All animal protocols complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval of the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.

HCC cell lines Hep3B, HepG2, HHCC, SMMC7721 and MHHC97H and immortalized human liver cells L02 were purchased from the Institute of Cellular Research of the Chinese Academy of Sciences (Shanghai, China). All of the cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% penicillin/streptomycin, and incubated at 37°C in a humidified 5% CO₂ atmosphere.

Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. First-strand cDNA was synthesized from total RNA with PrimeScript^® RT Master Mix (Takara Biotechnology Co., Ltd., Dalian, China). Real-time quantitative qPCR was performed (Bio-Rad Laboratories, Hercules, CA, USA) with SYBR^® Premix Ex Taq™ II (Takara Biotechnology). The cycling conditions were as follows: initial denaturation at 95°C for 30 sec, and then 40 cycles of denaturation at 95°C for 5 sec and annealing and elongation at 60°C for 30 sec. Bio-Rad CFX Manager 2.1 software was used for qPCR analysis. The GAPDH housekeeping gene was used as an internal control.

The PCR primers sets were as follows: i) CENP-H forward, 5′-CAGTCTAGTGTGCTCATGGAT-3′ and reverse, 5′-TCCATCTGTAGGTTTTGTCG-3′; ii) Bax forward, 5′-GGTTGTCGCCCTTTTCTACTTT-3′ and reverse, 5′-GTGAGGAGGCTTGAGGAGTCT-3′; and iii) Bcl-2 forward, 5′-TCCCATCAAAACTCCTGTCTTT-3′ and reverse, 5′-GCAAGTGAATGAACACCTTCTC-3′; iv) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-CAAGCTCATTTCCTGGTATGAC-3′ and reverse, 5′-CAGTGAGGGTCTCTCTCTTCCT-3′.

Total cell protein was extracted according to a previous study (15). After denaturation, equal amounts of total protein were separated electrophoretically on 10% SDS/polyacrylamide gels (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) using a tank transfer apparatus (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween (TBS-T) for 2 h, then incubated with primary antibodies overnight at 4°C. The following day, after the incubation with horseradish peroxidase-conjugated secondary antibodies, the blots were visualized with enhanced chemiluminescence horseradish peroxidase (HRP) substrate (Millipore) according to the manufacturer's instructions. The intensity of each band was assessed using Image Lab 4.0 (Bio-Rad). The following primary antibodies were used: anti-CENP-H (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-Bax (1:1,000), anti-Bcl-2 (both from Epitomics, Abcam, CA, USA, 1:2,000) and anti-cleaved caspase-3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA). To confirm consistent loading, mouse anti-β-actin antibody (Santa Cruz Biotechnology, Inc.) diluted 1:1,000 was used as a primary antibody. The secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. and were diluted 1:5,000.

To knock down CENP-H expression, 2 lentiviruses with different siRNA sequences were packaged using the LV-3 (pGLVH1/GFP + Puro) vector (GenePharma, Shanghai, China). The siRNA sequences were as follows: CENP-H1, 5′-TGGTTGATGCAAGTGAAGA-3′; and CENP-H2, 5′-GCTTGAGAAGAATGTTGACAT-3′. Concomitantly, the negative controls were also packaged. Puromycin (2.5 µg/ml; Sigma, St. Louis, MO, USA) was used to kill the uninfected Hep3B cells. Hep3B cells that were stably infected with CENP-H1 and CENP-H2 were named LV3-CENP-H1 and LV3-CENP-H2, respectively, and the negative control was named LV3-NC. Real-time PCR and western blotting were used to detect knockdown efficiency, and the virus with the highest knockdown efficiency was used in the subsequent experiments.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to detect cell proliferation. Hep3B, LV3-CENP-H1 and LV3-NC cells were separately seeded into 96-well plates at a density of 2×10³ cells/well (5 repeats/cell line), after culture for 0–7 days; MTT reagent (Sigma), which was freshly prepared, was added to each well. Following 4 h of incubation, the supernatant was discarded, and dimethyl sulfoxide (DMSO) (Sigma) was added to dissolve the crystals. All analyses were performed in triplicate. The absorbance was assessed at a test wavelength of 490 nm using an EnSpire™ Multilabel Reader (PerkinElmer, Waltham, MA, USA).

