Keratin 19 as a key molecule in progression of human hepatocellular carcinomas through invasion and angiogenesis

Tissue specimens were collected from 136 patients with HCC who underwent primary curative hepatectomy at the Nara Medical University Hospital, during the period between 2007 and 2012. No other treatments were given before resection. There were 103 men and 33 women with an age range of 29 to 84 (mean 69) years. Of the 136 HCC cases, 33 (24.3%) were positive for hepatitis B virus surface antigen (HBsAg), 62 (45.6%) were positive for hepatitis C virus antibody (HCVAb), and 43 (31.6%) were negative for both HBsAg and HCVAb. The follow-up period from surgical treatment until death due to HCC (16 cases) or the end of this study was 30 to 2550 days (mean 1100 days).

Tissues were fixed in 10% formalin, embedded in paraffin, cut into 3 μm sections, and mounted on silane-coated slides. One section from each tissue was stained with hematoxylin and eosin for histological examination. The diagnosis of HCC was based on WHO criteria [2]. Recurrence was diagnosed by biochemical tests (tumour marker; Alpha-fetoprotein, protein induced by vitamin K absence or antagonist-II), sonograms, computed tomography (CT) and magnetic resonance imaging (MRI). Written informed consent was obtained from all patients before treatment, according to our institutional guidelines. This study was approved by the institutional review board.

Immunohistochemical study was performed on paraffin sections using a BOND MAX Automated Immunohistochemistry Vision Biosystem (Leica Microsystems, Wetzlar, Germany). For antigen retrieval step, Bond Epitope Retrieval Solution 1 (citrate-based solution, pH 6.0) (Leica Biosystems, Nussloch, Germany) was used. Antibodies for immunohistochemistry are listed in Table 1. Double immunostaining was carried out following manufacturer’s protocols using K19 and the Bond Polymer Refine Detection kit (brownish colour, Leica Biosystems), and E-cadherin and the Bond Polymer Refine AP-Red Detection kit (red colour, Leica Biosystems). Bile ducts, liver, lymph nodes, vascular endothelium, and endothelial layer of the human placenta were used as positive control for K19, E-cadherin, Ki-67, CD31, and VASH1, respectively. Negative controls were carried out by substitution of the primary antibodies with non-immunized mouse serum, resulted in no signal detection (Additional file 1: Fig. S1). In this study, K19-positive HCC was defined as that in which > 5% of total carcinoma cells showed immunoreactivity against K19. E-cadherin, Ki-67, and VASH1 positive cells were counted in 1000 cancer cells from K19-positive and K19-negative areas in K19-positive HCC specimens. The number of blood vessels in K19-positive and K19-negative HCC specimens, identified by CD31 around cancer foci, was counted in 10 high-power fields (100×.).

The human HCC cell lines, HepG2, HuH-7, and PLC/PRF/5 were purchased from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and cultured in RPMI supplemented with 10% FBS.

The cells were seeded at 10⁵ cells per well in 6-cm plates, and transfected with 100 nmol/L control RNA (Santa Cruz bio, Dallas, TX, USA) or human K19 siRNAs using Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA, USA), in accordance with the manufacturer's protocol. After culturing for the indicated time, the samples were removed and homogenized.

Template cDNA was synthesised from 1 μg of total RNA using Primer Script RT reagent Kit (Takara, Shiga, Japan). The quantitative real-time PCR detection was performed using a SYBR® Premix Ex Taq kit (Takara). The amount of actin mRNA in each sample was used to standardise the quantity of each mRNA. The sequences of the primers used for PCR are shown in Table 2.

For the cell proliferation assay, the methane thiosulfonate (MTS) reagent was used as previously described [12‐14]. All the experiments were performed in triplicate.

In vitro invasion assays were performed using Matrigel invasion chambers (BD Biosciences, Bedford, MA, USA) as previously described [15]. Invading cells were counted under a light microscope. The experiment was repeated three times.

Cells were fixed at 70% confluence and then incubated at 37 °C overnight with staining solution containing X-gal substrate (Senescence Detection kit, BioVision, Milpitas, CA, USA). Cells were then observed under a microscope for the presence of blue stain [16].

