ANXA5 level is linked to

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ANXA5 level is linked to in vitro and in vivo tumor malignancy and lymphatic metastasis of murine hepatocarcinoma cell Boya Peng‡,1, Shuqing Liu‡,2, Chunmei Guo‡,1, Xujuan Sun1 & Ming-Zhong Sun*,1

Aim: To investigate ANXA5 overexpression on in vitro and in vivo malignancies of murine Hca-P cells. Materials & methods: Hca-P with low lymph node metastasis (LNM) potential was used as cell model. TEM, CCK-8 and Boyden transwell assays were performed for in vitro Hca-P behaviors. Hca-P-transplanted mouse model was established for in vivo experiment. Results: ANXA5-overexpressing monoclonal Anxa5-Hca-P-1, Anxa5-Hca-P-2 and Anxa5Hca-P-3 cells were obtained. ANXA5 upregulation alters the proliferation, morphology and rough endoplasmic reticulum of Hca-P cells, enhances in vitro migration and invasions of Hca-P, promotes in vivo malignant degree and LNM rate of Anxa5-Hca-P-3-transplanted mice. Conclusion: As a potential indicator for malignancy and lymphatic metastasis, ANXA5 overexpression increases in vitro migration and invasion of Hca-P cell, promotes in vivo malignancy, LNM rate and level of Hca-P-transplanted mice. First draft submitted: 17 June 2015; Accepted for publication: 20 October 2015; Published online: 30 November 2015

Hepatocarcinoma is characterized with high recurrence, metastasis and poor prognosis. Metastasis is a critical prognostic factor for hepatocarcinoma patients [1,2] . Lymph node metastasis (LNM), an initial metastasis step in epithelial carcinoma, is complex and dynamic [2,3] . The study on LNM helps in targeted new therapeutic strategies for hepatocarcinoma management. Encoded by a conserved multigene family and expressed in plants, vertebrates, invertebrates, fungi and protists, annexins are a superfamily of Ca 2+ -regulated phospholipid-binding proteins. There are 12 annexins common to vertebrates named as annexin A1–A11 and A13. The annexins are composed of two principal domains, a variable N-terminal domain from 11 to 196 residues and a conserved C-terminal core containing the Ca2+ - and membrane-binding sites. C-terminal cores of annexins are characterized by four homologous annexin repeats except for AnxA6, which has eight [4] . Each annexin repeat is composed by ∼70 highly conserved amino acid residues. A highly α-helical disc structure with a slight curvature is formed by these annexin repeats. Localizing on the concave side of annexin, the diversity of the flexible N-terminal domain is the principal criterion for distinguishing annexin subfamilies [4–7] . Annexins have been reported playing important roles in human diseases. The differential annexins expression and/or their subcellular redistributions regulate tumor cell p­roliferation, motility and apoptosis, tumor invasion and metastasis, angiogenesis and drug resistance [4,6–9] . ANXA5 is a member annexin family. In 1985, it was first identified as a vascular anticoagulant [5] . The deregulation of ANXA5 is closely associated with the tumor progression, invasion, metastasis,

Keywords

• ANXA5 • Hca-P • hepatocarcinoma • invasion • lymphatic metastasis • migration

Department of Biotechnology, Dalian Medical University, 9 West Section, Lvshun Southern Road, Dalian 116044, China Department of Biochemistry, Dalian Medical University, 9 West Section, Lvshun Southern Road, Dalian 116044, China *Author for correspondence: Tel.: +86 0411 8611 0445; Fax: +86 0411 8611 0218; [email protected]; [email protected] ‡ Authors contributed equally 1 2

