Open Access

The C-terminus domain of the hepatitis B virus x protein stimulates the proliferation of mouse foetal hepatic progenitor cells, although it is not required for the formation of spheroids

  • Authors:
    • Liming Yu
    • Shu Chen
    • Na Luo
    • Song He
  • View Affiliations

  • Published online on: June 14, 2017     https://doi.org/10.3892/ijmm.2017.3026
  • Pages: 400-410
  • Copyright: © Yu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The hepatitis B virus X (HBx) protein is an important factor in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). The C-terminal region of HBx plays a major role in the replication of HBV. Notably, HBx promotes the expansion and tumourigenesis of hepatic progenitor cells (HPCs) in mice. However, it remains unclear as to whether the C-terminal region of HBx is required for the stimulation fo the proliferation of mouse foetal HPCs (FHPCs). In our study, we used EpCAM+, CD133+ and CD49f+ FHPCs, which are bipotential clonogenic cells. These FHPCs transformed into mature hepatocytes and cholangiocytes when cultured under conditions that facilitate differentiation. Compared with the FHPCs grown as monolayers, spherical cell proliferation occurred more rapidly. Furthermore, spherically cultured FHPCs can grow in semi-solid agar and tend to maintain the morphology and characteristics of stem cells compared with growth in rat tail collagen. Notably, we also demonstrate that the C-terminus of HBx stimulates the proliferation of FHPCs, but is not required for the formation of spheroids, similar to hepatic cancer stem cells. These findings enhance our understanding of the HBx-induced tumourigenicity of FHPCs and may aid in the treatment of HCC.

Introduction

The hepatitis B virus (HBV) infects more than 350 million individuals worldwide and is the main cause of primary hepatocellular carcinoma (HCC). HBV-associated HCC is the fifth most common malignant disease worldwide (1). HCC is associated with a very high mortality rate, and the recurrence and metastasis of liver cancer remain a major concern. The hepatitis B virus X (HBx) protein is a low-molecular-weight (17-kDa), 154-amino-acid soluble protein that is a well-known risk factor for HBV-induced carcinogenesis (2). Although HBx, particularly the C-terminal region of HBx, is known to stimulate quiescent hepatocytes to undergo a G0/G1 transition and enter the cell cycle, thereby maximising the replication performance of HBV (3); however, the precise function of HBx in HBV-associated HCC remains unclear (4-6). It has been found that hepatic progenitor cells (HPCs) become activated in patients with chronic hepatitis C or B, HCC or other severe liver diseases, and damage occurs to liver cells and bile duct epithelial cells (7). A pluripotent progenitor cell in normal adult human liver has already been identified (8). It is well known that HCCs contain tumour-initiating stem-like cells (TISCs) (911), and microarray technologies utilizing integrative comparative genomic analysis of human HCC tissue samples, HCC cell lines and transgenic mouse models confirm that liver cancer stem cells and HPCs exhibit similar molecular characteristics (12). It has also been suggested that TISCs are derived from the malignant transformation of stem/progenitor cells (13). In addition, a study on the HBx-stimulated transformation of intrinsic cellular pathways to promote HPC proliferation and tumourigenicity also supports this claim (14). We thus hypothesised that the truncation of HBx is associated with the mechanism of HBx-induced proliferation.

The foetal and adult liver are common sources of HPCs. The activation of HPCs from adult liver has reportedly been induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (15,16), by choline-deficient or ethionine-supplemented diets (18), in transgenic mice [such as p53 null, methionine adenosyltransferase 1A (MAT1A)−/− or liver-specific phosphatase and tensin homolog deleted on chromosome 10 (Pten)−/− mice] (1922), and by reprogramming fibroblasts by defined factors (23). In addition, the foetal liver contains large numbers of HPCs, such as rat embryonic day (ED)14 foetal HPCs (FHPCs), which were purified to 95% homogeneity and are expected to be HPCs (24). In mice, over 60% of cells derived from ED14.5 Dlk+ cells are able to differentiate into both hepatocytes and biliary epithelial cells (25), and it has been shown that density gradient centrifugation can be used to purify mouse FHPCs (26). In the present study, we used ED14.5 mouse FHPCs to demonstrate that they display the characteristics of spherical growth and that the C-terminal region of HBx is required for the stimulation of cell proliferation. The findings of our study may enhance our understanding of the mechanisms through which HBx induces the tumourigenicity and proliferation of HPCs and may facilitate the development of future treatments for liver diseases.

Materials and methods

Animals

Primary hepatocytes were isolated from 6–8-week-old wild-type male C57BL/6 mice by a two-step collagenase perfusion (3), and FHPCs were isolated from ED14.5 wild-type C57BL/6 mice, as previously described (25). The animals were obtained from the Animal Experiment Center of Chongqing Medical University (Chongqing, China) and all animal experiments were performed according to the guidelines set by the National Health and Medical Research Council of China. All animal experiments were approved by the Ethics Committee of the Second Hospital of Chongqing Medical University (no. 2013-026).

