DEG analysis in liver hepatocellular carcinoma (LIHC) was performed using the GEPIA online database (http://gepia.cancer-pku.cn/detail.php). DEG was considered significant when log₂ fold of change (FC)>1 or log₂FC<-1 (P<0.05). Volcano plots of DEGs and their chromosomal locations were then illustrated. Survival analysis was conducted using Kaplan-Meier survival analysis (http://kmplot.com/analysis/) with log-rank test.

The human hepatoma cell lines Huh-7, MHCC97, Hep3B2.1-7, PLC/PRF/5 and BEL-7405 were purchased from iCell Bioscience, Inc. MHCC97 and Huh-7 cells were cultured in a standard Dulbecco modified Eagle medium (DMEM) (Wuhan Service bio Technology Co., Ltd.), while Hep3B2.1-7 and PLC/PRF/5 cells in standard minimum essential medium (MEM; Beijing Solarbio Science & Technology Co., Ltd.). Finally, BEL-7405 cells were cultured in standard RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.). All cells were grown at 37˚C in a humidified incubator with 5% CO₂.

Based on the expression levels of CRP-1 in HCC cell lines, BEL-7405 and Hep 3B2.1-7 cells were selected to establish stable CRP-1-overexpressing and silencing cells, respectively. The CRP-1 overexpression plasmid and the corresponding control vector or short hairpin RNA (shRNA) targeting CRP-1 and non-targeting shRNA (shNC) were transfected into BEL-7405 or Hep3B2.1-7 cells, respectively, using Lipofectamine^® 3000. Stably CRP-1-overexpressing or depleted cells were selected following treatment with 400 µg/ml G418.

For the inhibition experiments, the stable CRP-1-overexpressing cell lines were treated with 10 µM XAV-939 (Shanghai YuanYe Biotechnology Co., Ltd.) for 24 h for further analysis.

Cell viability was assessed using a CCK-8 kit (Nanjing KeyGen Biotech Co., Ltd.). Briefly, the stably transfected BEL-7405 and Hep3B2.1-7 cells were inoculated into 96-well plates at a density of 1x10⁴ cells/well. Cell viability was then determined at 0, 24, 48 and 72 h following seeding, according to the manufacturer's instructions.

Cell apoptosis was assessed using an Annexin V-FITC/PI Apoptosis Detection Kit (Nanjing KeyGen Biotech Co., Ltd.). Briefly, the stably transfected Hep3B2.1-7 cells were resuspended in 500 µl binding buffer and were then supplemented with 5 µl Annexin V-FITC and 5 µl PI. Subsequently, the samples were gently vortexed to mix reagents and incubated for 5-15 min at room temperature in the dark. Emitted fluorescence was quantified using the NovoCyte flow cytometer (ACEA Bioscience, Inc.).

To evaluate cell migration, HCC cells at a density of 1.5x10⁴ cells/well were cultured in the upper chamber of a Transwell insert (Anhui Labselect Technology Co., Ltd.). The lower chamber was filled with 800 µl medium supplemented with 10% FBS. Following incubation for 24 h, cells in the upper chamber were removed followed by fixing with 4% paraformaldehyde (PFA; Shanghai Aladdin regents Co. Ltd.) and staining with 0.1% crystal violet solution (Amresco, LLC). The migrated cells were counted and their images were captured under a light microscope (Olympus Corporation).

Cell migration was assessed using wound healing assays. The stably transfected BEL-7405 and Hep3B2.1-7 cells were pre-treated with serum-free medium supplemented with 1 µg/ml mitomycin C (MilliporeSigma) for 1 h. When cells reached 90% confluence, a wound was then created via scratching the cell monolayer with a 200-µl pipette tip. The average migration distance was measured at 24 h.

Stably transfected BEL-7405 and Hep3B2.1-7 cells were inoculated in 35-mm culture dishes at a density of 200 cells/dish for two weeks. The cells were then fixed with 4% PFA (MilliporeSigma) and the formed colonies were stained with Wright-Giemsa staining (Nanjing KeyGen Biotech Co., Ltd.).

Immunofluorescence staining was performed on PFA-fixed cell climbing sheets. Briefly, cells were permeabilized with 0.1% tritonX-100 (Beyotime Institute of Biotechnology) for 30 min. Following washing three times in PBS for 5 min each, cells were blocked with 1% BSA for 15 min, incubated with primary antibody against β-catenin (ABclonal Biotech Co., Ltd.) at 4˚C overnight, followed by incubation with secondary Cy3-conjugated anti-rabbit IgG (Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (Shanghai Aladdin regents Co. Ltd.).