Liquid based cytology (LBC) was used to prepare the cell lines for apoptosis assay by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) using the ApopTag in situ apoptosis detection kit (Oncor, Gaithersburg, MD, USA) [17]. We identified cells showing darkly stained nuclei or nuclear fragments as TUNEL-positive apoptotic cells, and counted those in several high-power fields.

Out of the total 136 HCC cases, 12 K19-positive HCC cases (8.8%) were examined in the present study (Fig. 1a). Results of an analysis of the relationship between K19 expression and various clinicopathological parameters are summarized in Table 3. K19-positive HCC predominantly occurred in young, female patients. K19-positive HCC was also associated with TNM stage, tumour differentiation (Fig. 1b), major vascular invasion, tumour-capsule formation as well as tumour necrosis. Early recurrence (within 6 months after surgery) frequently occurred in K19-positive cases. The percentages of extrahepatic recurrence were 41.7 and 10.5% in K19-positive and K19-negative cases, respectively (p = 0.002). Among the organs, metastasis to lung was most frequently observed in this study (p = 0.003). There was no significant difference between K19 expression and HBV or HCV infection. The non-HBV/non-HCV group and other pathological parameters such as microvascular invasion and fibrous stroma were not statistically correlated with K19 expression.

Survival analysis demonstrated that patients with K19-positive HCC had significantly poorer overall survival than did patients with K19-negative HCC (p < 0.01) (Fig. 1c). In contrast, there was no significant difference in disease-free survival (p = 0.573) unless the data were analysed during an early phase (Fig. 1d). The multivariate analysis demonstrated that tumour size and necrosis were independent predictors of overall survival, but this was not the case with K19 expression (Additional file 2: Table S1).

In the current study, we used three human HCC cell lines, HepG2, PLC/PRF/5, and HuH-7, which express K19 strongly, as determined by real-time PCR (Fig. 2a). K19 expression was successfully suppressed by transfection with K19 siRNA, followed by 72-h incubation (Fig. 2b). As shown in Fig. 2c, cell growth was significantly suppressed by K19 knockdown in PLC/PRF/5 and HuH-7 cells but not in HepG2 cells. When PLC/PRF/5 cells were transfected with K19 siRNA, senescence was induced, as assessed by SA-β-gal assay (Fig. 3d). Furthermore, K19 silencing upregulated mRNA levels of senescence-related genes such as p16 and p27 in PLC/PRF/5 cells (Fig. 3e). In LBC of HuH-7 cells, the number of apoptotic cells increased following K19 knockdown (Fig. 3f). Considered together, it appears that K19 knockdown induced apoptosis in HuH-7 cells and senescence in PLC/PRF/5 cells through the upregulation of p16 and p27 genes.

As stated above, cell growth was not significantly affected by K19 siRNA transfection of HepG2 cells. In the light of this result, we examined the effect of K19 knockdown on cancer invasion and angiogenesis. Figure 3a and c indicate that E-cadherin gene expression and matrigel invasion capacity of HepG2 cells increased and decreased, respectively, following K19 silencing. This suggests that K19 could enhance cancer invasion through decreased E-cadherin gene expression in HCC cells. Gene expression levels of Vimentin, N-cadherin, and snail were not affected by K19 knockdown (Fig. 3a). The expression of the angiogenesis-related genes VASH1 and FGFR1 decreased, while that of VASH2 increased following K19 knockdown in HepG2 cells (Fig. 3b). However, immunohistochemical analysis of HCC specimens indicated that VASH1 is strongly expressed not only in K19-positive HCC cells, but also in K19-negative HCC cells. VASH1 expression in HCC was not statistically correlated with K19 expression. Finally, we examined the E-cadherin expression and the HCC proliferative activity in both K19-positive and K19-negative areas using human K19-positive HCC specimens. Double immunohistochemical staining clearly showed that the percentages of cells positive for E-cadherin were 27.2% in K19-positive areas and 61.7% in K19-negative areas (p < 0.01) (Fig. 4a). In contrast, the Ki-67 proliferative index was higher in the K19-positive areas than in K19-negative areas (Fig. 4b). The Ki-67 proliferative index in the K19-negative area was similar to that observed in the K19-negative HCC specimens. Furthermore, the number of blood vessels around cancer foci was significantly higher in K19-positive HCC specimens than in K19-negative HCC specimens (Fig. 4c). These pathological data were thus in agreement with the results from the in vitro experiments.