Isolation of FHPCs

Pregnant C57BL/6 mice (14.5 days) were sacrificed and subjected to caesarean delivery. The livers were removed from the mouse foetuses and washed 2 times in cold phosphate-buffered saline (PBS), dissected into sections, digested with 0.1% collagenase IV and 0.05% DNAse (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12), and incubated for 30 min at 37°C, and a single cell-suspension was obtained via successive filtration through 150 and 75-μm cell strainers. Subsequently, the cell suspension was centrifuged at 1,000 × g for 10 min, and the cell pellet was collected and resuspended in 3 ml of PBS. Discontinuous Percoll (Amersham Pharmacia Biotech, Freiburg, Germany) gradients were prepared in 15-ml tubes by layering 3 ml each of 70% Percoll, 50% Percoll and 30% Percoll in sequence, after which the cell suspension was layered atop the Percoll solution. The tubes were centrifuged at 400 × g for 25 min. Depending on their different sizes and densities, cells aggregate at specific positions along the density gradient. The cell fraction at approximately 50% Percoll was collected and centrifuged again at 300 × g for 10 min, and the pelleted cells were collected as FHPCs. The trypan blue dye exclusion assay was used to assess cell viability. All steps were performed on ice to reduce the risk of cell damage.

Culture of FHPCs

The FHPCs were seeded onto type I collagen (Sigma-Aldrich)-coated 60-mm dishes at a density of 1×106 viable cells. The cells were maintained in DMEM/F-12 containing 1% penicillin/streptomycin, 10 μg/ml insulin (Sigma-Aldrich), 10% fetal bovine serum (FBS), 20 ng/ml epidermal growth factor (EGF; Peprotech, Inc., Rocky Hill, NJ, USA), 10 ng/ml leukemia inhibitory factor (LIF; Sigma-Aldrich) and 2 mM L-glutamine at 37°C with 5% CO2. The medium was changed after 24 h to remove dead and non-adherent cells, and the culture medium was subsequently changed every 1–2 days. The cells were observed daily under an inverted microscope (Nikon Eclipse Ti-U). The stellate cells were selectively detached from the dish by repeated differential digestion with TrypLE™ Express (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C for 2.5 min. The purified FHPCs were first passaged at a ratio of 1:2 at 5–7 days after plating and passaged every 2–3 days after a generation. The passaged FHPCs were serially diluted to a density of one cell/100 μl of culture medium and replated onto uncoated 96-well plates. Colonies containing >50 cells were quantified after 2 weeks using a binocular inverted microscope (Nikon Eclipse Ti-U).

Establishment of spherical FHPCs clonal cells

The cells were seeded into a 25 cm2 plastic culture bottle coated with a low concentration of type I collagen at a density of 2×105 viable cells/ml in serum-free medium; spherical cells (10,27) were collected by gentle centrifugation at 30 × g after 5–7 days and replated onto type I collagen-coated 6-well plates as FHPC clonal cells.

Periodic acid-Schiff (PAS) staining

After the attachment of spheroid cells on type I collagen for 7 days, glycogen storage was detected using a PAS staining kit (3), according to the manufacturer's instructions. The cells were fixed with 4% paraformaldehyde for 5 min, washed with distilled water for 1 min and incubated in periodic acid (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at room temperature for 10 min in the dark. The cells were then washed with distilled water for 5 min and incubated in Schiff's reagent (Nanjing Jiancheng Bioengineering Institute) for 15 min at room temperature. After a final wash in running distilled water for 1 min, the cells were dried at room temperature, incubated in methyl green solution for 30 sec, cleaned with double distilled water and subsequently observed under an inverted microscope (Nikon Eclipse Ti-U).

Alkaline phosphatase (AP) staining

AP staining is often used to identify stem cells (21) and cholangiocytes (23). Both spheroid and attached cells were fixed with 4% paraformaldehyde for 10 min and then washed 3 times with distilled water for 5 min each. BCIP/NBT working solution (Beyotime Institute of Biotechnology, Haimen, China) was prepared according to the manufacturer's instructions. The cells were incubated with BCIP/NBT solution for 30 min at room temperature in the dark, washed with distilled water for 30 sec and analysed under a light microscope.

Flow cytometry

The cells were harvested and washed twice with PBS, followed by staining in washing buffer (PBS containing 0.05% rat serum) at room temperature for 30 min. A total of 1×106 cells were incubated in 100 μl PBS with chromo-phore-conjugated antibodies at 4°C for 30 min. The following chromophore-conjugated antibodies were used: anti-mouse CD133 (prominin-1) PE (12-1331) (5 μl) and anti-mouse epithelial cell adhesion molecule FITC (EpCAM; 11-5791) (1 μl) (both from eBioscience, San Diego, CA, USA), anti-human and mouse CD49f PE (10 μl; 130-100-096; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Following incubation, the cells were washed twice and analysed on a flow cytometer (Becton Dickinson, San Jose, CA, USA). Isotype antibodies were used as negative controls.

Immunofluorescence

Cells grown on type I collagen-coated coverslips were fixed with 4% paraformaldehyde at 4°C for 30 min. The cells were then washed three times for 5 min with PBS, permeabilised with 0.1% Triton X-100 in PBS for 10 min and blocked with 6% rat serum in PBS for 30 min at room temperature. The cells were then incubated with chromophore-conjugated antibodies in PBS at 37°C in the dark for 30 min. The chromophore-conjugated antibodies were as described above. The cells were washed with PBS 3 times for 5 min in the dark. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Institute of Biotechnology) for 3 min in the dark. The cells were then examined under a fluorescence microscope (Olympus IX83).

Soft agar clone

The spheroid cells were collected and enzymatically (TrypLE™ Express; 8 min at 37°C) and mechanically dissociated into single cells. Agar (0.6%) in William's medium E (19) (WME; Sigma-Aldrich) supplemented as described above, kept at 40°C and poured into 6-well plates to form the lower layer. After the agar medium had solidified, either 1×103 single cells or spheroid cells in 1.0 ml of 0.36% agar at 40°C were plated onto the lower layer. Fresh culture medium was added weekly to keep the cultures moist. The cells were incubated with 5% CO2 at 37°C; growth potential and character were assessed after 3 weeks.

