FXYD6 overexpression in HBV-related hepatocellular carcinoma with cirrhosis

2.1 Clinical samples

Thirty-five fresh HBV-related HCC tissues with cirrhosis, 30 fresh cirrhosis tissues adjacent to HCC, and 10 normal fresh liver tissues distal to the surgically resected hepatic hemangioma without HBV infection were collected from the Department of Hepatobiliary and Pancreatic Surgery in Cangzhou Central Hospital (Cangzhou, China) between March 2014 and March 2018. The fresh tissues were stored in liquid nitrogen within 30 minutes after harvest and subsequently stored in a refrigerator at −80°C.

In addition, the formalin-fixed and paraffin-embedded tissue samples were obtained from 52 HBV-related HCC patients with cirrhosis who underwent radical surgery in the hospital between March 2012 and March 2017. HCC was staged according to the eighth edition of the HCC AJCC staging system. None of the patients received preoperative chemotherapy, radiotherapy, or biotherapy. Twenty-eight cirrhosis tissues matched with the primary tumor, and 15 normal liver tissues distal to the hepatic hemangioma in our hospital were also studied. Patients’ information, including gender, age, differentiation, tumor diameter, tumor number, integrity of tumor capsule or not, MVI, pathological stage, and AFP concentration in serum, was recorded at the hospital.

The mean age of the patients with HCC (44 males and 8 females) was 55.2 years, ranging from 30 to 78 years, and the median age was 56.5 years. Seven neoplastic specimens were well-differentiated carcinomas, 24 were moderately differentiated carcinomas, and 21 were poorly differentiated carcinomas. There were 22 patients with tumor 5 cm or less in diameter and 30 patients with tumor larger than 5 cm in diameter, 40 patients with single tumor and 12 patients with multiple tumors, 31 patients with integral tumor capsule and 21 patients with incomplete tumor capsule, 21 patients with MVI and 31 patients without MVI, 33 cases in stage I–II and 19 cases in stage III, and 38 cases with AFP < 400 ng/mL and 14 cases with AFP ≥ 400 ng/mL. The patients were reviewed at intervals of 1–3 months after surgery and recorded whether the carcinoma relapsed within 1 year. The patients received no treatment before recurrence.

1. Informed consent: Informed consent has been obtained from all individuals included in this study.

2. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies, and in accordance with the tenets of the Declaration of Helsinki and has been approved by the authors’ institutional review board or equivalent committee.

2.2 Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for FXYD6

Total RNA was isolated from tissue samples using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. cDNA was synthesized from 2 µg total RNA using an EasyScript Plus™ cDNA Synthesis Kit (ABM Inc., Massillon, OH, USA). According to the FXYD6 mRNA sequence published by GeneBank (ID: NM-022003), a pair of primers was designed to amplify the FXYD6 functional region sequence: the upstream primer was 5′-GAATTCAGTGCAGCTGAAAAGGAG-3′, and the downstream primer was 5′-CTCGAGTCAGTTCTCTGCTTTCTGG-3′. Meanwhile, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene used as a reference for normalization was also amplified. Its upstream primer was 5′-GGTGAAGGTCGGAGTCAACG-3′, and the downstream primer was 5′-CAAAGTTGTCATGGATGHACC-3′. Each PCR reaction contained 1 µL RT product, 20 pM of each primer, 800 µM dNTP (200 µM each), 1 unit Taq DNA polymerase, and nuclease-free H₂O up to 50 µL. Reactions were performed on a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). Amplification was conducted under the following thermal cycling conditions: 5 min at 95°C; 36 cycles of amplification consisting of 30 s at 95°C, 30 s at 53°C, and 30 s at 72°C; and a final extension at 72°C for 10 min. For analysis, the product was loaded in agarose gel, using GAPDH as a reference standard, electrophoresed, and photographed by a UV gel imaging analyzer (Bio-Rad, Hercules, CA, USA). The integral optical density (IOD) of the PCR product strip was detected using the Quantity One software (v4.6.2; Bio-Rad, Hercules, CA, USA). The ratio of the FXYD6 IOD to the corresponding GAPDH IOD was taken as the relative amount of FXYD6 mRNA in the tissues.

