Cancerous, para-cancerous and non-cancerous tissues were obtained from 30 patients who underwent surgery for HCC at the Affiliated Hospital of Nantong University (Nantong, China). Tissues were immediately frozen in liquid nitrogen and kept at 80°C until required. Cases included 22 males and 8 females and of these cases, 18 exhibited tumour sizes of ≥5 cm and 21 exhibited AFP levels of ≥ 50 ng/ml. Pathological examination with H&E staining showed that cancerous tissues were highly differentiated HCC. Of the para-cancerous tissues, 22 exhibited cirrhosis, 12 exhibited chronic hepatitis and 24 exhibited atypical hyperplasia. Of the non-cancerous tissues, 21 exhibited cirrhosis, 12 exhibited chronic hepatitis and 15 exhibited atypical hyperplasia. One portion of the specimen was for total RNA extraction and ANXA2 preparation and the other was fixed with 10% formalin for immunohistochemistry. Prior written informed consent was obtained from all patients according to the World Medical Association Declaration of Helsinki and the study received ethics board approval from the Affiliated Hospital of Nantong University.

shRNA corresponding to nucleotides 94–113 downstream of the transcription start site of the ANXA2 gene was synthesised according to methods previously described (22). The shRNA was inserted into BamHI- and HindIII-linearised pRNAT-U6.1/Neo shRNA expression vectors and fragments were conformed by sequencing and named as pRNAT-U6.1-shRNA or -negative, respectively. HepG2, SMMC-7721, SMMC-7402 and LO₂ cell lines were obtained from Biomics Biotechnologies (Nantong) Co., Ltd. (Nantong, China) and MHCC97-H cell lines were obtained from the Liver Cancer Institute, Fudan University (Shanghai, China). Cells were grown in DMEM with 10% fetal bovine serum at 37°C and 5% CO₂ and grown to 90–95% confluency. MHCC97-H cells were transfected with the pRNAT-U6.1-shRNA or -negative plasmids using PolyJet™ (SignaGen, Rockville, MD, USA). At 48 h, selection was performed with medium containing 400 μg/ml G418 for 14 days, followed by 200 μg/ml G418. Cell lines were named as the shRNA or negative groups and blank cells were named as the mock group.

RNA was isolated from 50 mg liver tissue using TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA integrity was examined by 1% agarose gel electrophoresis and quantity and purity was based on absorbance (A) at A₂₆₀ and the ratio at A_260/280 (Smartspec™ plus spectrophotometer; Bio-Rad, Hercules, CA, USA). ANXA2-cDNA was synthesised from 1 μg RNA using the 1^st strand cDNA synthesis kit (Fermentas Canada Inc., Burlington, ON, Canada). qPCR was performed using the StepOne™ system (Applied Biosystems, Foster City, CA, USA) with a solution containing 25 μl 2X SYBR Premix ExTaq (Takara Bio, Inc., Shiga, Japan), 2 μl primer mix, 1 μl 50X ROX Reference Dye I, 4 μl cDNA and 18 μl deionised water to 50 μl. The following primers were used: i) ANXA2 forward, 5′-TGAGCGGGATGCTTTGAAC-3′ and reverse, 5′-ATCCTGTCTCTGTGCATTGCTG-3′; and ii) β-actin forward, 5′-ATTGCCGACAGGATGCAGA-3′ and reverse, 5′-GAGTACTTGCGCTCAGGAGGA-3′ were used as control. In addition, a no template control was included in each run. The optimised conditions were as follows: 1 cycle at 95°C for 2 min, 40 cycles of 95°C for 10 sec, 62°C for 1 min and 60°C for 15 sec and analysis was performed using the 2^−ΔΔCt method.

Cells were cultured for 24 h on cover slips in 24-well plates, fixed with 4% paraformaldehyde and then blocked with PBS containing 3% BSA. Samples were incubated with rabbit anti-human ANXA2 antibody (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight. Following incubation with Cy3-labelled goat anti-rabbit IgG secondary antibody (1:500; Beyotime Institute of Biotechnology, Haimen, China), cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and sealed with 50% glycerin. Observations were performed under a microscope (IX71; Olympus Corp., Tokyo, Japan).

