Human liver HHL-5 cells and the HCC cell lines, SK-HEP-1, Hep3b and Huh-7, were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin and 100 U/ml penicillin (all Gibco; Thermo Fisher Scientific, Inc.), at 37˚C with 5% CO₂.

Hep3b cells were seeded into 96-well (2x10³ cells/well) or six-well (1x10⁵ cells/well) plates and transfected with short hairpin (sh) RNA-RPLP1 (100 nM) using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 12 h. Cells were cultured until they reached 60-70% confluence. RPLP1 shRNA-1 (5'-CATTAAAGCAGCCGGTGTAAATGTTGAGC-3') and RPLP1 shRNA-2 (5'-GAAGAAAGTGGAAGCATTCAAGAGATGCT-3') or the negative control RNA (non-targeting) were synthesized by Shanghai GeneChem Co., Ltd. Reverse transcription-quantitative (RT-q)PCR analysis was performed to assess transfection efficiency. Hep3b cells were used for subsequent experimentation 48 h post-transfection. RPLP1 expression was assessed by screening the Gene Expression Profiling Interactive Analysis database (http://gepia.cancer-pku.cn) to determine the association between RPLP1 expression and HCC progression. Moreover, the overall survival (OS) analyzed using the GEPIA database (http://gepia.cancer-pku.cn/) to investigate the role of RPLP1 in HCC progression and the UALCAN database (http://ualcan.path.uab.edu) was used to determine the association between RPLP1 expression and tumor grade of HCC.

Total RNA was extracted from transfected Hep3b cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The prepared reaction mixture was incubated at 37˚C for 60 min after brief centrifugation (14,000 x g for 5 min at 4˚C), followed by incubation at 85˚C for 5 min for RT. qPCR was subsequently performed using the SYBR Premix EX Taq™ II kit (Takara Bio, Inc.) on an ABI Prism 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Pre-denaturation at 95˚C for 5 min, followed by 40 cycles at 95˚C for 15 sec, 60˚C for 30 sec and 72˚C for 30 sec. The following primer sequences were used for qPCR: RPLP1, Forward: 5'-TGGCCTGGCTTGTTTGC-3' and reverse: 5'-CTCGGATTCTTCTTTCTTTGCTT-3'; and GAPDH, forward: 5'-CTCCTCCACCTTTGACGCTG-3' and reverse: 5'-TCCTCTTGTGCTCTTGCTGG-3'. Relative expression levels were calculated using the 2^-ΔΔCq method (16) and normalized to the internal reference gene GAPDH.

The CCK-8 (Dojindo Molecular Technologies, Inc.) assay was performed to assess the viability of transfected Hep3b cells, according to the manufacturer's instructions. Briefly, Hep3b cells transfected with or without RPLP1 shRNA-1 were seeded into 96-well plates at a density of 2x10³ cells/well and incubated for 0, 24, 48 and 72 h at 37˚C. Following incubation, 10 µl CCK-8 solution was added to each well for another 2 h and cell viability was analyzed at a wavelength of 450 nm using a microtiter plate reader (Bio-Rad Laboratories, Inc.).

Transfected Hep3b cells were seeded into six-well plates at a density of 1x10³ cells/well and cultured for 4 weeks at 37˚C, without disturbing. Cell colonies were subsequently fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 10 min at room temperature. Stained cells were counted in five randomly selected fields of view by eye and images were captured.

Total lysates were extracted from transfected Hep3b cells using RIPA buffer with protease inhibitors cocktail (both Sigma-Aldrich; Merck KGaA). Protein concentration was measured using the BCA assay kit (Bio-Rad Laboratories, Inc.) and 25 µg protein/lane was separated using 10% SDS-PAGE gels. The separated proteins were subsequently transferred onto PVDF membranes (EMD Millipore) and blocked with 5% skimmed milk at room temperature for 2 h. The membranes were incubated with primary antibodies against: Minichromosome maintenance (MCM; cat. no. #3228), proliferating cell nuclear antigen (PCNA; cat. no. #13110), vimentin (cat. no. #5741,), snail (cat. no. #3895), slug (cat. no. #9585) (all 1:1,000), β-catenin (1:500; cat. no. #9582), N-cadherin (cat. no. #13116), E-cadherin (cat. no. #5296) (all 1:500), claudin-1 (1:1,000, #4933, Cell Signaling Technology, Inc.), matrix metalloproteinase (MMP)-2 (cat. no. #4022,), matrix metalloproteinase (MMP)-9 (cat. no. #3852), tissue inhibitor of MMP-1 (TIMP-1; cat. no. #8946) (all 1:1,000) and GAPDH (1:2,000; cat. no. #5174) (all Cell Signaling Technology, Inc.) overnight at 4˚C. After washing with PBS, the membranes were incubated with secondary HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. #7074; Cell Signaling Technology, Inc.) at room temperature for 2 h, and then visualized using an ECL chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.). At last, the gray values of bands were detected using ImageJ software (version 1.48; National Institutes of Health) and normalized to GAPDH.

When Hep3b cells were cultured to the appropriate densities, cells in co-culture plates were fixed with 4% paraformaldehyde for 10 min at room temperature and subsequently permeabilized with 0.1% Triton X-100 for 10 min. Following blocking with 5% BSA and 10% horse serum (Sigma-aldrich; Merck KGaA) in PBST for 1 h at room temperature, Hep3b cells were incubated with primary antibody against Ki-67 (1:200; cat. no. #12075; Cell Signaling Technology, Inc.) overnight at 4˚C. Following the primary incubation, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; cat. no. ab150077; Abcam) for 1 h at room temperature. Cell nuclei were stained with DAPI for 5 min at room temperature. Hep3b cells were washed three times with PBST prior to observation under a light microscope at x200 magnification and images were captured (Olympus Corporation).