2.3 Immunohistochemistry

The HBV-related HCC with cirrhosis tissues, cirrhosis tissues adjacent to the carcinoma, and normal liver tissues were fixed in 10% formalin, embedded in paraffin, and serially sectioned at 4 µm. Each section was incubated at 60°C, deparaffinized, and rehydrated. Antigen retrieval was performed by placing slides in 10 mmol/L sodium citrate buffer (pH 6.0) and microwave treatment for 15 min. Then, the slides were allowed to cool down naturally in the buffer to room temperature. Endogenous peroxidase activity was eliminated by incubation in methanol with 0.3% H₂O₂ for 30 minutes, followed by washing with phosphate-buffered saline (PBS). Following non-specific reactions elimination with 5% normal horse serum for 1 h, the sections were incubated with mouse anti-human monoclonal FXYD6 prepared by Gao et al. [8] overnight at 4°C in a moist chamber. After washing in PBS, the sections were incubated with a biotinylated horse anti-mouse IgG antibody (ZB-2020; ZSGB-BIO, China) for 40 min at 37°C, washed again in PBS, and inculcated in horseradish peroxidase streptavidin (ZB-2404; ZSGB-BIO) for 40 min. The peroxidase reaction was developed in freshly prepared 3,3′-diaminobenzidine solutions (ZLI-9017; ZSGB-BIO), which was observed under a microscope (BX531; Olympus, Tokyo). Then, the sections were rinsed in water and counterstained using hematoxylin, dehydrated in ethanol, and mounted with xylene-based mounting medium. For negative controls, PBS was used instead of the FXYD6 monoclonal antibody under the same conditions.

The expression of FXYD6 in the tissues was evaluated with whole slide scanning under low magnification (×40) and then confirmed under high magnification (×100 and ×400). An immunoreactivity scoring system was applied. The extent of stained cells was given as follows: 0–5% = 0, 6–25% = 1, 26–50% = 2, 51–75% = 3, and 75–100% = 4. The intensity of color staining was defined by the following parameters: colorless, 0; whitish yellow, 1; yellow, 2; and brown, 3. The final immunoreactive score was determined by multiplying the intensity and extent of positivity scores of stained cells, with a minimum score of 0 and a maximum score of 12. The threshold for differentiating between final positive and negative immunostaining was set at 4 for interpretation. A negative staining was classified as having an immunostaining score of 0–3, whereas a positive staining was classified as having an immunostaining score of 4–12.

The slides were examined independently by two pathologists blinded to both clinical and pathological data, and all discrepancies were resolved by joint review of the slides in question.

2.4 Statistical analysis

Measurement data were expressed as mean ± SD, and statistical differences were determined by the independent samples t test. Classification data were shown as rate, and the comparison was analyzed by the chi-square test. These statistical analyses were all performed through SPSS software (v17.0; SPSS Inc., Chicago, IL, USA). For all analyses, p-values were two-tailed, and p < 0.05 was considered to indicate a statistically significant difference.

3.1 FXYD6 mRNA expression is markedly upregulated in HCC

The results of FXYD6 mRNA expression in HCC, cirrhosis, and normal liver tissues by semi-qualitative RT-PCR are shown in Figure 1 and Table 1. The relative IOD of FXYD6 mRNA expression was significantly higher in 35 HCC than that in 30 cirrhotic tissues distal to HCC (0.4461 ± 0.0344 vs. 0.2887 ± 0.0176; t = 23.723; p = 0.000) and significantly higher than that in 10 normal liver tissues without HBV infection (0.4461 ± 0.0344 vs. 0.2781 ± 0.0422; t = 12.959; p = 0.000). But there was no statistical difference between the relative IOD of FXYD6 mRNA expression in 30 cirrhosis tissues and that in 10 normal liver tissue (0.2887 ± 0.0176 vs. 0.2781 ± 0.0422; t = 0.771; p = 0.458).