The whole cell lysates and nuclear proteins were extracted from cells using a Western/IP lysis buffer and a nuclear protein extraction kit (both from Beyotime Institute of Biotechnology), respectively. The proteins were separated by SDS-PAGE (Beyotime Institute of Biotechnology) and were then blotted onto PVDF membranes (MilliporeSigma). Subsequently, membranes were blocked for 1 h in 5% non-fat milk, followed by incubation with primary antibodies overnight at 4˚C and then with the corresponding secondary antibodies for 45 min at 37˚C. The bands were visualized using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology). The primary antibodies used were as follows: Anti-CRP-1 (dilution, 1:500; cat. no. A7548), anti-β-catenin (dilution, 1:1,000; cat. no. A19657; both from ABclonal Biotech Co., Ltd.), anti-c-Myc (dilution, 1:500; cat. no. BA1284-2), anti- proliferating cell nuclear antigen (PCNA; dilution, 1:1,000; cat. no. BM0104), anti-cleaved caspase 3 (dilution, 1:1,000; cat. no. M00334-7), anti-E-cadherin (dilution, 1:3,000; cat. no. BM3903), anti-N-cadherin (dilution, 1:500; cat. no. BA0673), anti-vimentin (dilution, 1:1,000; cat. no. PB9359), anti-cyclinD1 (dilution, 1:1,000; cat. no. PB0403), anti-matrix metalloproteinase 7 (MMP-7; dilution, 1:1,000; cat. no. PB9037), anti-histone H3 (dilution, 1:1,000; cat. no. A12477-2), anti-β-actin (dilution, 1:1,000; cat. no. BA2305; all from Wuhan Boster Biological Technology, Ltd.) and anti-cleaved poly(ADP-ribose) polymerase (PARP; dilution, 1:1,000; cat. no. #5625; Cell Signaling Technology, Inc.). The secondary antibodies used were the following: Goat anti rabbit-IgG (dilution, 1:5,000; cat. no. A0208) and goat anti-mouse IgG (dilution, 1:5,000; cat. no. A0216; both from Beyotime Institute of Biotechnology).

Total RNA was extracted from cells using the TRIpure kit (Bioteke, Beijing, China) and its concentration was quantified by NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized using the BeyoRT II M-MLV reverse transcriptase kit (Beyotime Institute of Biotechnology). For qPCR analysis, the SYBR Green PCR Master Mix kit (Beijing Solarbio Science & Technology Co., Ltd.) was utilized. The relative gene expression levels were calculated using the 2^-ΔΔCq method (22). The primer sequences for CRP-1 were as follows: forward, 5'-AAGTGTCCCAAGTGCAACAA-3', and reverse, 5'-CGTCTTCCCACATTTCTCG-3'; β-actin: forward, 5'-GGCACCCAGCACAATGAA-3', and reverse, 5'-TAGAAGCATTTGCGGTGG-3'.

All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Inc.). The differences between two groups were compared using unpaired Student's t-test. The differences among more than 2 groups were compared using one-way ANOVA, followed by Tukey's multiple comparison. For cell viability results, a two-way ANOVA with Tukey or Sidak's post-hoc test was run. Each experiment was replicated at least 3 times. Data are expressed as the mean ± standard deviation (SD). P<0.05 was considered to indicate a statistically significant difference.

First, DEGs were analyzed in the GEPIA database. Therefore, a total of 2,224 DEGs, including 730 upregulated and 1,478 downregulated ones, were illustrated in volcano plots (Fig. 1A). The chromosomal location of DEGs is shown in Fig. 1D. Currently, the members of the LIM/double zinc finger domain family have gained tremendous attention due to their enriched cysteine LIM structure domain, which has been proved to be involved in tumor formation (23). Herein, the members of the LIM protein family were screened in the Pfam database (http://pfam.xfam.org/). The median expression levels of the LIM family members in tumor and normal tissues in the LIHC dataset are presented in Fig. 1B, while their log-fold-changes in Fig. 1C. Among them, CRP-1 exhibited the most obvious differential expression in the GEPIA database between tumor (n=369) and normal tissues (n=169; Fig. 1D).