Hep3b cells were seeded into six-well plates at a density of 1x10⁵ cells/well and transfected with or without shRNA-RPLP1. Once cells reached 100% confluence, the monolayers were scratched using 200-µl sterile pipette tips to create straight linear wounds. Cells were cultured in serum-free medium for 24 h at 37˚C, with 5% CO₂. Wounds were observed in five randomly selected fields using an inverted fluorescence microscope at 100x magnification and images were captured. The recovered wound area (%) at the indicated time point (24 h) was calculated according to the following formula: [(Wound width at 0 h)-(wound width at 24 h)]/wound width at 0 h.

Following transfection, Hep3b cells were resuspended in serum-free medium and plated in the upper chambers of Transwell plates precoated with Matrigel at 37˚C for 30 min. Complete medium (600 µl) was plated in the lower chambers. Following incubation for 24 h at 37˚C with 5% CO₂, cells in the upper chambers were gently removed using a wet cotton swab. Cells in the lower chambers were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 30 min, both at room temperature. Stained cells were counted in five randomly selected fields using an inverted light microscope and images were captured (magnification, x200).

Statistical analysis was performed using SPSS 13.0 software (SPSS Inc.). All experiments were performed in triplicate and data are presented as the mean ± standard error of mean (unless otherwise shown). A two-tailed unpaired Student's t-test was used to compare differences between two groups, while one-way ANOVA followed by Dunnett's post hoc tests were used to compare differences between multiple groups. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

RPLP1 expression was assessed by screening the Gene Expression Profiling Interactive Analysis database (http://gepia.cancer-pku.cn) to determine the association between RPLP1 expression and HCC progression. The results demonstrated that RPLP1 was highly expressed in HCC tissues compared with adjacent liver tissues (Fig. 1A) (529 cases; P=1.6x10^-12).

To further investigate the role of RPLP1 in HCC progression, the overall survival (OS) datasets was analyzed by GEPIA database (http://gepia.cancer-pku.cn/). As presented in Fig. 1B, high RPLP1 expression was associated with poor prognosis and high mortality, and the OS rate (log-rank P=0.091; hazard ratio=1.3) were less favorable in patients with high RPLP1 expression levels.

The UALCAN database (http://ualcan.path.uab.edu) was used to determine the association between RPLP1 expression and tumor grade of HCC. As presented in Fig. 1C, RPLP1 expression was positively associated with tumor grade. Taken together, these results suggested that RPLP1 may promote HCC development. In addition, RT-qPCR analysis was performed to detect RPLP1 expression in human liver HHL-5 cells and the HCC cell lines, SK-HEP-1, Hep3b and Huh-7. The results demonstrated that RPLP1 was highly expressed in the HCC cell lines compared with normal liver cells, and the most significant overexpression was observed in Hep3b cells (Fig. 1D). Thus, Hep3b cells were selected for subsequent experiments.

RPLP1 expression was downregulated using shRNA-RPLP1-1, shRNA-RPLP1-2 and shRNA-NC. Due to the low expression level induced by shRNA-RPLP1-1 (Fig. 2A), this shRNA was selected for subsequent experimentation. The results of the CCK-8 assay demonstrated that downregulation of RPLP1 significantly suppressed Hep3b cell viability (Fig. 2B). The results of the colony formation assay revealed that the proliferation rate of Hep3b cells transfected with shRNA-RPLP1-1 markedly decreased compared with the control and shRNA-NC groups (Fig. 2C). Furthermore, the expression levels of the standard markers of proliferation, Ki-67, PCNA and MCM (17) were assessed via western blotting and immunofluorescence. The results demonstrated that the expression levels of PCNA, MCM and Ki-67 decreased in the shRNA-RPLP1-1 group compared with the control and shRNA-NC groups (Fig. 2D and E). Taken together, these results suggested that downregulation of RPLP1 suppresses the proliferation of Hep3b cells.

To further investigate the effect of RPLP1 on cellular biological behaviors, proteins related to EMT were detected via western blot analysis. The results demonstrated that downregulation of RPLP1 significantly decreased the expression levels of vimentin, snail and slug, and increased β-catenin expression (Fig. 3A). In addition, N-cadherin expression (mesenchymal cell marker) (18) notably decreased following downregulation of RPLP1 expression, while the expression levels of E-cadherin (epithelial cell marker) (18) and claudin-1 significantly increased following downregulation of RPLP1 (Fig. 3B). Downregulation of RPLP1 also suppressed the expression levels of MMP-2 and MMP-9, and notably increased TIMP-1 expression (Fig. 4A). These results implied that downregulation of RPLP1 inhibited the EMT process of Hep3b cells.

The wound healing and Transwell assays were performed to confirm the effect of RPLP1 downregulation on the migratory and invasive abilities of Hep3b cells. As presented in Fig. 4B-E, the migratory and invasive abilities of Hep3b cells were significantly inhibited following downregulation of RPLP1. Taken together, these results suggested that downregulation of RPLP1 inhibits the potency of invasion and migration of Hep3b cells.