Hep3B, LV3-CENP-H1 and LV3-NC cells were seeded in fresh 6-well plates at a density of 500 cells/well in DMEM with 10% FBS. After 10 days, the cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min, and stained with 0.5% crystal violet for 20 min. Colonies were scored, and each experiment was repeated 3 times.

After 72 h of incubation, Hep3B, LV3-CENP-H1 and LV3-NC cells were harvested, washed twice with cold PBS, diluted in 1X binding buffer, stained with recombinant human Annexin V-Alexa Fluor 647 and PI using Annexin V-Alexa Fluor 647 apoptosis detection kit (Beijing 4A Biotech Co., Ltd, Beijing, China) according to the manufacturer's instructions, and then analyzed using a FACSCanto™ II Flow Cytometer (BD Biosciences, San Jose, CA, USA).

After 72 h of incubation, Hep3B, LV3-CENP-H1 and LV3-NC cells were harvested. The collected cells were first fixed in 2.5% glutaraldehyde for 2 h at 4°C, then rinsed in 0.1 M phosphate buffer (PB) (pH 7.4), and post-fixed in 1% osmium tetraoxide for 2 h. After being washed in PB, the cells were progressively dehydrated in a graded series of ethanol, and then cleared in QY-1 (Nissin EM, Tokyo, Japan). Then, embedding in epoxy resin 812 and sectioning were performed, and the ultrathin sections (60-nm thickness) were stained with both uranyl acetate and lead citrate. Finally, cellular ultramicroscopic structures were examined by TEM (Hitachi, Tokyo, Japan).

Male 4–6 week-old BALB/c nude mice were purchased from the Experimental Animal Center of Shanghai, and fed in specific pathogen-free (SPF) animal rooms with constant temperature and humidity at the Centre of Laboratory Animals of The Medical College of Xi'an Jiaotong University. After 4 days of adaptive feeding with an autoclaved chow diet with water, the mice were randomly divided into 2 groups (4 mice/group), and 2×10⁶ LV3-CENP-H/LV3-NC cells were subcutaneously injected into the right thigh of each mouse. The body weights of the mice and tumor volumes were assessed every 4–5 days, and the tumor volume was calculated using the following formula: Tumor volume = (tumor length × tumor width × tumor width)/2. After 4 weeks of observation of xenograft tumor formation, the mice were sacrificed, and the tumor tissues were obtained as soon as possible. After being washed with PBS, some of the tumor samples were fixed with 4% formalin and paraffin-embedded for histological studies, and others were immediately snap-frozen in liquid nitrogen and stored at −80°C for long-term preservation.

Xenograft samples were immediately fixed in 4% formalin once obtained from the mice. After paraffin-embedding, 4-µm thick paraffin sections were adhered to glass slides and prepared for immunohistochemical assay using a standardized streptavidin-peroxidase (SP) method as previously reported (15). Following deparaffinization, rehydration, antigen retrieval and blockage, the slides were incubated with primary antibodies overnight at 4°C. The following day, after secondary antibody incubation and dehydration, the slides were examined under a microscope (Olympus, Tokyo, Japan). The primary antibodies included anti-CENP-H (1:40; Santa Cruz Biotechnology, Inc.), anti-Bax, anti-Bcl-2 (1:100) (both from Epitomics), anti-cleaved caspase-3 (1:300) and anti-Ki-67 (1:200) (both from Cell Signaling Technology). The secondary antibodies were from Zhongshan Golden Bridge Biotechnology (Beijing, China).

The experimental data are presented as the mean ± SEM and were analyzed using the two-tailed Students t-test. P<0.05 was considered statistically significant. Statistical analysis was performed using SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL, USA).

