A total of 169 tumor tissue samples and 151 adjacent non-tumorous liver tissue samples were collected at surgery from patients with HCC at Zhejiang Provincial People's Hospital (Zhejiang, China) between January 2010 and December 2016. The patient cohort included 142 males and 27 females, with an age range of 25–90 years old and a mean age of 58 years old. All tissues were fixed with 4% formalin for 24 h at room temperature, paraffin-embedded, and diagnosis was confirmed by pathologists at Zhejiang Provincial People's Hospital (Zhejiang, China). Information regarding tumor size, number, location, Edmondson Grade and tumor metastasis were collected from patient medical records. Overall survival (OS) was defined as the time from the date of surgery to the date of mortality or the last day of follow up. The research was approved by the Zhejiang Provincial People's Hospital Ethics Committee (Zhejiang, China), and informed consent was obtained from each participant.

Using the paraffin-embedded specimens, two tissue microarrays were designed and constructed by BioChip Company (Shanghai, China, www.shbiochip.com). Briefly, two tissue microarrays in (5-µm thick) were incubated at 70°C for 2 h, then deparaffinized in xylene 10 min for three times, prior to rehydration in a graded ethanol series (100, 95, 85 and 75% for 5 min). For antigen retrieval, the microarrays were boiled at 120°C with TE buffer (1.21 g/l Tris, 0.37 g/l EDTA, 0.5 ml/l Tween-20) at high pressure for 3 min. The sections were then treated with 3% hydrogen peroxide for 15 min at room temperature to eliminate endogenous peroxidase activity, and blocked with 10% goat serum (reagent A; Histostain-plus Bulk kit; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at room temperature for 20 min. The sections were then incubated with a CRHBP primary antibody (dilution, 1:200, AF2796; R&D Systems, Inc., Minneapolis, MN, USA) overnight at 4°C. Following 3 washes with PBS, the sections were incubated with a biotinylated second antibody (reagent B; Histostain-plus Bulk kit) for 15 min, prior to the addition of streptavidin-peroxidase (reagent C, Histostain-plus Bulk kit) for 15 min at room temperature, according to the manufacturer's protocol. Finally, a chromogenic reaction was performed using a DAB color-substrate solution (OriGene Technologies, Inc., Rockville, MD, USA), according to the manufacturer's protocol. Color development was stopped when the brown was observed obviously in the tissue microarrays. Hematoxylin (cat. no. C0107; Beyotime Institute of Biotechnology, Haimen, China) staining was performed for 3–5 min. The process was completed with dehydration in 75, 85, 95, and 100% ethyl alcohol for 5 min respectively, transparency three times in xylene for 10 min and mounting with gelatin resin. All procedures were performed at room temperature unless otherwise specified.

The results of immunohistochemical CRHBP staining were interpreted by 2 pathologists of the Pathology Department of Zhejiang Provincial People's Hospital, considering the staining intensity and the proportion of stained cells. The staining intensity was scored as follows: 0, negative staining; 1, weak staining; 2, medium staining, and 3, strong staining. The proportion of stained cells were scored as follows: 0, no cells stained; 1, 1–25% cells stained; 2, 26–50% cells stained; 3, 51–75% cells stained, and 4, >75% cells stained. We multiplied the staining intensity and proportion scores to confirm the level of CRHBP expression: 0 was considered to indicate negative expression, and ≥1 was considered to indicate positive expression.

Human liver cancer cell lines HCCLM3, MHCC97H, Huh7, Hep3B, HepG2 were obtained from cell bank of Shanghai Academy of Sciences (Shanghai, China), and cultured in DMEM (SH30243.01; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (16000–044; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% penicillin-streptomycin solution (SV30010; Hyclone). All cells were maintained in a humidified incubator with 5% CO₂ at 37°C.

CRHBP expression was analyzed in three paired HCC and adjacent non-cancerous tissues and five liver cancer cell lines (HCCLM3, MHCC97H, Huh7, Hep3B, HepG2). Proteins were extracted with RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and concentration determined by BCA Kit (cat. no. P0009; Beyotime Institute of Biotechnology). Then, proteins were heat-inactivated at 100°C 10 min, 12% SDS-PAGE (20 µg protein/lane) and transferred to polyvinylidene fluoride membranes by electrophoresis. The membranes were blocked in 5% skim milk for 1.5 h at room temperature. A CRHBP primary antibody (dilution, 1:1,000, AF2796; R&D) was incubated with the membranes overnight at 4°C. The membranes were washed in Tris-buffered saline with Tween three times prior to addition of a horseradish peroxidase conjugated-goat anti-Mouse IgG secondary antibody (dilution, 1:5,000, HA1006; HuaAn Biotechnology Co., Ltd., Hangzhou, China) for 1.5 h at room temperature. Finally, protein expression levels were analyzed via grey level was using Quantity One software (version 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total RNA was isolated from five liver cancer cell lines (HCCLM3, MHCC97H, Huh7, Hep3B, HepG2) using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNAs were synthesized from 1 µg of DNase-treated total RNA using the PrimeScriptTM RT reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan), under 65°C, 5 min, 42°C, 60 min and 70°C, 10 min of reverse transcription. The primers of CRHBP were 5′-CACACCAGCATCGAAACTGC-3′ (forward) and 5′-TGAAGACCATTTACGTGTCCCA-3′ (reverse). The Primers for GAPDH were 5′-TGAAGGTCGGAGTCAACGG-3′ (forward) and 5′-CTGGAAGATGGTGATGGGATT-3′ (reverse). qPCR was carried out using a Bio-Rad CFX Connect Real-Time system (Bio-Rad Laboratories, Inc) using the thermal cycling condition: 98°C for 2 min followed by 39 amplification cycles at 98°C for 15 sec and 60°C for 15 sec. At the end of the PCR cycles, melting curve analysis were performed. The expression of CRHBP was normalized to GAPDH using 2^−ΔΔCq method (17).