Colony formation assay

Spheroid cells and single cells were plated onto type I collagen-coated 6-well plates at a density of 1×103 viable cells, and colonies containing >50 cells were quantified after 2 weeks. The single cells were seeded onto type I collagen-coated 24-well plates at a density of 1×104 viable cells, and cell numbers were quantified over 6 sequential days using a binocular inverted microscope (Nikon Eclipse Ti-U) and plotted to generate a cell proliferation curve.

Differentiation into hepatocytes and cholangiocytes in vitro

The clonally expanded cells (5×103 viable cells/cm2) were cultured in standard medium and differentiation medium supplement with 10 ng/ml hepatocyte growth factor (HGF; Peprotech, Inc.), 1×10−7 mol/l dexamethasone and 1% dimethyl sulfoxide (DMSO) (both from Amresco LLC, Solon, OH, USA), but without LIF. After 2 weeks, the cells were collected and subjected to PAS staining as described above, as well as immunofluorescence (28). The primary antibodies used for immunofluorescence included albumin (ALB; 1:50, sc-50536), cytokeratin 19 (CK19; 1:50, sc-25724) and hepatocyte nuclear factor (HNF)4α (1:50, sc-8987) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The tetraethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit antibody (SA00007-2) was obtained from Proteintech Group, Inc. (Chicago, IL, USA). The FHPC clones were seeded onto type I collagen (29) and fibronectin (28), cultured in standard medium without LIF for 2 weeks, and observed for morphological changes using an inverted fluorescence microscope (Olympus IX83).

Relative transcription-quantitative polymerase chain reaction (RT-qPCR)

RT-qPCR was performed as previously described (23). Total RNA was isolated from cell lines and clinical samples using TRIzol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Reverse transcription reactions were conducted with oligo (dT) 18 primers and random primers according to the instructions of the manufacturer of the M-MLV Reverse Transcriptase kit (Invitrogen). Quantitative PCR (qPCR) was performed with SYBR Premix Ex Taq™ (TaKaRa, Tokyo, Japan) using the Step One Plus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The primer sequences used for PCR are listed in Table I. The gene expression levels were calculated relative to the expression of β-actin in tumor cell lines or clinical samples using the 2−ΔΔCt method.

Table I

Primer sequences used for real-time PCR.

Table I

Primer sequences used for real-time PCR.

GenesForward (5′→3′)Reverse (5′→3′)
AFP ACCTTCCTGTCTCAGTCATTCT CCTGACATCCAGGTAGATTTCCA
CK19 GTTCAGTACGCAGGGTCAGT GAGGACGAGGTCACGAAGC
CFTR CTCAGCCTTCTGTGGCCGG TCCGGGTCATTTTCAGCTCCAC
GAPDH AGGTCGGTGTGAACGGATTTG GGGGTCGTTGATGGCAACA

[i] AFP, alpha fetoprotein; CK19, cytokeratin 19; CFTR, cystic fibrosis transmembrane conductance regulator; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Plasmid DNA

The pGEMHBV and pGEMHBVΔx plasmids both encode a more-than-unit-length HBV genome (pay-w1.2), though the latter contains a stop codon at amino acid 7 of the HBx ORF (payw1.2*7) (30). The plasmids pSI-HA-x, pSI-HA-x1-101 and pSI-HA-x43-154 were generated by PCR cloning into the pSI vector. According to the previous studies by Hodgson et al (31) and Luo et al (3), anine-amino-acid HA epitope tag was cloned at the N-terminus of HBx and its truncation mutants.

Transfection of the FHPCs

The medium was changed 1 h prior to transfection. HPCs were transfected with PolyJet (SignaGen® Laboratories, Ijamsville, MD, USA) according to the manufacturer's instructions, and a green fluorescent protein (GFP)-expressing plasmid was co-transfected at a proportion of 1:1 in order to assess the transfection efficiency. GFP expression was observed daily, and the cells were collected at the highest efficiency of expression.

Western blot analysis and immunoprecipitation

At 4 days post-transfection, the cells were collected and lysed on ice in RIPA Lysis Buffer (Beyotime Institute of Biotechnology). The primary antibodies included anti-HBcAg (1:400; B0586; DakoCytomation, Glostrup, Denmark) and anti-HA (1:200, sc-805; Santa Cruz Biotechnology, Inc.). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:5,000; ZB-2010) and the anti-β-actin antibody (1:500; TA-09) were from Beijing Zhongshan Golden Bridge Biotechnology, Co. (Beijing, China). Western blot analysis was performed as previously described (3), and protein bands were quantitated using the Quantity One Image analysis system. HBx immunoprecipitation was performed prior to western blot analysis. Equal amounts of total protein from cells transfected with pSI-HA-x, pSI-HA-x1-101, pSI-HA-x43-154 and pSI-control were incubated with protein A/G Plus-agarose beads (Beyotime Institute of Biotechnology) and primary anti-HA antibody. Specific operations were performed according to the manufacturer's instructions.

FHPC proliferation following transfection in vitro

The FHPCs transfected with pSI-HA-x, pSI-HA-x1-101, pSI-HA-x43-154 and pSI-control were plated at a density of 1,000 small cell clusters onto type I collagen-coated 6-well plates as described above. Colonies containing 10–50 and >50 cells were quantified after 1 week using a binocular inverted microscope (Nikon Eclipse Ti-U).

Statistical analyses

The data are presented as the means ± SD. Statistical analyses were carried out using one-way analysis of variance (ANOVA) and Tukey's test facilitated with GraphPad software version 5.0 (San Diego, CA, USA). A value of P<0.05 was considered to indicate a statistically significant difference.

