

(b)





5 Conclusions


Abbreviations


reaction


Blank control

Mig

ratio

n

pmiR-NC pmiR-HPSE-1

Inva

sion

(a)


120

100

80

60

40

20

0

HCC

LM3

cell

num

bers

lowastlowast


(b)



20

40

60

80

100

120

lowast

Adhe

renc

e rat

e (

)





Disclosure






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















ence

ofhu

man

heparanase

gene

Oligon

ucleotide

Sequ

enceso

fmiRNAoligo

Correspon

ding

sequ

ence

ofmRN

Apm

iR-H

PSE-1-F

51015840-TGCT

GTA

TCCT

GGTT

GAC

TTGAG

ATTG

TTTT

GGCC

ACTG

ACTG

ACAAT

CTCA

ACAAC

CAGGAT

A-31015840

356ndash

376b

ppm

iR-H

PSE-1-R

51015840-C

CTGTA

TCCT

GGTT

GTT

GAG

ATTG

TCAG

TCAG

TGGCC

AAAAC

AAT

CTCA

AGTC

AAC

CAGGAT

AC-31015840

pmiR-H

PSE-2-F

51015840-TGCT

GTA

CAGAG

CTTC

TTGAG

TAGGTG

TTTT

GGCC

ACTG

ACTG

ACAC

CTAC

TCGAAG

CTCT

GTA

-31015840

490ndash

570b

ppm

iR-H

PSE-2-R

51015840-C

CTGTA

CAGAG

CTTC

GAG

TAGGTG

TCAG

TCAG

TGGCC

AAAAC

ACCT

ACTC

AAG

AAG

CTCT

GTA

C-31015840

pmiR-H

PSE-3-F

51015840-TGCT

GTA

TACT

CGAAG

CTTC

CTTC

TCGTT

TTGGCC

ACTG

ACTG

ACGAG

AAG

GAC

TTCG

AGTA

TA-31015840

1280ndash1301b

ppm

iR-H

PSE-3-R

51015840-C

CTGTA

TACT

CGAAG

TCCT

TCTC

GTC

AGTC

AGTG

GCC

AAAAC

GAG

AAG

GAAG

CTTC

GAG

TATA

C-31015840

pmiR-H

PSE-4-F

51015840-TGCT

GAAAG

CTGGCA

AGCC

CAGTG

AAG

TTTT

GGCC

ACTG

ACTG

ACTT

CACT

GGTT

GCC

AGCT

TT-31015840

1560ndash1580b

ppm

iR-H

PSE-4-R

51015840-C

CTGAAAG

CTGGCA

ACCA

GTG

AAG

TCAG

TCAG

TGGCC

AAAAC

TTCA

CTGGGCT

TGCC

AGCT

TTC-

31015840


LO-2

cell

Hep

G2

cell

BEL-7402

cell

HCC

LM3

cell

Hep

G2

cell

BEL-7402

cell

HCC

LM3

cell

HPSE

GAPDH

mRNA Protein

LO-2

cell

(a)


HPS

E re

lativ

e exp

ress

ion

leve

ls

8

7

6

5

4

3

2

1

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast^^

lowastlowast^^

mRNAProtein

(b)










3 Results



110 120 130 140 150 160 170


(a)

110 120 130 140 150 160


(b)

170110 120 130 140 150 160


(c)

110 170120 130 140 150 160


(d)







4 Discussion




(a) (b)

(c) (d)

(e) (f)

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4



Tran

sfect

ion

effici

enci

es o

f pla

smid

s 10

08

06

04

02

0

(g)



mRN

APr

otei

n

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4

HPSE

HPSE

GAPDH

GAPDH

(a)H

PSE

expr

essio

ns aft

er tr

ansfe

ctio

n

8

7

6

5

4

3

2

1

0

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4





mRNAProtein

(b)





5 Conclusions


Abbreviations


reaction


Blank control

Mig

ratio

n

pmiR-NC pmiR-HPSE-1

Inva

sion

(a)


120

100

80

60

40

20

0

HCC

LM3

cell

num

bers

lowastlowast


(b)



20

40

60

80

100

120

lowast

Adhe

renc

e rat

e (

)





Disclosure






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LO-2

cell

Hep

G2

cell

BEL-7402

cell

HCC

LM3

cell

Hep

G2

cell

BEL-7402

cell

HCC

LM3

cell

HPSE

GAPDH

mRNA Protein

LO-2

cell

(a)


