A total of 24 formalin-fixed, paraffin-embedded, surgically resected cases of HCC and adjacent normal tissues were retrieved from The First Affiliated Hospital of China Medical University General Surgery (Shenyang, China), which were collected from May 1st, 2011 to September 1st, 2013. A total of 12 fresh specimens of HCC and adjacent normal tissue were stored at −80°C until use. The human hepatoma cell line Bel7402 was derived from the Chinese Department of Medical Genetics. The current study was approved by the ethics committee of The First Affiliated Hospital of China Medical University General Surgery (Shenyang, China). All patients gave written signed informed consent at the time of hospitalization.

Concentrated Pin1 polyclonal antibodies, monoclonal antibodies cyclin D1 and gradient protein were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Ready-to-use immunohistochemistry hypersensitivity, DAB chromogenic enzyme substrate kit and β-catenin type and concentration of an anti-horseradish peroxidase-labeled goat anti-rabbit immunoglobulin (Ig)G secondary antibody were purchased from Beijing Biotechnology Co. (Beijing, China) Total RNA extraction kits were purchased from Huamei Co. (Beijing, China) and agarose was obtained from Promega Corp. (Madison, WI, USA). The polymerase chain reaction (PCR) robot thermocycler was purchased from Biometra Co. (Goettingen, Germany). The ultraviolet spectrophotometer UA-1201 was from Shimadzu, (Kyoto, Japan) and the GL212Pro Gel imaging analysis system was purchased from (Carestream Health, Inc., Rochester, NY, USA).

The expression of Pin1 and β-catenin was determined by streptomycin avidin-peroxidase (SP) connection. The expression of Pin1 protein was then mapped to the cytoplasm and (or) the nucleus. Staining intensity was scored the following: 0, indicated no staining; 1, pale yellow; 2, brown and 3, tan (dark brown). The average percentage of positive cells was converted to a four-tier numeric score (0, <25%; 1, 25–49%; 2, 50–74%; 3, ≥75). The final score was determined by multiplying the above two scores (≥3, overexpression). Positive expression of β-catenin in normal epithelial tissue was mapped to the cell membranes, but rarely to the cytoplasm. A score ≥10% in the cytoplasm and (or) the nuclei of cells was noted as positive expression to the extend of abnormal accumulation. The scanned slides were independently analyzed by two high senior pathologists, and when their findings were inconsistent, consensus was reached through re-evaluation and discussion. Cyclin D1 protein was located in the cytoplasm and (or) the nucleus. A lack of significantly positive cells was regarded as negative (-), <20% positive cells was regarded as weakly positive (+) and >20% positive cells was regarded as positive (++).

The analysis buffer (50 mM Tris-HCl; pH 7.4, 150 mM NaCl, 1% NP-40 and 0.1% SDS) was overlaid five times on top of a fresh sample, which was then cut into sections and subjected to ultrasonic treatment. The supernatant protein was extracted using high-speed centrifugation, electrophoresis, transfer, adding of primary (β actin) and secondary (horseradish peroxidase-labeled goat anti-rabbit IgG) antibodies and DAB chromogenic reagent kit (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China. As a result, the DNA bands were able to be viewed clearly on the transfer film.

Total RNA was extracted from exponentially growing cells using TRIzol™ reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). A total of 5 μg total RNA was reverse transcribed using M-MLV reverse transcriptase (N8080018; Invitrogen Life Technologies) and then Pin1, β-catenin and cyclin D1 were amplified. GAPDH was amplified simultaneously as an internal reference. Each sample was repeated at least three times. First-strand cDNA was synthesized from 2 μl total RNA by adding 10 μl 2X buffer, 4 μl MgSO₄, 0.5 μl dNTP (10 mmol), 0.5 μl oligo(dT) 15, 0.25 μl double-distilled (dd)H₂O and 0.25 μl RNase-inhibitor. The denaturation step of the asymmetric PCR was performed at 94°C for 30 sec. A total of 35 cycles of denaturation (30 sec, 94°C), annealing (30 sec, 55°C) and extension (20 sec, 70°C) were performed. The annealing temperature of the internal reference gene GAPDH was 62°C. PCR amplification was performed in a total volume of 3 μl cDNA, consisting of 2.5 μl of 10X PCR buffer (50 mM Tris-HCl; pH 7.4, 150 mM NaCl, 1% NP-40 and 0.1% SDS), 2 μl dNTP (2.5 mM), 1.5 μl sense and antisense of each primer, 1 μl TaqDNA polymerase (1 U/μl) and 17.1 μl ddH₂O.

Following amplification, the PCR products were scanned by the Gel oc1000 imaging system (Bio-Rad, Hercules, CA, USA), analyzed using agarose gel electrophoresis analysis (10 g/l metaphor agarose gel) and stained with ethidium bromide (EB). The comparison between the integral A value of the sample bands and the corresponding β-actin mRNA represented the relative amount of mRNA in the sample (Table I). Electrophoresis was scanned using a BioDoc-It Imaging System (UVP, Inc., Upland, CA, USA).