Results

Isolation and culture of FHPCs

Recently, more than half of FHPCs enriched by Percoll discontinuous gradient centrifugation (PDGC) were found to be positive for CD133, CD49f and CD90 (26) in rats. We hypothesised that PDGC may also be used to enrich HPCs in mouse foetuses at ED14.5. In agreement with our hypothesis, the enriched FHPCs were concentrated in the 50% Percoll™ layer, after plating onto type I collagen for 24 h, and the erythrocyte and anchorage-independent cell population was removed by washing and replacing the medium. Primary cultures of the purified FHPCs formed a variety of colonies after 36–48 h. Those cells derived from the upper part of 50% Percoll™ cell layer appeared larger and were arranged in a paving-stone-like pattern (Fig. 1A–C), while those from the lower part of the 50% Percoll™ cell layer formed denser colonies that were more homogenous (Fig. 1D) and proliferated rapidly. After 5 days of primary culture, the colonies mixed together (Fig. 1E and F) and presented a high nucleus/cytoplasm ratio. The cells were digested at 7–10 days in primary culture and split at a 1:2 ratio, and the cells proliferated rapidly after passaging (Fig. 1G). To obtain single clonal cell lines, we performed limiting dilution as previously described by Conigliaro et al (28) by seeding cells in a 96-well plate at a concentration of 1 cell/well. Small colonies were visible at 5 days and 2 weeks after plating, and 27.98% of wells contained colonies with >50 cells (data not shown).

Spheroid FHPC clonal cells

In our study, single FHPCs proliferated very slowly; changing the concentration of type I collagen coated onto the plates and culturing in serum-free medium led the HPCs to also form spheroids. When the concentration was at 4–5 μg/cm2, over the next 3–5 days, a subpopulation of cells detached and grew in suspension as spheroid cells (Fig. 1H). When the spheroid FHPCs were replated onto 8–10 μg/cm2 type I collagen in DMEM/F12 medium containing FBS (32,33), they attached immediately and formed dense cell colonies in one day (Fig. 1I). After 5 days, the periphery of the colonies consisted of cells forming a cordlike morphology (Fig. 1J) as previously described by Schmelzer et al (34). We classified the colonies as a spheroid FHPC line, given their high proliferative potential and maintenance of a dense colony morphology following continuous passage in culture (Fig. 1K).

Culture of FHPCs on collagen-coated dishes

We then performed immunophenotyping profiling of the FHPCs in spheroid cells and after attachment. We assessed the expression of EpCAM, CD133, CK19 and CD49f in our FHSCs by immunofluorescence staining. At 7 days after being seeded onto type I collagen, the spheroids and attached cells were both found to co-express CK19 and CD49f (Fig. 2A–C). EpCAM expression was high in the spheroids (Fig. 2A), but was low in the attached cells (Fig. 2B). EpCAM also became downregulated after the attachment of the spheroid FHPCs. To further verify these results, culturing was prolonged to 13 days. CD133 was expressed in both the spheroid and attached cells (Fig. 2D and E), and EpCAM expression was identical to that described above. In the single cells and extensions from the periphery of the spheroids, the expression of CD133, EpCAM, CK19 and CD49f was lower than that in the centre of the spheroids. Notably, EpCAM and CD133 were expressed not only at the surface of the attached cells, but also in the cytoplasm, though the entire sphere expressed both EpCAM and CD133 in the spheroid cells (Fig. 2D and E). FCM analyses of the colonies revealed that 98.98±0.20, 93.24±4.14 and 71.67±12.29% of the attached cells were positive for CD133, CD49f and EpCAM expression, respectively (Fig. 2F), while only 55.44±22.46% of the cells co-expressed CD133 and EpCAM, indicating an overlap between CD133-positive and EpCAM-positive cells; a subpopulation of cells was only positive for EpCAM. Thus, almost all the spheroid cells, as well as the majority of the attached cells, expressed markers of FHPCs. The spheroid FHPCs cultured on collagen-coated dishes for 7 days were negative for PAS staining (Fig. 3A), while red glycogen particles were visible in the cytoplasm of cells at the centre of the attached colonies (Fig. 3B), indicating that the spheroids had less stem cell characteristics and differentiated into hepatocytes following attachment. Consistent with these findings, spheroid human hepatic stem cells (hHpSCs) initially transition into hepatoblasts on plastic and then give rise into hepatocytic and cholangiocytic lineages (17), while induced hepatic stem cells (iHepSCs) from mouse embryonic fibroblasts form aggregates of approximately 100 mm in diameter and show significant glycogen storage staining by PAS when plated on Matrigel (23). Similarly, staining the spheroids for AP revealed that the entire sphere was stained purple brown (Fig. 3C), and only the centre of the attached cells was AP-positive (Fig. 3D). Cholangiocytes (23) and human embryonic stem cells (35) have previously been shown to stain positively for AP, and our findings with spheroid FHPCs are also consistent with these results.

