The present study was performed in accordance with the reporting recommendations for tumor marker prognostic studies guidelines (17). The institutional ethics committee of Southern Hospital (Guangzhou, China) approved the protocol, and all enrolled subjects provided written informed consent.

Fresh HCC tissue samples and matched adjacent non-tumorous tissues were collected from 65 HCC patients who underwent resection at the Digestive Disease Research Institute of Southern Hospital between March 2008 and March 2011. The enrolled patients i) had a conclusive pathologic diagnosis of HCC; ii) had received curative resection, which was defined as macroscopically complete removal of the tumor; and iii) had available detailed clinicopathological data. Patients were excluded if they had received adjuvant chemotherapy or radiotherapy prior to surgery, or if there was evidence of other malignancies. The detailed clinicopathological characteristics of the HCC patients included in the current study are presented in Table I.

Patients were followed up until August 31, 2014. Among the 65 patients, 9 (13.8%) were lost to follow-up. Tumor recurrence confirmation was based on typical appearances on magnetic resonance imaging and/or computed tomography scans, as well as elevated α-fetoprotein protein (AFP) levels. The median follow-up period was 31 months (range, 1–71 months). Tumor differentiation was based on the criteria proposed by Edmondson and Steiner (18). Tumor stage was defined according to the American Joint Committee on Cancer (AJCC)/International Union against Cancer tumor node metastasis classification system (19).

Immunostaining was performed on 4-µm sections of paraffin-embedded tissue specimens. The sections were deparaffinized with xylene and rehydrated in a graded alcohol series. Antigen retrieval was carried out in a microwave oven in a sodium citrate solution (pH 8.0). Endogenous peroxidase was inactivated by incubating the samples in 3% H2O2 at room temperature for 20 min. Upon blocking with goat serum (Wuhan Boster Biological Engineering Co., Ltd.) at room temperature for 30 min, the samples were incubated with rabbit polyclonal anti-TP53INP1 antibody (catalog no. AP11890b; 1:50; Abgent, Inc., San Diego, CA, USA) at 4°C overnight in a moist chamber. They were then washed thoroughly with PBS and incubated with secondary antibodies (catalog no. HSP0007; 1:200; Shanghai Mjol Biological Technology Co., Ltd.) at 37°C for 30 min, conjugated to peroxidase (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). Staining (which was brown-colored) was visualized using a 3,3′-diaminobenzidine kit (Zhongshan Golden Bridge Biotechnology Co., Ltd.). Upon counterstaining with hematoxylin, the samples were dehydrated in a graded alcohol series and mounted. Negative controls were prepared in the absence of primary antibody.

Immunohistochemical staining was evaluated by two independent observers who were blinded to the clinical data. Concordance was achieved in 94% of the cases, and disagreements were resolved by consensus (20). Each sample was scored according to the intensity of the staining (no staining=0, weak staining=1, moderate staining=2 and strong staining=3) and the percentage of stained cells (<5%=0, 5–25%=1, 26–50%=2, 51–75%=3 and 76–100%=4). The percentage of cells at each intensity was multiplied by the corresponding intensity value to obtain an immunostaining score ranging from 0 to 12. The scores were combined to obtain an overall mean score. Using this assessment system, the optimal cutoff values were as follows: 0–3 (low) and 4–12 (high).

Proteins were extracted in radioimmunoprecipitation buffer (EMD Millipore, Billerica, MA, USA). Protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Samples with equal amounts of total protein were separated on 12% SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Upon blocking in TBS/Tween-20 containing 5% non-fat milk powder at room temperature for 5 min with agitation, the membranes were incubated for 1 h with anti-TP53INP1 rabbit polyclonal antibody (catalog no. AP11890b; 1:250; Abgent, Inc.) and anti-β-actin mouse monoclonal antibody (catalog no. CW0096; 1:1,000; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) at 4°C overnight. Upon incubation of the membranes with secondary antibodies (catalog no. HSP0007; 1:200; Shanghai Mjol Biological Technology Co., Ltd.) at room temperature for 3 h, immunoreactive bands were visualized by enhanced chemilumiscence using a GeneGnome HR Bioimaging System (Syngene, Frederick, MD, USA).

