Between December 2011 and December 2013, samples of blood and liver tissues were collected from 60 patients (44 males and 36 females; mean age, 55.4 years; range, 18–76 years) who had been diagnosed with HCC, based on criteria used by the American Association for the Study of Liver Diseases (www.aasld.org). The protocol for the present study was approved by the ethics committee of Longgan Hospital (Shenzhen, China), which also waived the requirement to obtain informed consent from the tissue donors.

Samples of blood (10 ml) were collected from patients with HCC and normal control subjects, placed into labeled tubes and allowed to clot for 30 min at room temperature. The blood was centrifuged for 5 min at 3,000 g and 4°C, following which, 0.5 ml aliquots of serum were transferred into tubes and labeled. The serum was then frozen immediately, ideally within 10 min of aliquoting, and stored at −80°C until further use.

Tissue samples were homogenized in 50 mM Tris-HC1 (pH 7.5) and centrifuged at 2,500 × g for 5 min. TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) was used to isolate total RNA from the samples of HCC cancer tissue and the serum of patients with HCC, according to the manufacturer's protocol. Control sample RNA was purchased from Ambion, Inc. (Austin, TX, USA) and BioChain Institute, Inc. (Newark, CA, USA). RNA quality was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc. Palo Alto, CA, USA), and RNA samples with integrity scores >5 were subjected to RT-qPCR analysis.

cDNA templates of different genes were obtained by RT of the RNA using Super M-MLV RT (BioTeke Corporation, Beijing, China), and the final reaction mixture of volume 20 µl contained 10 µl of 2X Taq PCR Master-mix (BioTeke Corporation), 1 µl of each primer (GIMAP5, forward 5′-CATGTTAGGGAAGCTCAGTC-3′, reverse 5′-GAAGGGTTCTACTGTGTCTCA-3′; GIMAP6, forward 5′-TGGATGCTCTGGATGTTGCA-3′, reverse 5′-TCCTGCTCATCCCCTTGTG-3′; hypoxanthine phosphoribosyltransferase 1 (HPRT1), forward 5′-GACCAGTCAACAGGGGACAT-3′, reverse 5′-GTGTCAATTATATCTTCCACAATCAAG-3′), 1 µl cDNA template and 7 µl of RNase free H₂O. Thermal cycling parameters for the amplification were as follows: Denaturation step, 95°C for 5 min; followed by 36 cycles at 95°C for 20 sec, 52°C for 20 sec and 72°C for 30 sec; and the reaction was stopped by a step at 25°C for 5 min. The relative expression level of the targeted genes was normalized to HPRT1 according to the 2^−ΔΔCq method (13).

The samples of the HCC and matched normal tissues were fixed in formalin and embedded in paraffin. Subsequently, 4-µm sections of the 60 paraffin-embedded HCC tumor tissue samples and matched non-tumor tissue samples were deparaffinized in xylene, rehydrated in an alcohol series and washed in distilled water. The sections were then treated by microwave for antigen retrieval for 8 min at 450 W, and incubated with serum-free blocking reagent (Dako, Carpentaria, CA, USA) for 10 min at room temperature to inhibit non-specific staining. Subsequently, the slides mounted with the tissue sections were incubated with rabbit anti-GIMAP5 (1:200; cat. no. ab74575; Abcam, Cambridge, MA, USA) or rabbit anti-GIMAP6 (1:200; cat. no. ab126067; Abcam) antibodies in a humid chamber overnight at 4°C. After three 5 min washes with 0.01 M phosphate-buffered saline (PBS), goat anti-rabbit secondary antibody (1:200; cat. no. A0277; Beyotime Institute of Biotechnology, Jiangsu, China) was added to the sections and incubated at 37°C for 30 min prior to five PBS washes. Peroxidase activity was detected using the enzyme substrate, 3,3 N-diaminobenzidine tertrahydrochloride. Scores of the immunohistochemical assay was determined by scanning the slides using an Aperio ScanScope GL (Leica Microsystems GmbH, Wetzlar, Germany) at ×400 magnification. ImageScope software (Leica Microsystems GmbH) was then used to assess the scanned images based on the percentage of positively stained cells and the staining intensity. Scores for the expression levels of GIMAP5 and GIMAP6 were based on the staining intensity and the percentage of positively-stained cells, as follows: 0, 0–9% of cells stained positive; 1, 10–29% of cells stained positive; 2, 30–69% of cells stained positive; 3, 70–100% of cells stained positive.

All data were analyzed using Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA). All values are presented as the mean ± standard error of the mean, obtained from analyses of three independent experiments. Differences between groups were analyzed using the non-paired Student's two-tailed t-test. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1A, the mRNA expression of GIMAP5 was significantly downregulated in the 30 HCC tissue samples, compared with the 30 samples of adjacent matched non-tumor tissues (P<0.001). Similar results were shown for the expression of GIMAP6 in another set of 30 HCC tissues and 30 samples of adjacent non-cancerous tissue (P<0.001; Fig. 1B). These results indicated that GIMAP5 and GIMAP6 were dysregulated at the mRNA level in HCC tissues, and this dysregulation may be involved in the pathogenesis of HCC.

Dysregulation of mRNA may or may not be reflected in the blood of patients. Therefore, the present study examined the mRNA expression levels of GIMAP5 and GIMAP6 in the serum of blood from patients with HCC and healthy control subjects. It was found that the mRNA expression levels of GIMAP5 and GIMAP6 were significantly downregulated in the blood of the patients with HCC, compared with their expression levels in the blood of the normal healthy subjects (P<0.001; Fig. 2).

The present study subsequently examined whether the dysregulation observed in the mRNA expression of GIMAP5 and GIMAP6 were also observed in the respective proteins. The IHC examinations of the protein expression levels of GIMAP5 and GIMAP6 revealed results that were consistent with the results for mRNA expression, with the protein levels of GIMAP5 and GIMAP6 being downregulated in the HCC tissue samples, compared with their levels in the samples of adjacent normal tissue (P<0.001; Fig. 3). These results suggested that the GIMAP5 and GIMAP6 proteins may offer potential as diagnostic biomarkers for HCC.

The present study also examined the protein expression of GIMAP5 and GIMAP6 in the blood of patients with HCC using ELISA. It was found that the GIMAP5 and GIMAP6 proteins were expressed at lower levels in the blood of the patients with HCC, compared with the expression levels in the blood of healthy control subjects (P<0.001; Fig. 4), providing further confirmation of the lower expression levels of GIMAP5 and GIMAP6 in the tissues and blood of patients with HCC.