Self-renewal of FHPCs in collagen and soft agar

We then determined whether single cells taken from spheroids could still form spheroid colonies. Therefore, we dissociated spheroids by mechanical pipetting and enzymatic digestion to obtain single cells, which were then replated at a density of 1,000 viable cells/well onto 6-well plates coated with collagen or soft agar. Spheroids were also seeded as a control. After 3 weeks of subculture, the single cells grown in soft agar formed dense cell colonies (Fig. 3E), while no spheroid cells were observed in collagen (Fig. 3F), and spheroid cells in soft agar tended to balloon (Fig. 3G), whereas the spheroids seeded on collagen attached and expanded within 3 days (Fig. 3H). We then replated equivalent numbers of attached cells, following dissociation by enzymatic digestion into small cell clusters, and spheroids on collagen for subculturing. Over the next 2 weeks, the spheroids formed 3-fold more colonies than the attached cells (Fig. 3I). We then collected 104 attached cells as described above and seeded them on collagen-coated 24-well plates to generate a growth curve (Fig. 3J). Thus, we concluded that the cells were able to maintain their viability and ability to form new spheroids in soft agar, but lost this capacity on collagen. Furthermore, this colony-forming and self-renewal potential was downregulated in small cell clusters derived from attached cells, yet these small cell clusters still retained robust proliferation. Furthermore, the study by Yamashita et al (10) indicated that spheroid HCC cells maintained greater EpCAM expression than attached cells, and spheroid FHPCs also shared this feature.

FHPC differentiation into hepatocytes in vitro

To address whether the established spheroid FHPCs could differentiate into hepatocytes in vitro, we cultured the FHPCs in differentiation medium supplemented with 10 ng/ml HGF, 1% DMSO and 10−7 mol/l dexamethasone, as well as in standard medium. HGF, DMSO (29,36) and dexamethasone (37) have been shown to facilitate hepatocyte differentiation in vitro. The FHPCs were cultured in differentiation medium for 2 weeks; the cell size increased, whereas the nucleus/cytoplasm ratio decreased, and compared to culture in standard medium (Fig. 4A), glycogen particles accumulated distinctly (Fig. 4B), as shown by immunofluorescence staining (Fig. 4C). In addition, real-time PCR (Fig. 4D) revealed that the expression of FHPC marker genes (AFP), as well as the co-expression of FHPCs and cholangiocyte marker genes (CK19) (17), was distinctly downregulated, but the expression of cholangiocyte marker genes (CFTR) was upregulated. Immunofluorescence staining showed that FHPCs expressed ALB, but were negative for HNF4α. Consistent with these findings, a portion of cell aggregates that had formed after spheroid attachment disintegrated into larger-sized individual cells within 24 h in standard medium (data not shown), indicating that the FHPCs colonies spontaneously differentiated in the presence of cytokine and collagen.

FHPC differentiation into cholangiocytes in vitro

Upon the addition of EGF, but without LIF, for 2 weeks, colonies with branch structures at the periphery were consistently observed (Fig. 4E). Under similar conditions using collagen, colonies with branches (Fig. 4F) and bile duct structures also formed in culture. Moreover, immunofluorescence staining revealed that the expression of FHPC markers (CD133 and EpCAM) was downregulated in these bile duct structures (Fig. 4G–I). Taken together, these results indicated that the FHPCs differentiated into cholangiocytes in vitro.

The C-terminal region of HBx is required for the HBx-induced proliferation of FHPCs

Given that full-length HBx promotes the expansion and tumourigenicity of HPCs in DDC-treated mice (14), we considered whether HBx would also promote the proliferation of FHPCs, as well as the ability of HBx truncation mutants to maintain HBx activity. FHPCs transfected with pSI-HBV, pSI-HBVΔx, pSI-HA-x, pSI-HA-x1-101, pSI-HA-x43-154 and pSI-control were examined at 4 days post transfection (Fig. 5A). It was worth noting that in the FHPCs transfected with full-length HBx, spheroid colonies formed in culture, as observed in previous studies (14), whereas no spheroids were observed with the HBx truncation mutants. The transfected cells were collected, and HBc expression was analysed by western blot analysis. HBx expression was detected using a combination of immunoprecipitation and western blotting. We considered whether the HBx protein is possibly influenced by other HBV proteins. Of note, the HBc protein levels showed no change in the pSI-HBV-and pSI-HBVΔx-transfected HPCs (Fig. 5B) and were consistent with previous results in mouse hepatocytes (3). Notably, we found that HBx protein levels were higher after HBx transfection when the protein amount to 200 μg. The levels of HBx43-154 reach approximately the same level as full-length HBx, but the expression of HBx1-101 was lower than that of full-length HBx (Fig. 5C). These results are consistent with the findings of previous studies (3,38) on mouse hepatocytes and HepG2 cells, which showed that C-terminal deletion result in lower levels of HBx expression, whereas N-terminal deletion had no significant effects. In addition, FHPCs transfected with pSI-HA-x and pSI-HA-x43–154 permitted subculturing for 1 month, with a minimum of 5 passages. Collectively, these findings indicated that the C-terminal region of HBx was required for HBx proliferation, but was not necessary for the formation of spheroids.

Full-length and N-terminally deleted HBx promote clone formation in FHPCs

Next, 1,000 small cell clusters transfected with pSI-HA-x, pSI-HA-x1–101, pSI-HA-x43–154 and pSI-control were subcultured in 6-well plates. Small cell clusters attached and extended after 1 week, and compared to the pSI-control (Fig. 5D), the colonies formed by the pSI-HA-x transfected FHPCs (Fig. 5E) were larger in size and greater in number. Moreover, colonies were small and growed slowly after pSI-HA-x1–101 (Fig. 5F) transfection; however, the colonies formed by the PSI-HA-x43-154-transfected cells (Fig. 5G) exhibited no significant difference compared to the pSI-HA-x-transfected cells, which indicated that the C-terminal region of HBx was necessary for HBx to promote the proliferation of FHPC colonies. However, it remains unclear precisely how the C-terminal region affects the function of HBx in FHPCs and whether the C-terminal region specifically participates in signalling pathways [interleukin-6 (IL-6)/STAT3 activities, and Wnt/β-catenin] that enable the proliferation and tumourigenicity of FHPCs.

