This study conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki-Ethical Principles for Medical Research Involving Human Subjects and has been approved by the Institutional Review Board of Nagoya University, Japan. Written informed consent for usage of clinical samples and data, as required by the institutional review board, was obtained from all patients.

The human HCC cell lines Hep3B, HepG2, PLC/PRF/5 and SK-Hep1 were purchased from the American Type Culture Collection (Manassas, VA, USA). HLE, HLF, HuH1 and HuH7 were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) (16). HuH2 cells were a gift from Aichi Cancer Center (Nagoya, Japan). Cells were stored at −80°C and cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) at 37°C in an atmosphere containing 5% CO₂. Primary HCC tissues and corresponding noncancerous tissues appropriate for extraction of total RNA were collected from 144 patients undergoing liver resection for HCC at Nagoya University Hospital between January 1998 and January 2012. None of the patients underwent preoperative treatment including chemotherapy or interventional radiology. Tissue samples were frozen immediately in liquid nitrogen and stored at −80°C. RNA was extracted from HCC tissue samples ~5-mm² that contained >80% tumor cells and lacked necrotic tissue (17,18). The corresponding noncancerous tissue samples were collected from areas >2 cm away from the tumor edge. Specimens were classified histologically according to the TNM Classification of Malignant Tumors, 7th Edition (19). Clinicopathological parameters and the patient follow-up data were collected from medical records.

PRMT5 mRNA levels were determined using qRT-PCR. Total RNAs (10 μg) were extracted from nine HCC cell lines and 144 pairs of clinical samples and were amplified using primers specific for PRMT5 as follows: sense 5′-TCTCATGGTTTCCCATCCTC-3′ in exon 16 and antisense 5′-CCTTCTTGGAATTGCTGCAT-3′ in exon 17, which generate a 102-bp product. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (TaqMan, GAPDH control reagents; Applied Biosystems, Foster City, CA, USA) was quantified in each sample for standardization. qRT-PCR was performed using the SYBR-Green PCR Core reagents kit (Applied Biosystems) as follows: one cycle at 95°C for 10 min, 40 cycles at 95°C for 5 sec, and 60°C for 60 sec. The expression levels of each sample were determined in triplicate, and the data are presented as the PRMT5 value divided by the GAPDH value (20,21). Patients were stratified into PRMT5-low and PRMT5-high groups, respectively, according to the median value of the PRMT5 mRNA expression level in cancer tissues of 144 patients as follows: high (higher than the median value) and low (the median value or lower).

The expression and location of PRMT5 protein was investigated by immunofluorescence staining using three HCC cell lines, Hep3B, HLE and HuH7, expressed relatively high levels of PRMT5. HCC cells were fixed on microscope slides by methanol. Slides were cooled at −10°C for 5 min and incubated with mouse monoclonal antibody raised against PRMT5 (sc-376937 FITC; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), diluted 1:50 in phosphate-buffered saline (PBS). The nuclei were counter-stained with 1 μg/ml DAPI. The slides were observed under a fluorescence microscope, FSX100 (Olympus, Tokyo, Japan).

Cells were incubated in RIPA lysis buffer, and the lysates were stored at −30°C. Total lysate protein (15 μg/well) was electrophoretically transferred onto nitrocellulose membranes that were blocked using 5% skim milk and incubated at 4°C overnight with a rabbit anti-PRMT5 polyclonal antibody (Cell Signaling Technology, Beverly, MA, USA) diluted 1:1,000. The membrane was then washed and probed with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (Cell Signaling Technology). After the final wash, immune complexes were visualized using an LAS-4010 image analyzer (GE Healthcare, Pittsburgh, PA, USA). β-actin served as an endogenous control (22).

Small interfering RNAs (siRNAs) specific for PRMT5 (siPRMT5), siPRMT5-1, 5′-CAGCCACUGAUGGACAAUCUGGAAU-3′ and siPRMT5-2, 5′-CCGGCUACUUUGAGACUGUGCUUUA-3′ (Hokkaido System Science, Sapporo, Japan) were used to transfect HLE and HuH7 cells that express relatively high levels of PRMT5. AccuTarget Negative Control siRNA fluorescein-labeled (Cosmo Bio Co., Ltd., Tokyo, Japan) served as a control nontargeting siRNA (siControl). HLE and HuH7 cells (4×10⁵ and 2×10⁶ cells/dish, respectively) were seeded into 10 cm dishes containing 10 ml of antibiotic-free DMEM with 10% FBS, and were transfected with 400 pmol of siControl or siPRMT5-1 and 2 in the presence of 40-μl LipoTrust EX Oligo (Hokkaido System Science) 24 h after cell seeding. After 72 h incubation following siRNA transfection, total RNA and protein were extracted, and cells were treated with EDTA-trypsin and used for functional assays.