HPS

E re

lativ

e exp

ress

ion

leve

ls

8

7

6

5

4

3

2

1

0

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast^^

lowastlowast^^

mRNAProtein

(b)










3 Results



110 120 130 140 150 160 170


(a)

110 120 130 140 150 160


(b)

170110 120 130 140 150 160


(c)

110 170120 130 140 150 160


(d)







4 Discussion




(a) (b)

(c) (d)

(e) (f)

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4



Tran

sfect

ion

effici

enci

es o

f pla

smid

s 10

08

06

04

02

0

(g)



mRN

APr

otei

n

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4

HPSE

HPSE

GAPDH

GAPDH

(a)H

PSE

expr

essio

ns aft

er tr

ansfe

ctio

n

8

7

6

5

4

3

2

1

0

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4





mRNAProtein

(b)





5 Conclusions


Abbreviations


reaction


Blank control

Mig

ratio

n

pmiR-NC pmiR-HPSE-1

Inva

sion

(a)


120

100

80

60

40

20

0

HCC

LM3

cell

num

bers

lowastlowast


(b)



20

40

60

80

100

120

lowast

Adhe

renc

e rat

e (

)





Disclosure






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















110 120 130 140 150 160 170


(a)

110 120 130 140 150 160


(b)

170110 120 130 140 150 160


(c)

110 170120 130 140 150 160


(d)







4 Discussion




(a) (b)

(c) (d)

(e) (f)

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4



Tran

sfect

ion

effici

enci

es o

f pla

smid

s 10

08

06

04

02

0

(g)



mRN

APr

otei

n

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4

HPSE

HPSE

GAPDH

GAPDH

(a)H

PSE

expr

essio

ns aft

er tr

ansfe

ctio

n

8

7

6

5

4

3

2

1

0

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4





mRNAProtein

(b)





5 Conclusions


Abbreviations


reaction


Blank control

Mig

ratio

n

pmiR-NC pmiR-HPSE-1

Inva

sion

(a)


120

100

80

60

40

20

0

HCC

LM3

cell

num

bers

lowastlowast


(b)



20

40

60

80

100

120

lowast

Adhe

renc

e rat

e (

)





Disclosure






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e) (f)

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4



Tran

sfect

ion

effici

enci

es o

f pla

smid

s 10

08

06

04

02

0

(g)



mRN

APr

otei

n

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4

HPSE

HPSE

GAPDH

GAPDH

(a)H

PSE

expr

essio

ns aft

er tr

ansfe

ctio

n

8

7

6

5

4

3

2

1

0

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4





mRNAProtein

(b)





5 Conclusions


Abbreviations


reaction


Blank control

Mig

ratio

n

pmiR-NC pmiR-HPSE-1

Inva

sion

(a)


120

100

80

60

40

20

0

HCC

LM3

cell

num

bers

lowastlowast


(b)



20

40

60

80

100

120

lowast

Adhe

renc

e rat

e (

)





Disclosure






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















mRN

APr

otei

n

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4

HPSE

HPSE

GAPDH

GAPDH

(a)H

PSE

expr

essio

ns aft

er tr

ansfe

ctio

n

8

7

6

5

4

3

2

1

0

Blan

k co

ntro

l

pmiR

-NC

pmiR

-HPS

E-1

pmiR

-HPS

E-2

pmiR

-HPS

E-3

pmiR

-HPS

E-4





mRNAProtein

(b)





5 Conclusions


Abbreviations


reaction


Blank control

Mig

ratio

n

pmiR-NC pmiR-HPSE-1

Inva

sion

(a)


120

100

80

60

40

20

0

HCC

LM3

cell

num

bers

lowastlowast


(b)



20

40

60

80

100

120

lowast

Adhe

renc

e rat

e (

)





Disclosure






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Blank control

Mig

ratio

n

pmiR-NC pmiR-HPSE-1

Inva

sion

(a)


120

100

80

60

40

20

0

HCC

LM3

cell

num

bers

lowastlowast


(b)



20

40

60

80

100

120

lowast

Adhe

renc

e rat

e (

)





Disclosure






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Disclosure






Acknowledgments


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
















Research Article Downregulation of Heparanase Expression ...

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