Figure 1

The expression of FXYD6 mRNA in HCC with HBV-associated cirrhosis, paracancerous cirrhosis, and normal liver tissues. There was high expression of FXYD6 mRNA in HCC and low expression of FXYD6 mRNA in cirrhosis and normal liver tissues. N: normal live tissues, C: cirrhosis tissues, and T: HCC with HBV-related cirrhosis tissues.

3.2 FXYD6 protein is highly expressed in HCC

We examined FXYD6 protein immunohistochemically in 52 HBV-related HCC with cirrhosis tissues, 28 distal non-cancerous cirrhosis tissues, and 15 normal liver tissues. This study showed that there was negative immunostaining of FXYD6 in most cirrhosis tissues and in most normal liver tissues. FXYD6 negative reactivity was observed in 10/15 (66.7%) and 18/28 (64.3%) in normal liver slides and cirrhosis slides, respectively (Table 2). However, the positive expression rate of FXYD6 was 4/7 (57.1%) for well-differentiated HCC, 20/24 (83.3%) for moderately differentiated HCC, and 17/21 (81.0%) for poorly differentiated HCC. In all, the positive expression rate of FXYD6 in HCC was 41/52 (78.8%), which was significantly higher than that in normal tissues 5/15 (33.3%) or that in cirrhosis tissues 10/28 (35.7%).

3.3 Expression of FXYD6 correlates with MVI, pathological stage, and early recurrence

The expression level of FXYD6 in HCC was related to MVI and pathological stage. We examined the expression of FXYD6 protein in HCC, cirrhosis, and normal liver and found that the protein is mainly located in the cytoplasm but not in the nucleus as shown in Figure 2. The correlation between FXYD6 expression and various clinicopathological factors was also analyzed. As shown in Table 3, FXYD6 protein expression was positively correlated with MVI, and the positive expression rate of FXYD6 in HCC with MVI (20/21, 95.2%) was higher than that without MVI (21/31, 67.7%). In our studies, the FXYD6 expression was also associated with the pathological stage. Increased FXYD6 expression was found to significantly correlate with the degree of pathological stage of HCC. The positive expression rate of FXYD6 in HCC with pathological stages I–II (22/33, 66.7%) was significantly lower than that with pathological stage III (18/19, 94.7%). But there was no significant correlation between the increased expression of FXYD6 and other clinicopathological factors, including gender (X² = 0.000, p = 1.000), age (X² = 0.002, p = 0.968), histological differentiation (X² = 2.322, p = 0.313), tumor diameter (X² = 1.610, p = 0.204), tumor number (X² = 0.983, p = 0.321), integrity of tumor capsule or not (X² = 2.028, p = 0.154), and AFP concentration in serum (X² = 0.000, p = 1.000).

Figure 2

Representative immunohistochemical staining in normal liver, cirrhosis, and HCC. (A) Negative signal of FXYD6 was detected in normal liver tissues. (B) Most hepatocytes negatively expressed FXYD6, but a few hepatocytes next to portal area positively expressed FXYD6 in cirrhosis. (C and D) Positive expression of FXYD6 was found in moderately differentiated HCC, and FXYD6 was also highly expressed in the tumor cells (the red arrow) infiltrating the microvessel (the black arrow). Magnification (A, B, and C) ×100 and (D) ×400.

We also statistically analyzed the relationship between FXYD6 protein expression and early recurrence in postoperative patients with HCC. We followed up these 52 HCC patients who underwent curative hepatectomy and received no treatment before the HCC recurrence was discovered and found that the expression level of FXYD6 in HCC was related to early recurrence. The recurrence rate within 1 year in the high FXYD6 protein expression group was obviously higher than that in the low FXYD6 protein expression group (22/41, 53.7% vs. 2/11, 18.2%; X² = 4.392, p = 0.036).