10.2217/fon.15.289 © 2016 Future Medicine Ltd

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ISSN 1479-6694

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Research Article  Peng, Liu, Guo, Sun & Sun drug resistance and treatment [3–4,6–9] . However, the correlation between ANXA5 and tumor LNM is poorly understood. We previously reported ANXA5 was linked to the lymphatic metastasis of hepatocarcinoma [3,8–9] . ANXA5 was upregulated over twofold in Hca-F cells with high LNM potential than in Hca-P cells with low LNM potential [3,8–9] , suggesting its important role in tumorigenicity and ­lymphatic metastasis. Hca-P is a murine hepatocarcinoma cell line with low LNM rate that specifically metastasizes to lymph node (LN) without dissemination to other organs [3,8–14] . We selected Hca-P as the cell model both for the study on the malignancy as well as the initial (low) and specific lymphatic metastasis of hepatocarcinoma. By upregulating ANXA5 level Hca-P cells using PcDNA3.1/V5-HisB-Anxa5 overexpression vector, the investigation of ANXA5 expression level on the in vitro and in vivo malignant behaviors and lymph node metastasis capacity of Hca-P was conducted in current work. Material & methods ●●Cell culture, animals & ethics statement

Hca-P cell line was maintained in our laboratory. Hca-P cells were incubated in RPMI-1640 (Gibco, NY, USA) containing 15% fetal bovine serum (FBS) (PAA, Australia) at 37°C with 5% CO2 . Inbred Chinese 615 mice (6–8 weeks, 18–22 g) were provided and maintained at the SPF Experimental Animal Center, Dalian Medical University. Mice were treated and sacrificed in accordance with the protocols approved by Experimental Animal Ethical Committee of Dalian Medical University (permit number: L2012012).

RT-PCR Kit (Takara, Japan). RT-PCR and PCR were performed on a MyCycle™ Thermal Cycler (Bio-Rad, CA, USA) using PrimerScript RTase and PrimeSTAR HS DNA polymerase. PCR products were analyzed by 1% agarose gel electrophoresis. Construction of pMD®19-T-Anxa5 cloning vector

Anxa5 PCR product was purified using universal DNA purification kit (Tiangen, China) and inserted with an A base tail using Ex Taq® HS kit (TaKaRa, Japan). Repurified using above kit, the A-tailed Anxa5 gene was linked to pMD®19-T simple vector (TaKaRa) using T4 DNA Ligase (TaKaRa) at 16°C over night. The recombinant pMD®19-T-Anxa5 plasmid was transformed into Escherichia coli DH5α (TaKaRa) and amplified in LB medium with 100 μg/ml ampicillin (Sigma, MO, USA). The positive recombinant plasmid was extracted by alkaline-lysis method, digested by BamH I/EcoR I and analyzed by 1% agarose gel electrophoresis. Construction of pcDNA3.1-V5/HisB-Anxa5 vector

PcDNA3.1/V5-HisB-Anxa5 expression vector was constructed. The recombinant pMD®19-TAnxa5 cloning vector was digested with BamH I and EcoR I. The cleaved insert was subcloned into a pcDNA3.1/V5-HisB vector (CPG Biotech, China) using T4 DNA Ligase (TaKaRa) at 16°C over night. The ligation was transformed into competent E. coli DH5α cells and amplified for plasmid extraction by E.Z.N.A.® EndoFree Plasmid Mini Kit (OMEGA, USA). The plasmid was validated by PCR assay, restriction enzyme digestion and sequencing analysis.

●●Primer design & recombinant plasmid

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construction Primer design & PCR amplification

Anxa5 stable transfection & monoclonal Axna5-Hca-P cells screening

Ba sed on sequence (GeneBa nk ID : BC003716.1), the forward (F) and reverse (R) primers of Anxa5 were designed by Oligo7 (F: 5′-CGGGATCC ­A TCATGG C TACG AG AGGC AC TGTG AC -3′; R : 5′-GGAATTC­TGTCATCC­TCGCCCCCG CAGAGC-3′). The F primer contains a BamH I and the R primer contains an EcoR I restriction cleavage site. Total RNA was extracted from Hca-P cells using Trizol™ reagent (Life Technologies, CA, USA) and amplified by RT-PCR using high fidelity PrimeScript

Hca-P cells were inoculated in 85% RPMI 1640 supplemented with 15% FBS for 3 days, and seeded into a 24-well plate at the density of 105/500 μl for 24 h at 37°C with 5% CO2. The concentrations of recombinant and empty plasmids were determined by Thermo Scientific NanoDrop 2000 (Thermo Fisher Scientifc, DE, USA). Each of the plasmid extractions were mixed with Lipofectamine® 2000 reagent (Invitrogen, CA, USA) at the ratio of 0.5:1.5 (μg:μl), then mixed well with 98 μl RPMI-1640 medium at room temperature (RT) for 20 min.