Subsequently, the expression levels of CRP-1 were also compared between tumor and normal liver tissues in the UALACAN database. The results revealed that CRP-1 was remarkably upregulated in HCC tissues compared with adjacent normal tissues (Fig. 2A). Kaplan-Meier survival analysis showed that patients with high CRP-1 expression levels had worse overall survival compared with those with low levels (Fig. 2B). In addition, western blot analysis and RT-qPCR analysis demonstrated that CRP-1 was differentially expressed in five HCC cell lines, namely BEL-7405, PLC/PRF/5, Huh-7, Hep3B2.1-7 and MHCC97. Therefore, Hep3B2.1-7 cells expressed the highest CRP-1 levels, BEL-7405 cells the lowest, and PLC/PRF/5, Huh-7 and MHCC97 cells moderate ones (Fig. 2C). Subsequently, Hep3B2.1-7 and BEL-7405 cells were transfected with specific shRNAs targeting CRP-1 (sh-Control; sh-CRP-1#1/2) and CRP-1 overexpression plasmids, respectively. The transfection efficiency was verified by RT-qPCR and western blot analysis (Fig. 2D and E).

CRP-1-overexpressing BEL-7405 cells and CRP-1-depleted Hep3B2.1-7 cells were employed to investigate the biological function of CRP-1 in HCC in vitro. Therefore, CRP-1 silencing reduced the viability of Hep3B2.1-7 cells, as verified by CCK-8 assays. The opposite trend was observed in CRP-1-overexpressing BEL-7405 cells (Fig. 3A). To gain further insights into the mechanism underlying the effect of CRP-1 on HCC cell proliferation, the protein expression levels of PCNA and c-Myc were detected using western blot analysis. The results showed that the expression levels of both PCNA and c-Myc were notably decreased in CRP-1-depleted HCC cells. By contrast, CRP-1 overexpression yielded the opposite results (Fig. 3B). Furthermore, colony formation assays demonstrated that the number of colonies was significantly reduced in CRP-1-silenced Hep3B2.1-7 cells compared with the control group. However, the number of formed colonies was markedly higher in CRP-1-overexpressing BEL-7405 cells compared with control cells (Fig. 3C and D). In addition, the apoptosis rate of CRP-1-silenced Hep3B2.1-7 cells was significantly higher compared with that of control cells (Fig. 3E). The above finding was also supported by the protein expression levels of cleaved caspase 3 and cleaved PARP (Fig. 3F). The aforementioned results suggested that CRP-1 upregulation could promote the proliferation and survival of HCC cells.

The wound healing assay results showed that CRP-1 knockdown attenuated wound healing compared with control cells, while CRP-1 overexpression enhanced the migration ability of BEL-7405 cells (Fig. 4A). In line with the above results, Transwell assays confirmed that CRP-1 silencing suppressed the invasion of Hep3B2.1-7 cells (Fig. 4B). By contrast, CRP-1-overexpressing cells exhibited a more aggressive invasion potential compared with control cells (Fig. 4B). Subsequently, the association between CRP-1 and EMT in HCC cells was investigated. The results demonstrated that CRP-1 overexpression promoted EMT process in BEL-7405 cells, accompanied by E-cadherin downregulation, and N-cadherin and vimentin upregulation. However, CRP-1 knockdown reversed these effects (Fig. 4C).

It has been reported that the aberrant activation of the Wnt/β-catenin pathway plays a key role in the development of HCC (24). Therefore, the present study aimed to investigate whether CRP-1 was involved in the regulation of Wnt/β-catenin signaling in HCC cells. The effect of CRP-1 overexpression or silencing on enhancing or inhibiting the nuclear localization of β-catenin in HCC cells was verified by immunofluorescence staining (Fig. 5A). Therefore, CRP-1 overexpression significantly enhanced the nuclear expression β-catenin and that of the Wnt/β-catenin signaling-related downstream target-genes, cyclin D1 and MMP-7 (Fig. 5B). However, the expression levels of the above proteins were reduced in CRP-1-depleted HCC cells (Fig. 5B). To determine whether the Wnt/β-catenin pathway was involved in the CRP-1 induced HCC cell proliferation and migration, cells were treated with XAV-939, a Wnt/β-catenin signaling inhibitor, to impair its activation. The results demonstrated that treatment with XAV-939 significantly inhibited the CRP-1 overexpression-induced proliferation and migration of BEL-7405 cells (Fig. 5C and D).