Discussion

HCC is associated with a very high mortality rate, and the recurrence and metastasis of HCC have not been targeted by specific treatments. In recent years, TISCs have been found in HCC (10,11,13,22,39), and the association between stem cells and TISCs has attracted increasing attention (10,22,3841). Rat ED14 FHPCs exhibited 95% homogeneity, as well as the cell culture and gene expression characteristics of HPCs (24). Early studies also indicated that over 60% of cells derived from E14.5 Dlk+ foetal mice were also positive for both ALB and CK19, which became downregulated during liver development (25). In our study, enriched FHPCs were concentrated in the 50% Percoll™ layer following PDGC, whereas the 20% Percoll™ layer contained the hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented (CDE) diet (18).

Human mammary epithelial cells proliferate in suspension, forming non-adherent mammospheres (27) that are enriched in progenitor/stem cells. Hepatic spheroids suspended in serum-free Kubota's medium (KM) (34,42) and seeded onto tissue culture plastic or embryonic stromal cell feeders can form cell colonies with two distinct morphologies: hepatoblasts and hHpSCs (34), and the hHpSCs can transition to hepatoblasts (17). Notably, it has been demonstrated that EpCAM+ HCCs can also form spheroids efficiently (10). Consistent with these observations, we demonstrated that the FHPCs formed spheroids at low concentrations of collagen. Furthermore, these spheroids were positive for EpCAM, CD133, CD49f, CK19 and ALB. Previous studies have shown that the hHpSC phenotypic profile includes neural cell adhesion molecule (NCAM), CD133 and CK8,/18/19 (17,34). Mouse HPCs express CD49f (18,42), CD133, Dlk/Pref-1, CK19 (25) and TROP2 (36), but are negative for Thy-1, CD11b, CD31, CD34, c-kit and CD45 (16,33). EpCAM was considered in all parenchymal cells and biliary epithelial cells, as a marker identifying HPCs in humans (17,34), TISCs in HCCs (10) and HPCs in adult mice administered a DDC diet (14,36) or a partial hepatectomy (PH) model (29).

Compared with FHPCs attached as a monolayer, the spherical cells proliferated more rapidly. The spherical FHPCs could also grow on semi-solid agar and tended to retain the morphology and characteristics of stem cells compared with growth in rat tail collagen. It is known that hepatic oval cells from adult mice stop growing on semi-solid agar (18), which seems to be in conflict with the findings of our study; there may exist some diversity in terms of the tumourigenicity of adult mice hepatic oval cells and FHPCs.

FHPCs converted into mature hepatocytes and bile duct epithelial cells when cultured under conditions that facilitate differentiation. Our data further suggested that FHPCs differentiated into both hepatocytes and cholangiocytes upon exposure to EGF, HGF and DMSO. Matrigel has been used to maintain and differentiate cholangiocytes (23,29,33,35). Of note, collagen also shared this ability, but few colonies tended to form biliary epithelial cell under these conditions.

The study by Wang et al (14) showed that HBx prompted intrinsic cellular transformation by stimulating the expansion and tumourigenicity of HPCs. Lizzano et al (41) found that the C-terminal region of HBx was crucial for the stability and function of HBx, and Hodgson et al (31) demonstrated that the carboxyl region was required for maximal HBV replication in HepG2 cells both in vitro and in vivo. Notably, we confirmed that the C-terminus of HBx is essential for its capacity to stimulate the proliferation of FHPCs, but is not necessary for the formation of spheroids, similar to hepatic cancer stem cells; we conjecture that the truncation mutants of HBx downregulate its tumourigenicity in FHPCs. Collectively, our study provides preliminary evidence of the relevance between C-terminus and full-length HBx in FHPCs and lays the foundation for further studies of the exact mechanism and signalling pathways through which the C-terminus of HBx stimulates proliferation and tumourigenicity in FHPCs.

Acknowledgments

This study was supported by a grant from Key Program of Medical Science of Chongqing Health Bureau (no. 2013-1-019).

References

1 

Marrero JA: Hepatocellular carcinoma. Curr Opin Gastroenterol. 22:248–253. 2006. View Article : Google Scholar : PubMed/NCBI

2 

Benhenda S, Cougot D, Buendia MA and Neuveut C: Hepatitis B virus X protein molecular functions and its role in virus life cycle and pathogenesis. Adv Cancer Res. 103:75–109. 2009. View Article : Google Scholar : PubMed/NCBI

3 

Luo N, Cai Y, Zhang J, Tang W, Slagle BL, Wu X and He S: The C-terminal region of the hepatitis B virus X protein is required for its stimulation of HBV replication in primary mouse hepatocytes. Virus Res. 165:170–178. 2012. View Article : Google Scholar : PubMed/NCBI

4 

Bouchard MJ, Wang LH and Schneider RJ: Calcium signaling by HBx protein in hepatitis B virus DNA replication. Science. 294:2376–2378. 2001. View Article : Google Scholar : PubMed/NCBI

5 

Keasler VV, Hodgson AJ, Madden CR and Slagle BL: Hepatitis B virus HBx protein localized to the nucleus restores HBx-deficient virus replication in HepG2 cells and in vivo in hydrodynamically-injected mice. Virology. 390:122–129. 2009. View Article : Google Scholar : PubMed/NCBI

6 

Tsuge M, Hiraga N, Akiyama R, Tanaka S, Matsushita M, Mitsui F, Abe H, Kitamura S, Hatakeyama T, Kimura T, et al: HBx protein is indispensable for development of viraemia in human hepatocyte chimeric mice. J Gen Virol. 91:1854–1864. 2010. View Article : Google Scholar : PubMed/NCBI