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ANXA5 deregulation on murine hepatocarcinma lymphatic metastasis  Fifty microliter of plasmid-lipofectamine® mixture was added into each well for transfection at 37°C with 5% CO2 for 48 h. The transfected cells were continuously incubated in medium with 400 μg/ml G418 (Sigma) until the survived cells were completely resistant to G418. Monoclonal Anxa5- and empty vector-transfected Hca-P cells were obtained using limiting dilution method in G418 at 200 μg/ml. Three monoclonal Anxa5-transefced and one empty vector-transfected Hca-P cells were named as Anxa5-Hca-P-1, Anxa5-Hca-P-2, Anxa5Hca-P-3 and Control-Hca-P. ANXA5 levels were measured by western blotting assay. ●●Transmission electron microscopy

characterization

Control-Hca-P and Anxa5-Hca-P-3 cells were fixed in 2.5% glutaraldehyde at 4°C over night, post-fixed with 1% osmium tetroxide for 2 h, dehydrated in a graded ethanol series (50, 70, 80, 90 and 100%), infiltrated with propylene oxide at 4°C for 4 h and embedded in Epon 812. Ultrathin sections were obtained using an Ultramicrotome (Leica EM UC6, Germany). All sections, mounted onto copper grids (150mesh squares), were contrasted using uranyl acetate and lead citrate before being visualized using a transmission electron microscope (JEM2000EX, Japan).

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●●SDS-PAGE & western blotting

Cell pellets were obtained by centrifuging at 1000 × g rpm for 5 min, suspended and ultrasound sonicated in ice-cold lysis buffer (KeyGEN, China) with 1 μl 100 mM phenylmethanesulfonyl fluoride (PMSF), 2 μl phosphate inhibitors and 0.2 μl protease inhibitor. The supernatants were collected at 12,000 rpm for 15 min at 4°C. Protein concentrations were measured using Bradford assay. Equal amounts of protein samples from each group were separated by 12% SDS-PAGE. Protein bands were transferred onto nitrocellulose (NC) membrane (Pall Corporation, NY, USA). Being blocked in 5% skim milk (in Tris-buffered saline with Tween-20 [TBST] buffer) for 3 h at RT, the NC membrane was incubated with primary antibodies shaking with 90 rpm at 4°C overnight. The primary antibodies were rabbit anti-ANXA5 (Epitomics, USA, 1:2000) and mouse anti-βactin (Proteintech, China, 1:5000). The NC membranes were washed with TBST for three times, incubated with peroxidase-conjugated goat anti-rabbit/anti-mouse IgG (ZSGB-Bio, China, 1:1000) at 37°C for 1 h. Being extensive washed by TBST 4 × 10 min, protein bands were visualized by ECL (Adavansta, CA, USA) and detected by Bio-Rad ChemiDoc™ MP imaging system (Bio-Rad, USA). Protein levels were normalized and quantified against β-actin.

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Figure 1. Identifications of Anxa5 cds and recombinant plasmid. (A) PCR of Anxa5 cds. Lane M: DNA marker, lane 1: anxa5 PCR product; (B) Anxa5 cloning plasmid pMD®19-T-Anxa5. Lane M: marker, 1: pMD®19-T-Anxa5, 2: pMD®19-T-Anxa5 cleavage by BamHI, 3: pMD®19-T-Anxa5 cleavage by BamHI and EcoRI, 4: PCR product; (C) Identification expression plasmid pcDNA3.1-V5/HisB-Anxa5. Lane M: marker, 1: pcDNA3.1-V5/HisB-Anxa5, 2: pcDNA3.1-V5/HisB-Anxa5 cleavage by BamHI, 3: pcDNA3.1-V5/HisB-Anxa5 cleavage by BamHI and EcoRI, 4: PCR product.