Cell proliferation was evaluated using a cell counting kit-8 (CCK-8; Beyotime Institute of Biotechnology). Cells and blank controls were seeded in 96-well plates (2×10³ cells/well with 100 μl medium; n=5) and cultured for 24 h. Next, 10 μl CCK-8 solution was added to the culture medium for 2 h and the absorbance was recorded at A₄₅₀ by a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). This was repeated 5 times at various time points. The cell cycle assay was performed using a cell cycle and apoptosis kit (Beyotime Institute of Biotechology). Cells were seeded at 1.0×10⁶ cells/well (6-well plate) and cultured for 24 h. Subsequently, cells were digested with trypsin and fixed for 24 h at 4°C in pre-cooled 70% ethanol. Cells were stained with propidium iodide and analysed by a flow cytometry (BD FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) for cell cycle analyses.

Cells were plated at 1.0×10⁵ cells/well in 0.5 ml serum-free medium in 24-well Matrigel-coated Transwell units with polycarbonate filters (CoStar Group Inc., Washington, DC, USA) containing 8-μm pores. The outer chambers were filled with 0.5 ml medium containing 10% FBS. At 24 h, the cells were fixed in methanol and stained with crystal violet. The top surface of the membrane was gently scrubbed with a cotton bud and cells invading through the membrane filters were counted on glass slides. Images were captured using an inverted microscope equipped with a CCD camera (IX71; Olympus Corp.).

The protocol was approved by the ethics review committee for animal experimentation (Nantong University). In total, 16 BALB/C nude mice (SPF; 6 weeks-old; body weight, 20±3 g; Shanghai Super-B&K Laboratory Animal Co., Ltd., Shanghai, China) were randomly divided into mock, negative, shRNA and control groups (4 mice in each group). Cells (2×10⁷/mouse) suspended in 0.2 ml DMEM were injected subcutaneously into the right flank of the nude mice. Tumour size was measured using calipers at the indicated time points and their volume was calculated according to the formula: Volume = (length × width²)/2. Mice were sacrificed on day 21 following injection. The following formula was used to calculate the rate of tumourigenicity inhibition: Tumourigenicity inhibition rate = [(tumour weight control - tumour weight shRNA)/tumour weight control] × 100.

Pathological examination was performed by H&E staining. Immunohistochemistry (streptavidin-peroxidase method) with anti-human ANXA2 antibody (1:500; Santa Cruz Biotechnology, Inc.) was performed and negative controls were treated with non-specific mouse IgG. ANXA2 staining was assessed using the immunoreactive score. In brief, the percentage of positive cells was semi-quantitatively classified as follows: Diffuse positive (+++), >50% of total cells; moderate (++), 16–50%; and weak (+), 5–15%; and negative (−), <5%. Cells were evaluated by two independent pathologists and any differences in interpretation were resolved by consensus. Duplicate tissue cores for each tumour showed high levels of homogeneity for staining intensity and percentage of positive cells. The higher score was used as the final score in cases with a difference between duplicate tissue cores.

Tissues were homogenised in an ice-cold homogenisation buffer and centrifuged at 800 × g. Total proteins were determined by bicinchoninic acid assay. Each 20 mg of protein was separated by 15% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes and blocked with 5% BSA in Tris-buffer. Membranes were immunoblotted overnight with anti-ANXA2 or anti-β-actin antibodies (Santa Cruz Biotechnology, Inc.), followed by respective horseradish peroxidase-conjugated secondary antibodies. Bands were subsequently visualised by a chemiluminescence detection system (Millipore, Billerica, MA, USA) and density was determined by an image analyser. ANXA2 levels were presented as relative ratio (RR) and calculated using the formula for signal intensity (SI) of ANXA2 and β-actin: RR = SI_ANXA2/SI_β-actin.

MHCC97-H cells and xenograft tumours in nude mice were divided into shRNA, negative and mock groups. Statistical evaluation of results was performed by the least significant difference or Newman-Keuls test, Student’s t-test, χ² test and Fisher’s exact test. P<0.05 was considered to indicate a statistically significant difference.