The proliferation of HLE and HuH7 cells was evaluated using a Premix WST-1 Cell Proliferation assay system (Takara Bio Inc., Kusatsu, Japan). After transfection of siPRMT5, HLE and HuH7 (3×10³ cells/well) cells were seeded into 96-well plates in DMEM containing 2% FBS. The optical density of each well was measured 1 h after the addition of 10 μl of WST-1 0, 1 and 3 days after seeding (23). The growth rate indicated an incremental rate of the optical density from the cell seeding day.

The ability of cells to invade Matrigel was determined using BioCoat Matrigel invasion chambers (BD Biosciences, Bedford, MA, USA) according to the manufacturer's protocol. HLE and HuH7 cells (2.5×10³ cells/well) were suspended in serum-free DMEM and seeded in the upper chamber. After an appropriate incubation time, cells present on the surface of the membrane were fixed, stained and counted using a microscope in eight randomly selected fields (24).

The migration of HLE and HuH7 cells was determined using a wound-healing assay. After transfection, HLE (2×10⁴ cells/well) and HuH7 (5×10⁴ cells/well) were seeded into each well of a 35 mm dish with culture insert (Ibidi GmbH, Martinsried, Germany) in DMEM containing 2% FBS. After 24 h, the insert was removed, and the width of the wound was measured at 100-μm intervals (20/well, ×40 magnification) at cell-dependent time intervals.

Differences between data of two groups were evaluated using the Mann-Whitney test. The χ² test was used to analyze the significance of the association between the expression levels of PRMT5 mRNA and the patient clinicopathological parameters. Overall survival rates were calculated using the Kaplan-Meier method, and the differences in survival curves were evaluated using the log-rank test. All statistical analyses were performed using JMP 10 software (SAS Institute Inc., Cary, NC, USA). A p-value <0.05 was considered statistically significant.

The levels of PRMT5 mRNA differed among the nine HCC cell lines regardless of their differentiated phenotypes (Fig. 1A). HuH7 (differentiated) and HLE (undifferentiated) cells expressed relatively high levels of PRMT5 mRNA and were used in the functional analyses described below. As shown in Fig. 1B, PRMT5 protein was distributed in the cytoplasm, especially in the perinuclear compartment.

The specificity of siPRMT5s were checked using BLAST in the NCCI database (25). Then, the total scores and E-values of the two siPRMT5s were 50.1 and 5e⁻⁶, and the query cover rates of transcriptomes other than PRMT5 mRNA were <70%. The knockdown effects of siPRMT5-1 and 2 were determined by qRT-PCR and western blot analysis (Fig. 2A). The knockdown effect by single siPRMT5 sequence was not enough for functional analysis, we transfected HCC cells with both the siPRMT5s in following analysis. To determine the functions of PRMT5 in HCC, we used HLE and HuH7 cells transfected with siPRMT5-1 and -2 sequences for evaluation of the proliferation, invasion, and migration. In HuH7 cells, PRMT5 knockdown significantly decreased cell proliferation on days 1 and 3 compared with the untransfected and siControl groups (Fig. 2B). Second, the number of cells that invaded Matrigel markedly decreased when PRMT5 expression was inhibited (Fig. 3A). Third, the migration of HuH7 cells decreased significantly after transfection with the siPRMT5 compared with the untransfected and siControl-transfected cells at 16 and 24 h (Fig. 3B). Similarly, invasion and migration of HLE cells were markedly reduced in siPRMT5-transfected cells (data not shown).

The ages of the 144 patients ranged from 34 to 84 years (median, 65.5 years), the male-to-female ratio was 121:23, and 37 and 80 patients were infected with hepatitis B or C virus, respectively. The numbers of patients with normal liver function, chronic hepatitis and cirrhosis were 10, 82 and 52, respectively. The median duration of patient follow-up was 40.1 months (range, 2.3–145 months); 90, 37 and 17 patients were diagnosed with stage I, II or III HCC, respectively.

There were no significant differences in PRMT5 mRNA levels in noncancerous tissue samples from patients with normal liver function, chronic hepatitis, and cirrhosis. In contrast, the mean level of PRMT5 mRNA in HCC tissues was significantly higher compared with that of the corresponding noncancerous tissues (p=0.001) (Fig. 4A). When patients were divided according to TNM stage, PRMT5 mRNA expression levels in patients with stage III HCC were significantly higher compared with patients with stage II HCC (Fig. 4B).

To evaluate the prognostic significance of PRMT5 expression, patients were categorized into low- and high-PRMT5 groups (n=72 each) according to their median levels of PRMT5 mRNA expression in HCC tissue described in Materials and methods. High levels of PRMT5 mRNA were significantly associated with advanced TNM stage, independent of background liver status, tumor size or vascular invasion (Table I). The members of the high-PRMT5 group were more likely to have a worse prognosis compared with those in the low PRMT5 group (5-year overall survival rates, 53 and 76%, respectively; p=0.021) (Fig. 4C). However, there were no significant differences between the groups in relapse-free survival rate, and high levels of PRMT5 were not an independent prognostic factor in multivariate analysis.