To investigate the protein and gene expression levels of CENP-H in different HCC cell lines and immortalized human liver cells, total protein and mRNA were extracted from Hep3B, HepG2, HHCC, SMMC7721, MHHC97H and L02 cells, separately. Western blotting revealed that the CENP-H protein levels in all HCC cell lines were higher than that in the L02 cells (Fig. 1A and B). Real-time PCR analysis produced similar results as the western blotting assay (Fig. 1C). These results demonstrated that CENP-H was upregulated at both the protein and mRNA levels in HCC cell lines and suggest that it may play an important role in HCC progression. Additionally, considering the high endogenous expression of CENP-H in the Hep3B cell line, the Hep3B cell line was chosen for the following knockdown experiments.

To elucidate the biological function of CENP-H in HCC cells, we packaged lentiviruses to infect Hep3B cells in order to construct stable shRNA-mediated CENP-H knockdown. After 72 h of incubation, the viral infection efficiency was >90% using fluorescence microscopy (Fig. 2A). To further validate the knockdown efficiency, real-time PCR and western blotting were performed. The results confirmed that compared with the negative control, both the siRNA sequences significantly eliminated the endogenous CENP-H expression in the Hep3B cells (Fig. 2B-D). Taking knockdown efficiency into account, we chose LV3-CENP-H1 for the subsequent biological experiments.

The MTT and colony formation assays were used to detect the changes in the proliferative capacity of Hep3B after CENP-H knockdown. The MTT results revealed that LV3-CENP-H1 cells had a lower proliferative ability than the Hep3B and LV3-NC cells. In addition, the proliferative abilities of the Hep3B and LV3-NC cells did not significantly differ (Fig. 3A). CENP-H knockdown suppressed the colony formation ability of the Hep3B cells (Fig. 3B). Both the MTT and colony formation assay results demonstrated that CENP-H knockdown inhibits Hep3B proliferation.

Flow cytometric and TEM assays were used to detect cell apoptosis. The apoptotic index was increased from 3.13±0.42% in the LV3-NC cells to 19.96±0.5% in the LV3-CENP-H1 cells after CENP-H knockdown (Fig. 4A). Through TEM, we observed chromatin condensation, mitochondrial swelling and apoptotic bodies in the LV3-CENP-H1 cells (Fig. 4B). Both the flow cytometric and TEM results indicated that CENP-H knockdown induced apoptosis of the Hep3B cells. Based on western blotting, we found that cleaved caspase-3 expression was increased in the LV3-CENP-H1 cells compared with the level noted in the Hep3B and LV3-NC cells (Fig. 5A).

Based on swollen mitochondria observed using TEM, the apoptosis-related factors involved in the mitochondrial apoptotic pathway were detected. Both protein and mRNA expression levels of Bax were increased in the LV3-CENP-H1 cells compared to these levels in the Hep3B and LV3-NC cells (Fig. 5B and D). Conversely, the protein and mRNA expression levels of Bcl-2 were downregulated after CENP-H knockdown (Fig. 5C and E).

The Hep3B xenograft model was used to determine whether CENP-H knockdown affects tumor growth in vivo. The LV3-CENP-H1 group of animals formed much smaller tumors from 20 days after cell inoculation than the control group. After sacrifice, we confirmed that the average tumor weight in the LV3-CENP-H1 group was lighter than in the control. In addition, the average tumor volume in the LV3-CENP-H1 group was also smaller than in the control (Fig. 6). Immunohistochemistry for CENP-H confirmed the decreased staining in the LV3-CENP-H1 group of tumors compared with the LV3-NC tumors. Similarly to the in vitro experiment results, the LV3-CENP-H1 tumors exhibited increased staining for cleaved caspase-3 and Bax, compared with the LV3-NC tumors. In addition, the expression of Bcl-2 decreased in the LV3-CENP-H1 group. Immunohistochemistry for Ki-67 was used to detect the proliferative activities of both the LV3-CENP-H1 and LV3-NC tumors. As expected, the LV3-CENP-H1 tumors had a lower Ki-67 expression than the LV3-NC tumors (Fig. 7).