7 

Newsome PN, Hussain MA and Theise ND: Hepatic oval cells: helping redefine a paradigm in stem cell biology. Curr Top Dev Biol. 61:1–28. 2004. View Article : Google Scholar : PubMed/NCBI

8 

Herrera MB, Bruno S, Buttiglieri S, Tetta C, Gatti S, Deregibus MC, Bussolati B and Camussi G: Isolation and characterization of a stem cell population from adult human liver. Stem Cells. 24:2840–2850. 2006. View Article : Google Scholar : PubMed/NCBI

9 

Rountree CB, Senadheera S, Mato JM, Crooks GM and Lu SC: Expansion of liver cancer stem cells during aging in methionine adenosyltransferase 1A-deficient mice. Hepatology. 47:1288–1297. 2008. View Article : Google Scholar : PubMed/NCBI

10 

Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, Jia H, Ye Q, Qin LX, Wauthier E, et al: EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology. 136:1012–1024. 2009. View Article : Google Scholar : PubMed/NCBI

11 

Ji J, Yamashita T, Budhu A, Forgues M, Jia HL, Li C, Deng C, Wauthier E, Reid LM, Ye QH, et al: Identification of microRNA-181 by genome-wide screening as a critical player in EpCAM-positive hepatic cancer stem cells. Hepatology. 50:472–480. 2009. View Article : Google Scholar : PubMed/NCBI

12 

Marquardt JU and Thorgeirsson SS: Stem cells in hepatocarcinogenesis: evidence from genomic data. Semin Liver Dis. 30:26–34. 2010. View Article : Google Scholar : PubMed/NCBI

13 

Rountree CB, Mishra L and Willenbring H: Stem cells in liver diseases and cancer: recent advances on the path to new therapies. Hepatology. 55:298–306. 2012. View Article : Google Scholar

14 

Wang C, Yang W, Yan HX, Luo T, Zhang J, Tang L, Wu FQ, Zhang HL, Yu LX, Zheng LY, et al: Hepatitis B virus X (HBx) induces tumorigenicity of hepatic progenitor cells in 3,5-diethoxy-carbonyl-1,4-dihydrocollidine-treated HBx transgenic mice. Hepatology. 55:108–120. 2012. View Article : Google Scholar

15 

Rountree CB, Barsky L, Ge S, Zhu J, Senadheera S and Crooks GM: A CD133-expressing murine liver oval cell population with bilineage potential. Stem Cells. 25:2419–2429. 2007. View Article : Google Scholar : PubMed/NCBI

16 

Dorrell C, Erker L, Schug J, Kopp JL, Canaday PS, Fox AJ, Smirnova O, Duncan AW, Finegold MJ, Sander M, et al: Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice. Genes Dev. 25:1193–1203. 2011. View Article : Google Scholar : PubMed/NCBI

17 

Turner R, Lozoya O, Wang Y, Cardinale V, Gaudio E, Alpini G, Mendel G, Wauthier E, Barbier C, Alvaro D, et al: Human hepatic stem cell and maturational liver lineage biology. Hepatology. 53:1035–1045. 2011. View Article : Google Scholar : PubMed/NCBI

18 

Tirnitz-Parker JE, Tonkin JN, Knight B, Olynyk JK and Yeoh GC: Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet. Int J Biochem Cell Biol. 39:2226–2239. 2007. View Article : Google Scholar : PubMed/NCBI

19 

Dumble ML, Croager EJ, Yeoh GC and Quail EA: Generation and characterization of p53 null trans-formed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma. Carcinogenesis. 23:435–445. 2002. View Article : Google Scholar : PubMed/NCBI

20 

Ding W, Mouzaki M, You H, Laird JC, Mato J, Lu SC and Rountree CB: CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis. Hepatology. 49:1277–1286. 2009. View Article : Google Scholar

21 

Rountree CB, Ding W, He L and Stiles B: Expansion of CD133-expressing liver cancer stem cells in liver-specific phosphatase and tensin homolog deleted on chromosome 10-deleted mice. Stem Cells. 27:290–299. 2009. View Article : Google Scholar

22 

Galicia VA, He L, Dang H, Kanel G, Vendryes C, French BA, Zeng N, Bayan JA, Ding W, Wang KS, et al: Expansion of hepatic tumor progenitor cells in Pten-null mice requires liver injury and is reversed by loss of AKT2. Gastroenterology. 139:2170–2182. 2010. View Article : Google Scholar : PubMed/NCBI

23 

Yu B, He ZY, You P, Han QW, Xiang D, Chen F, Wang MJ, Liu CC, Lin XW, Borjigin U, et al: Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors. Cell Stem Cell. 13:328–340. 2013. View Article : Google Scholar : PubMed/NCBI

24 

Oertel M, Menthena A, Chen YQ, Teisner B, Jensen CH and Shafritz DA: Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver. Gastroenterology. 134:823–832. 2008. View Article : Google Scholar : PubMed/NCBI

25 

Tanimizu N, Nishikawa M, Saito H, Tsujimura T and Miyajima A: Isolation of hepatoblasts based on the expression of Dlk/Pref-1. J Cell Sci. 116:1775–1786. 2003. View Article : Google Scholar : PubMed/NCBI

26 

Liu WH, Li R and Dou KF: Convenient and efficient enrichment of the CD133+ liver cells from rat fetal liver cells as a source of liver stem/progenitor cells. Stem Cell Rev. 7:94–102. 2011. View Article : Google Scholar