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Research Article  Peng, Liu, Guo, Sun & Sun

β-actin

Anxa5 relative expression level

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Figure 2. Western blotting of ANXA5 levels. (A) Gel images for Control-Hca-P, Anxa5-Hca-P-1, Anxa5-Hca-P-2 and Anxa5-Hca-P-3 cells; (B) relative ANXA5 levels in Control-Hca-P, Anxa5-Hca-P-1, Anxa5-Hca-P-2 and Anxa5-Hca-P-3. The differences were statistically significant (p < 0.05). ●●Cell proliferation assay

The effect of ANXA5 overexpression on Hca-P proliferation was evaluated using Cell Counting Kit-8 (CCK-8, Solarbio, Japan) assay. ControlHca-P, Anxa5-Hca-P-1, Anxa5-Hca-P-2 and Anxa5-Hca-P-3 cells were seeded in a 96-well plate 1500 cells/well in 100 μl RPMI-1640 with 15% FBS, incubated at 37°C with 5% CO2 for 0, 24, 48, 72 and 120 h, respectively, in prior to 10 μl of CCK-8 reagent was added into each

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well for further incubation at 37°C for 2 h. The absorbances at 450 nm were recorded using a Multiscan Go Spectrophotometer (Thermofisher Scientific, MA, USA). ●●In vitro cell migration assay

Migration was performed using 24-well transwell units with 8 μm id polyester membrane plates (Corning, NY, USA). The filter surfaces were coated with 6 μl fibronectin (Millipore, MA,

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ANXA5 deregulation on murine hepatocarcinma lymphatic metastasis  USA). 500 μl RPMI 1640 containing 20% FBS (PAA, Australia) was added into each lower compartment of the chambers. 5 × 104 cells in 100 μl serum-free RPMI-1640 were seeded in the upper chamber and incubated in 5% CO2 at 37°C for 24 h. Nonmigrated cells on the upper surface filter were wiped with cotton swabs. Migrated cells on the lower filter surface were fixed in 4% paraformaldehyde for 20 min, stained in 0.1% crystal violet for 20 min and washed with PBS. Cells were counted under a light microscope at a magnification of 100×. The data were represented as the averaged cell number of five fields from each of triplicate experiments.

Research Article

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●●In vitro cell invasion assay

The filter surfaces of 24-well transwell units were coated with 30 μl of ice-cold ECM gel (BD, USA; 1:8 dilution with serum-free RPMI1640), incubated at 37°C for 5 h, dried at RT. Then 50 μl of serum-free RPMI-1640 was added into the well for hydration for 30 min to generate an artificial basement membrane. The rest of procedures were the same as described in cell migration assay.

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●●In vivo tumorigenicity & lymph node

metastasis rate assays

Twenty inbred mice were randomly divided into two groups. Control-Hca-P and Anxa5-Hca-P-3 cells were subcutaneously inoculated into the left footpads of mice (1 × 106 cells per mouse). In 25 days after inoculation, the mice were sacrificed. The sizes and volumes of solid tumors were measured, calculated and compared. The popliteal, inguinal, iliac arterial, pararenal and axillary lymph nodes from tumor-bearing mice were collected, stained by hematoxylin–eosin and examined by light microscope. LNM rates of tumor cells were then calculated.

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●●Data processing & statistical analysis

All the results were presented as mean ± standard deviations. Statistical analyses were performed using SPSS 17.0 software by one-way analysis of variance, repeated measures analysis of variance and Wilcoxon test. Statistical significance was set at p < 0.05. Results ●●Recombinant Anxa5 overexpression

plasmid is successfully constructed

PCR product of Anxa5 showed a band with the size matching its theoretical length of 960 bp

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Figure 3. ANXA5 upregulation on Hca-P shape and ultrastructure. (A) Morphology observation by microscope at a magnification of 400; (B) transmission electron microscopy of Control-Hca-P at magnifications of 10,000 (scale: 2 μm) and 25,000 (0.5 μm, enlarged insert plot); (C) transmission electron microscopy of Anxa5-Hca-P-3 at magnifications of 10,000 (2 μm) and 25,000 (0.5 μm, enlarged insert plot).