Bel7402 cells were cultured and supplemented with 10% fetal calf serum, 100 μg/ml streptomycin, 100 international U/ml penicillin and 0.286 g/ml glutamine at (Gibco-Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified 5% CO₂ atmosphere. The medium was changed every day depending on the cell growth and the cells were detached with 0.25% trypsin until cell growth was stable at the logarithmic phase. Morphological changes of cultured cells were observed under an inverted microscope. The resulting DNA was used to transfect plasmid IPO 2000. Pin1 plasmids and primers were synthesized and identified by Takara Bio, Inc. (Shiga, Japan). The primers of β-catenin and cyclin D1 were synthesized by Shanghai Sangon Co., Ltd. (Shanghai, China).

The cultured endothelial cells were grown on culture plates on a sterilized coverslip for 48 h following transfection for 2–24 h. The cells were then rinsed three times with phosphate-buffered saline (PBS) and fixed in 4% (v/v) paraformaldehyde/PBS by incubation for 15 min at 37°C with 3% hydrogen peroxide to block endogenous peroxidase. Next, they were rinsed three times with PBS by incubation for 1 h at room temperature and were then incubated with the primary antibody (β actin) at 4°C overnight. The cells were rinsed three times for 10 min with PBS by incubation for 30 min with the secondary antibody. Then, they were rinsed three times for 10 min with PBS and two times for 5 min with DW2, and mounted in glycerol. Finally, the cells were observed under a Olympus BX60 fluorescent microscope (Olympus Corporation, Tokyo, Japan).

The cells were rinsed two or three times with PBS and then the PBS buffer was removed. Next, RIPA lysis buffer (200 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride, 9 nM pepstatin, 9 nM antipain, 10 nM leupeptin, and 10 nM chymostatin) was added (10⁷ cells/ml) and cells were scraped and collected in centrifuge tubes and incubated at 4°C for 15 min in lysis buffer. The solution in each tube was centrifuged at 14,000 × g for 15 min and the supernatant, which contained the protein, was then removed from the new tubes. Then, 100 μl agarose beads were added to the cellular proteins, agitated at 4°C for 2 h, centrifuged at 14,000 × g for 10 min, and the supernatant was collected to detect the protein concentration. Each sample was adjusted to the same protein concentration (10 μg/ml), the primary antibody (β actin) was added to 500 μl cell lysates and agitated at 4°C for 2 h. Subsequently, 100 μl 50% sepharose beads were added and agitated at 4°C for 2 h. The sepharose beads were collected by centrifugation at 14,000 rpm for 5 sec and the supernatant was discarded. The sepharose beads were rinsed three times with 800 μl PBS and they were then collected, after which they were suspended in 60 μl sample buffer (50 mM Tris-HCl; pH 7.4, 150 mM NaCl, 1% NP-40 and 0.1% SDS) and centrifuged following 5 min in a boiling water bath, to run electrophoresis. Finally, clear bands appeared at 19 and 34 kDa, which indicated the precipitation of immune complexes.

All statistical analyses were performed using the SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). A χ² test was used to compare the frequencies. One-way analysis of variance and Student’s t-test were used for the normally distributed variables, whereas the Mann-Whitney U test was used for the non-normally distributed variables. Comparisons between the two groups for nominal variables were performed using Fisher’s exact test. All of the tests of statistical significance were two-sided. P<0.05 was considered to indicate a statistically significant difference between values.

The expression of Pin1, cyclin D1 and β-catenin in HCC and adjacent normal tissues are shown in Fig. 1. Pin1 demonstrated no expression in the adjacent normal tissues, but a strongly positive expression in the cytoplasm and nuclei of HCC cells. Out of 24 cases of HCC specimens analyzed, 66.7% (16/24) demonstrated a strong positive Pin1 expression and 58.3% (14/24) revealed a nuclear expression. However, β-catenin was weakly expressed in the cytoplasm and nuclei of HCC cells. No expression of β-catenin was identified in the adjacent normal tissues. Similarly, cyclin D1 also demonstrated a weak expression in the cytoplasm and nuclei of HCC cells, but no expression in the adjacent tissues. A total of 79.2% (19/24) of the HCC specimens demonstrated a weakly positive expression of cyclin D1, including 33.3% (8/24) in the nuclei of HCC cells.

The expression levels of Pin1, cyclin D1 and β-catenin mRNA in HCC cell lines were significantly higher than those in the adjacent normal tissues (Fig. 2 and Table II). The associations between Pin1, β-catenin and cyclin D1 are summarized in Tables III–V. The results revealed that there were no significant difference between Pin1, β-catenin and cyclin D1 expression levels (all P>0.05).

Immunoprecipitation analyses demonstrated two clear bands at 19 and 34 kDa and a brown band at 55 kDa as expected (Fig. 3). Immunofluorescence analysis of HCC cells indicated that Pin1 was present in the cytoplasm and nucleus, and β-catenin and cyclin D1 were present in the nucleus.

Immunofluorescence images of HCC cells showed that Pin1 demonstrated green fluorescence in the cytoplasm and nuclei, while β-catenin and cyclin D1 revealed red fluorescence in the nuclei (Figs. 4 and 5).