Total RNA was extracted from tissues using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and complementary DNA libraries were generated from total RNA using a High-Capacity cDNA Archive kit according to the manufacturer's protocol (Applied Biosystems; Thermo Fisher Scientific, Inc.). RT-qPCR was performed in triplicate using the SYBR-Green system on a LightCycler 480 Real-Time PCR System (Roche Diagnostics GmbH, Mannheim, Germany). Relative mRNA levels were calculated according to the quantification cycle (Cq) values corrected for GAPDH expression using the 2-ΔΔCq method as follows: ΔΔCq=ΔCq (treatment)-ΔCq (control) or ΔCq=Cq (target genes)-Cq (GAPDH). The primer sequences were as follows: TP53INP1, 5′-GCACCCTTCAGTCTTTTCCTGTT-3′ (forward) and 5′-GAGAAAGCAGGAATCACTTGTATC-3′ (reverse); and GAPDH, 5′-GAAGGTGAAGGTCGGAGT-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse).

All statistical analyses were carried out using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). Differences between two independent groups were analyzed using the Student's t-test. The clinicopathological features of HCC were analyzed using the Pearson's χ2 test. Recurrence-free survival (RFS) and overall survival (OS) were calculated using the Kaplan-Meier method, and significance was assessed using the log-rank test. RFS was defined as the interval between the date of surgery and the date of detection of a recurrent tumor. OS was defined as the interval between the date of surgery and the date of mortality or last follow-up. Independent prognostic factors for OS and RFS were identified using the Cox proportional hazards regression model. Data are presented as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference.

TP53INP1 expression was significantly decreased in HCC tissues compared with adjacent non-tumorous tissues, as determined via western blotting and immunohistochemistry. TP53INP1 was predominantly localized in the cytoplasm of hepatic cells, with little staining was present in the nuclei, as visualized via immunohistochemistry. Heavy TP53INP1 staining was observed in the epithelial cells in normal-appearing mucosa adjacent to HCC cells, whereas TP53INP1 staining in HCC cells was faint or absent (Fig. 1). In western blot analyses, TP53INP1 expression was lower in 8 of 9 HCC tissue samples than in matched adjacent non-tumorous tissue (Fig. 2).

Notably, TP53INP1 mRNA expression was significantly lower in HCC tissues (0.4103±0.03674) than in adjacent non-tumorous tissues (0.6851±0.05825, P=0.0001) (Fig. 3).

Expression of TP53INP1 and its clinicopathological relevance in hepatic tissues. To investigate the significance of TP53INP expression in HCC, the association between TP53INP mRNA levels and the clinicopathological characteristics of 65 HCC patients were evaluated in the present study. TP53INP1 mRNA expression was categorized as high or low. As shown in Table II, low expression of TP53INP1 mRNA closely correlated with AJCC stage (P=0.014) and vascular invasion (P=0.024). There were no significant differences in other clinical characteristics between the high and low expression groups.

The potential association between TP53INP1 expression and survival (RFS and OS) was retrospectively evaluated via Kaplan-Meier analysis. RFS (Fig. 4A) and OS (Fig. 4B) were significantly worse in HCC patients expressing low TP53INP1 levels compared with those expressing high TP53INP1 levels, with median survival times of 10 and 38 months, respectively (P=0.003).

Twelve clinicopathological variables were included in a univariate analysis, including age, gender, serum hepatitis B surface antigen (HBsAg) levels, serum AFP levels, liver cirrhosis, vessel invasion, intrahepatic metastasis, tumor size, tumor number, tumor differentiation, AJCC stage and TP53INP1 expression. Of these, tumor differentiation, AJCC stage, vascular invasion and TP53INP1 expression were significant prognostic factors of RFS and OS in univariate analysis (Table III). In addition, multivariate analysis (Table IV) revealed that TP53INP1 was also an independent prognostic factor for both RFS [hazard ratio (HR)=2.284, 95% confidence interval (CI)=1.157–4.511, P=0.017] and OS (HR=2.680, 95% CI=1.087–6.608, P=0.032) (Table III). Thus, low expression of TP53INP1 may serve as a prognostic indicator for patients with HCC.