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Research Article  Peng, Liu, Guo, Sun & Sun (Figure 1A) . Cleaved by BamH I/EcoR I restriction enzymes, recombinant pMD®19-T-Anxa5 cloning vector showed two bands on gel (1%) with the sizes ∼2700 and ∼1000 bp (Figure 1B) , matching the theoretical fragments of empty pMD®19-T vector (2692 bp) and Anxa5 (960 bp). PCR amplification using pMD®19-TAnxa5 plasmid as the template gave a band of ∼960 bp (Figure 1B) . Electrophoresis analyses of the single and double enzymatic cleavage product of pcDNA3.1/V5-HisB-Axna5 expression plasmid and PCR amplication product using it as the template demonstrated Axna5 fragment was sucessfully inserted into pcDNA3.1/V5-HisB vector (5500 bp) (Figure 1C) . Sequencing analysis (data unshown) of the recombinant plasmid was the same as Anxa5 sequence in GenBank (BC003716.1). PcDNA3.1/V5-HisB-Anxa5 overexpression vector was established. ●●Stable overexpression of ANXA5 in

monoclonal Anxa5-transfecetd-Hca-P cells

ANX A5 levels in Anxa5-Hca-P-1, Anxa5Hca-P-2, Anxa5-Hca-P-3 and Control-Hca-P cells were determined by WB assay (Figure 2A) . Compared with the Control-Hca-P, ANXA5 levels in Anxa5-Hca-P-1, -2 and -3 cells increased by ∼15.0, 20.3 and 67.7% (Figure 2B) . The stable cellular upregulation of ANXA5 ensures the feasibility of studying the effect of its overexpression on the biological properties of Hca-P cells. ●●ANXA5 overexpression induces

morphology & ultrastructure changes of Hca-P

Compared with the Control-Hca-P cells, inverted optical microscope images suggested morphology alteration for Anxa5-Hca-P-3 cells (Figure 3A) . The cell sizes, volumes and contents increased for Anxa5-Hca-P-3 cells following ANXA5 overexpression. The cell membrane surface became rougher (Figure 3A) . ANXA5 overexpression affects Hca-P ultrastructure.

Transmission electron microscopy showed more mitochondria (marked by white arrow, Figure 3B & C) and more autophagic vacuoles presenting in Anxa5-Hca-P-3 than Control-Hca-P cells. Following ANXA5 upregulation, the number of intracellular rough endoplasmic reticula (RER) increased, while in the contrast, the mitochondrial crista and matrix density decreased in Hca-P cells (Figure 3) . ●●ANXA5 level is linked to Hca-P cell

proliferation

CCK-8 assay was performed to measure the effect of ANXA5 upregulation on Hca-P proliferation. As shown in Table 1 & Figure 4, Anxa5Hca-P-1 and Anxa5-Hca-P-2 showed enhanced proliferation abilities, while Anxa5-Hca-P-3 showed reduced cell proliferation capacity compared with the Control-Hca-P cells (Figure 4) . ANXA5 upregulation inconsequently and controversially increases or decreases the proliferation of Hca-P cells, which might depend on the upregulation level and reflects the complicated and mysterious functions of protein function in tumors. ●●ANXA5 upregulation enhances the in vitro

migration & invasion potentials of Hca-P cells

ANXA5 upregulation enhanced the in vitro invasion and migration capacities of HcaP. Following ANX A5 over-expression, the migration and invasion capacities of Hca-P cells increased (Figure 5A) . The migrated cell numbers for Control-Hca-P, Anxa5-Hca-P-1, Anxa5-Hca-P-2 and Anxa5-Hca-P-3 were 46.0 ± 7.2, 77.3 ± 4.2, 73.7 ± 11.1 and 123.0 ± 12.8 (Figure 5A & B) . Compared to ControlHca-P cells, the relative migration capacities of Anxa5-Hca-P-1, -2 and -3 cells increased by ∼68.0%, 60.2% and 167.4% due to ANXA5 overexpression. The invaded cell numbers for Control-Hca-P, Anxa5-Hca-P-1, -2 and -3 were

Table 1. CCK-8 assay for the proliferations of Hca-P cells. Time (h)

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Absorbance at 450 nm of each cell group (mean ± standard deviation) 

 