The intensity of ANXA2 expression in HCC, adjacent and distant cancerous specimens are shown in Table I. ANXA2 expression was markedly higher in HCC compared with that of the adjacent or distant cancerous tissues. ANXA2 was localised to the cell membrane and cytoplasm of HCC tissue (100%) and mainly in the cytoplasm of the matched adjacent tissue (90%), however, the distant cancerous tissue was not ANXA2-positive. Although no significant difference in the rate of ANXA2 expression (χ²=3.518; P=0.070) was found between the HCC and adjacent groups, the intensity of ANXA2 expression in the HCC group was significantly higher compared with that of the adjacent (Z=6.113; P<0.001) or distant cancerous groups (Z=7.328; P<0.001). A significant difference (F=498.221; P<0.001) was found among the various groups and ratio of ANXA2.

Alterations in the expression of ANXA2 in the hepatoma cell lines with low or high metastatic potential following transfection with shRNA are shown in Fig. 1 and Table II. ANXA2 expression was significantly higher (P<0.05) in hepatoma cells with a 5–8 fold increased expression compared with that of the LO₂ cells (Figs. 1A and 1B). ANXA2-mRNA levels in MHCC97-H cells were significantly higher (F=286.254; P<0.001) compared with that of the other cell lines (Table II). The MHCC97-H cells with the highest ANXA2-mRNA levels were selected to observe the effects of shRNA targeting ANXA2 on the proliferation and invasion of cells. ANXA2-mRNA was markedly downregulated (q=71.993; P<0.001) following transfection with stably expressing shRNA at the protein level (Fig. 1C). The ratio of ANXA2 to β-actin (Fig. 1D) demonstrated that ANXA2 expression in the shRNA group was significantly downregulated (P<0.001), with an evidently weaker red immunofluorescence signal (Fig. 1E) in the membrane and cytoplasm. Decreasing immunofluorescence was not noted in the negative or mock groups. However, the mock cells marked with DAPI dye showed a number of fragmented nuclei with condensed chromatin.

The effect of ANXA2 suppression on the proliferation and cell cycle of MHCC97-H cells following transfection with specific shRNA is shown in Fig. 2. The cell proliferation ability (Fig. 2A) in the shRNA group was significantly decreased (P<0.05) compared with that of the mock and negative groups, but no significant difference was found between the mock and negative groups. Analysis of the cell cycle (Fig. 2B) indicated that the ratio of cells in S phase in the shRNA group was significantly decreased compared with the mock group [27.76 and 36.14%, respectively (P<0.05)] compared with that of the mock group, whereas the ratio of cells in G₀–G₁ and G₂-M phases were increased compared with that of the mock group (P<0.05). The growth of shRNA cells was markedly inhibited in vitro and the MHCC97-H cell invasive potential was significantly downregulated (Fig. 2D; P<0.05) in the shRNA group compared with that of the mock cells (percentage of invading cells, 52.16 and 86.14%, respectively; Fig. 2D).

The effect of silencing ANXA2 in xenograft tumours on tumour growth in vivo is shown in Fig. 3. Tumourigenicity (Fig. 3A) showed a marked reduction in tumour size in the shRNA group compared with that of the mock or negative groups and the rate of tumour inhibition was 38.24%. The tumour growth curve (Fig. 3B) over 21 days indicates that silencing ANXA2 reduced the tumourigenic potential (P<0.05) in vivo. The mice appeared emaciated and tumour weight was 1.89±0.16 g in the shRNA group (Fig. 3C.), significantly lower compared with that of the mock (3.06±0.14 g) and negative (3.07±0.17 g) groups. Pathological analysis of the tumours showed no clear morphological alterations (H&E; Fig. 3D) and the distribution of ANXA2 was mainly localised to the cell membrane in the shRNA group and the cell membrane and cytoplasm in the mock and negative groups. ANXA2 intensity in tumours was significantly lower (Z=2.530; P=0.011) in the shRNA group (3/4; +) compared with that of the mock (4/4; ++ and +++) and negative (4/4; +++) groups.