27 

Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ and Wicha MS: In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 17:1253–1270. 2003. View Article : Google Scholar : PubMed/NCBI

28 

Conigliaro A, Colletti M, Cicchini C, Guerra MT, Manfredini R, Zini R, Bordoni V, Siepi F, Leopizzi M, Tripodi M, et al: Isolation and characterization of a murine resident liver stem cell. Cell Death Differ. 15:123–133. 2008. View Article : Google Scholar

29 

Li WL, Su J, Yao YC, Tao XR, Yan YB, Yu HY, Wang XM, Li JX, Yang YJ, Lau JT, et al: Isolation and characterization of bipotent liver progenitor cells from adult mouse. Stem Cells. 24:322–332. 2006. View Article : Google Scholar

30 

Melegari M, Scaglioni PP and Wands JR: Cloning and characterization of a novel hepatitis B virus X binding protein that inhibits viral replication. J Virol. 72:1737–1743. 1998.PubMed/NCBI

31 

Hodgson AJ, Hyser JM, Keasler VV, Cang Y and Slagle BL: Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication. Virology. 426:73–82. 2012. View Article : Google Scholar : PubMed/NCBI

32 

Chen Q, Kon J, Ooe H, Sasaki K and Mitaka T: Selective proliferation of rat hepatocyte progenitor cells in serum-free culture. Nat Protoc. 2:1197–1205. 2007. View Article : Google Scholar : PubMed/NCBI

33 

Tsuchiya A, Heike T, Fujino H, Shiota M, Umeda K, Yoshimoto M, Matsuda Y, Ichida T, Aoyagi Y and Nakahata T: Long-term extensive expansion of mouse hepatic stem/progenitor cells in a novel serum-free culture system. Gastroenterology. 128:2089–2104. 2005. View Article : Google Scholar : PubMed/NCBI

34 

Schmelzer E, Zhang L, Bruce A, Wauthier E, Ludlow J, Yao HL, Moss N, Melhem A, McClelland R, Turner W, et al: Human hepatic stem cells from fetal and postnatal donors. J Exp Med. 204:1973–1987. 2007. View Article : Google Scholar : PubMed/NCBI

35 

Tanimizu N, Miyajima A and Mostov KE: Liver progenitor cells develop cholangiocyte-type epithelial polarity in three-dimensional culture. Mol Biol Cell. 18:1472–1479. 2007. View Article : Google Scholar : PubMed/NCBI

36 

Okabe M, Tsukahara Y, Tanaka M, Suzuki K, Saito S, Kamiya Y, Tsujimura T, Nakamura K and Miyajima A: Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver. Development. 136:1951–1960. 2009. View Article : Google Scholar : PubMed/NCBI

37 

Bi Y, Huang J, He Y, Huang J, He Y, Zhu GH, Su Y, He BC, Luo J, Wang Y, Kang Q, Luo Q, et al: Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells. J Cell Biochem. 108:295–303. 2009. View Article : Google Scholar : PubMed/NCBI

38 

Yang XR, Xu Y, Yu B, Zhou J, Qiu SJ, Shi GM, Zhang BH, Wu WZ, Shi YH, Wu B, et al: High expression levels of putative hepatic stem/progenitor cell biomarkers related to tumour angio-genesis and poor prognosis of hepatocellular carcinoma. Gut. 59:953–962. 2010. View Article : Google Scholar : PubMed/NCBI

39 

Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A, Nakauchi H and Taniguchi H: Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology. 44:240–251. 2006. View Article : Google Scholar : PubMed/NCBI

40 

Lee JS, Heo J, Libbrecht L, Chu IS, Kaposi-Novak P, Calvisi DF, Mikaelyan A, Roberts LR, Demetris AJ, Sun Z, et al: A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nat Med. 12:410–416. 2006. View Article : Google Scholar : PubMed/NCBI

41 

Lizzano RA, Yang B, Clippinger AJ and Bouchard MJ: The C-terminal region of the hepatitis B virus X protein is essential for its stability and function. Virus Res. 155:231–239. 2011. View Article : Google Scholar

42 

Kubota H and Reid LM: Clonogenic hepatoblasts, common precursors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proc Natl Acad Sci USA. 97:12132–12137. 2000. View Article : Google Scholar : PubMed/NCBI

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August-2017
Volume 40 Issue 2

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Spandidos Publications style
Yu L, Chen S, Luo N and He S: The C-terminus domain of the hepatitis B virus x protein stimulates the proliferation of mouse foetal hepatic progenitor cells, although it is not required for the formation of spheroids. Int J Mol Med 40: 400-410, 2017
APA
Yu, L., Chen, S., Luo, N., & He, S. (2017). The C-terminus domain of the hepatitis B virus x protein stimulates the proliferation of mouse foetal hepatic progenitor cells, although it is not required for the formation of spheroids. International Journal of Molecular Medicine, 40, 400-410. https://doi.org/10.3892/ijmm.2017.3026
MLA
Yu, L., Chen, S., Luo, N., He, S."The C-terminus domain of the hepatitis B virus x protein stimulates the proliferation of mouse foetal hepatic progenitor cells, although it is not required for the formation of spheroids". International Journal of Molecular Medicine 40.2 (2017): 400-410.
Chicago
Yu, L., Chen, S., Luo, N., He, S."The C-terminus domain of the hepatitis B virus x protein stimulates the proliferation of mouse foetal hepatic progenitor cells, although it is not required for the formation of spheroids". International Journal of Molecular Medicine 40, no. 2 (2017): 400-410. https://doi.org/10.3892/ijmm.2017.3026