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0 24 48 72 96 120

0.198 ± 0.006 0.232 ± 0.004 0.300 ± 0.003 0.444 ± 0.019 0.714 ± 0.022 1.211 ± 0.010

0.191 ± 0.012 0.254 ± 0.007 0.333 ± 0.007 0.490 ± 0.023 0.786 ± 0.049 1.326 ± 0.115

0.193 ± 0.012 0.269 ± 0.009 0.346 ± 0.014 0.576 ± 0.020 0.881 ± 0.046 1.408 ± 0.010

0.188 ± 0.009 0.204 ± 0.004 0.251 ± 0.002 0.373 ± 0.003 0.543 ± 0.023 0.955 ± 0.049

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ANXA5 deregulation on murine hepatocarcinma lymphatic metastasis 

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Figure 4. Effect of Anxa5 overexpression on Hca-P cell growth. The proliferations of Anxa5-Hca-P-1, Anxa5-Hca-P-2 and Anxa5-Hca-P-3 cells were of significant differences (p < 0.05) compared with Control-Hca-P cells.

measured as 35.3 ± 6.0, 52.0 ± 10.1, 55.7 ± 8.5 and 70.7 ± 7.0 (Figure 5A & C) , respectively. The invasion capacities of Anxa5-Hca-P-1, -2 and -3 cells increased by ∼47.3%, 57.8% and 100.3% (Figure 5A & C) following ANXA5 upregulations. ●●ANXA5 promotes the in vivo tumor

malignancy grade & lymph node metastasis potential of Hca-P

The effect of ANXA5 overexpression on tumorigenicity of Hca-P cells was investigated. Anxa5Hca-P-3 cells were transplanted into the mice left footpads. On the 25th day after inoculation, the sizes and volumes of solid tumors were measured and calculated. Volume is defined as V = πabc/6, where a, b and c were the length, width and height of the tumor in centimeters. Although three more Anxa5-Hca-P-3-transplanted mice showed tumor volume >1.545 cm3 than the control, ANXA5 upregulation showed no statistical significance on the size of primary tumor induced by Hca-P cells (Table 2) . Based on the definition of apparent malignant levels as illustrated in Figure 6A & Table 2, ANXA5 upregulation increased the malignant grade of primary tumor (Table 2) and the in vivo LNM potential of Hca-P cells (Figure 6B & Table 3) significantly. The invaded tumor cells in metastastical LNs (Figure 6B4 & B5) were in the same morphology

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of Hca-P cells in primary tumor (Figure 6B2) . Based on the classification of LNM levels as illustrated in Figure 6B & Table 2, we found the in vivo LNM rate and malignant grades were enhanced for Anxa5-Hca-P-3 cells. The LNM rate of Anxa5-Hca-P-3 was ∼20.6% higher than that of Control-Hca-P. The percentage of LNM with ++ and +++ levels of Anxa5-Hca-P-3 was ∼18.1% higher than that of Control-Hca-P cells (Table 2) . Discussion Lymphatic metastasis is a complex and dynamic process for tumor malignancy [2–3,15] . Hepatocarcinoma occurs with high recurrence, high metastasis and poor prognosis. Metastasis is a critical prognostic factor for hepatocarcinoma patients [1,2] . However, the lymphatic metastasis of hepatocarcinoma is poorly understood. ANXA5 upregulation was correlated with bladder cancer progression toward nodal positivity, with higher tumor stage, liver metastasis, increased recurrence and lower overall survival of colorectal cancer, and with the migration and invasion of oral carcinoma cells [16–18] . Previously, we linked ANXA5 to the lymphatic metastasis of hepatocarcinoma [3,8–9,19] . In current work, we selected Hca-P as an ideal cell model both for hepatocarcinoma maligancy and

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Migration

Research Article  Peng, Liu, Guo, Sun & Sun

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Figure 5. The effect of ANXA5 expression on Hca-P migration and invasion. (A) Images of migrated and invaded cells of ControlHca-P, Anxa5-Hca-P-1, Anxa5-Hca-P-2 and Anxa5-Hca-P-3 at the magnification of 100×. (B) Comparative migration capacities of 4 group cells. The increased migration of Anxa5-Hca-P cells were of statistical significances compared with Control-Hca-P cells (p