CRHBP mRNA expression in HCC and normal tissues was compared using the online Oncomine database (https://www.oncomine.org) with the following filtering conditions: gene, CRHBP; analysis type, cancer vs. normal; cancer type, hepatocellular carcinoma; data type, mRNA. The 10-year survival analysis was performed using OncoLnc (http://www.oncolnc.org/). Gene enrichment analysis of CRH was performed using STRING (https://string-db.org/) as there was not enough CRHBP data in the online database, and the top 20 enriched genes analyzed to investigate its biological function. Target signaling pathway was searched on KEGG database (www.genome.jp).

Statistical analysis was performed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA). χ^{2 test} was used to analyze the association between CRHBP expression and clinicopathological parameters. The overall survival curve was generated using the Kaplan-Meier method and further analyzed using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

The majority of the patients who participated in the present study had a history of hepatitis B virus infection (79.3%). Upon the last day of follow-up, 84 patients were alive and 41 mortalities had occurred. A total of 44 patients were lost to follow-up (Table I).

Positive immunohistochemical staining of CRHBP was detected in 84/169 (49.7%) HCC tissues and 142/151 (94.0%) adjacent non-tumorous tissues. The expression of CRHBP in tumor tissues was significantly lower than that in adjacent non-tumorous tissues (P<0.001; Table II; Fig. 1A and B). A bar chart illustrating the score values of the immunostaining of CRHBP expression in HCC tissues (Fig. 1C).

Tumor diameters ≥5 cm were significantly associated with low expression of CRHBP (P=0.013). Low CRHPB expression was also significantly associated with Edmondson Grade (P=0.002), high α-fetoprotein (AFP) levels (P=0.014) and hepatitis B virus (HBV) infection (P=0.020). No significant association was identified between CRHBP expression and age, sex, tumor location, metastasis status, microvascular invasion or cirrhosis (P>0.05; Table I).

To evaluate the prognostic value of CRHBP expression, Kaplan-Meier curves were constructed. The 5-year overall survival rate was significantly different between patients exhibiting low CRHBP expression (39.20 months) and patients exhibiting high CRHBP expression (49.18 months) (P=0.012; log-rank test; Fig. 2). Thus, it was inferred that patients exhibiting low CRHBP expression in the liver tissue had a reduced survival time compared with those exhibiting high CRHBP expression.

To further confirm the expression pattern of CRHBP in HCC, the protein expression level of CRHBP was determined in paired HCC and adjacent non-cancerous liver tissues by western blotting. Compared with the adjacent non-tumor tissues, significant downregulation of CRHBP protein expression was observed in HCC tissues (Fig. 3A and B). Furthermore, CRHBP levels were lower in highly metastatic liver cancer cell lines, including HCCLM3 and MHCC97H cells, than in those with low metastatic potential, including Huh7 and Hep3B cells, and was highest in HepG2 hepatic cancer cells as determined by western blotting and RT-qPCR (Fig. 3C and D). These results suggest that reduced CRHBP expression is common in patients with HCC, and that this may be associated with tumorigenesis.

A total of 5 datasets: Wurmbach Liver Statistics (18), Chen Liver Statistics (19), Roessler Liver Statistics, Roessler Liver 2 Statistics (20) and Mas Liver Statistics (21), were selected to analyze the expression of CRHBP in HCC vs. normal tissues using Oncomine databases. It was demonstrated that the expression of CRHBP mRNA was significantly lower in HCC tissues than in normal tissues (Fig. 4; P<0.01). OncoLnc was used to reveal that patients exhibiting low expression of CRHBP had a shorter 10-year survival time (Fig. 5; log-rank, P=0.0122). STRING was used to identify the top 20 biological processes of CRH gene enrichment (Table III) and a part of an important signaling pathway of cAMP was generated on KEGG database (Fig. 6).